University of Southern California Dissertations and Theses

The role of a novel integrin activator in the maintenance of stem cell niche and activity-induced synaptic structural modifications in Drosophila

(USC Thesis Other)

The role of a novel integrin activator in the maintenance of stem cell niche and activity-induced synaptic structural modifications in Drosophila

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content

THE ROLE OF A NOVEL INTEGRIN ACTIVATOR IN THE MAINTENANCE
OF STEM CELL NICHE AND ACTIVITY-INDUCED SYNAPTIC
STRUCTURAL MODIFICATIONS IN DROSOPHILA

By
Joo Yeun Lee

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirement for the Degree
DOCTOR OF PHILOSOPHY
(NEUROSCIENCE)

MAY 2017

Copyright 2017 Joo Yeun Lee
ii

ACKNOWLEDGMENTS
First of all, I would like to thank my supportive mentor Dr. Karen Chang who is
my role model in pursuing research career. I have always admired her inspiring ideas
and determination toward science, which have greatly influenced my intellectual
development. As science comprises ways in which unexpected discoveries are made,
there were positive findings but also numbers of downfall that sometimes come with
frustration. However, under her tireless guidance and encouragement even with a mind-
bending critique from reviewers, now I am at the last chapter of my Ph.D. journey. She
has always shown commitment to the project even at hardship and that trust allowed
me to be persistent and become an independent researcher. I would like to express my
sincere gratitude to Dr. Chang for incredible mentorship and support for shaping my
research to the completion.
I am also thankful for my Dissertation Committee members Dr. Jeannie Chen, Dr.
Alexandre Bonnin, Dr. Dion Dickman, and Dr. Wange Lu for offering productive
feedback and critical evaluation. Their insightful scientific inputs were invaluable to bring
my project fulfillment. I would also like to thanks the team of Chang lab, Dr. Junhua
Geng, Juhyun Lee, Dr. Jillian Shaw, Liping Wang, and Dr. Derek Sieburth and his lab
members who have been a great resource for discussions and compelling insights.
I am also grateful for our team members of Knowing Neurons, Kate Fehlhaber,
Anita Ramanathan and Dr. Jillian Shaw who provided me with a platform to pursue my
passion in artistic creation alongside being a neuroscientist. I am privileged to be a part
of creative neuroscience community and a recipient of the Next Generation Award from
the Society for Neuroscience.
Last but not least, I would like to send special thank to my family for their
iii

incredible patience, support and faith even when I have doubted myself. I couldn’t have
come this far without their unconditional love and wise advices that left indelible impact
on me and guided me to find my passion in science and art. I am grateful for endless
encouragement and laughter that remind me everyday how lucky I am to have them in
my life. I have gained a measure of strength and perseverance from my family day by
day that will carry me across the finish line of any goal that I set for myself. I am forever
in your debt for all the support. Thank you!

iv

TABLE OF CONTENTS

ACKNOWLEDGMENTS ii
LIST OF FIGURES vii
ABSTRACT ix
CHAPTER 1: Introduction 1
1.1 DNAJB11, ERDJ3, Shriveled 1
1.2 Stem Cell Niche 2
1.2.1 Stem Cell Niche Functions: Self Renewal and Differentiation 2
1.2.2 Drosophila testes as a Model for Niche Studies 6
1.2.3 Adhesion in the Stem Cell Niche: Integrin and Cadherin 9
1.3 Synaptic Plasticity 13
1.3.1 Activity-dependent Synaptic Remodeling 13
1.3.2 Integrin Signaling in Synaptic Plasticity 17
1.3.3 Drosophila Neuromuscular Junction as a Model System for
Studying Synaptic Plasticity 21
1.4 Summary 22

CHAPTER 2: Maintenance of Stem Cell Niche Integrity by a Novel Activator
of Integrin Signaling 25
2.1 Abstract 25
2.2 Introduction 26
2.3 Materials and Methods 29
2.4 Results 35
2.4.1 Deterioration of Hub Architecture in shriveled mutants 35
2.4.2 Shriveled Encodes a Conserved Protein 40
2.4.3 Shriveled Interacts with Integrin to Control Hub Anchoring 45
2.4.4 Shriveled is a Secreted Protein that Activates Integrin Signaling
in vitro 48
2.4.5 Secretion of Shriveled Activates Integrin Signaling and
Modulates E-cadherin Levels in vivo 53
2.4.6 Upregulation of Shv Preserves Stem Cell Niche During Aging 61
2.5 Discussion 64
v

2.5.1 Summary of Results 64
2.5.2 Maintenance of Stem Cell Niche Structure by Extracellular
Presence of Shv 64
2.5.3 Shv Interacts and Activates Integrin Signaling 65
2.5.4 Shv-dependent Signaling Modulates E-cadherin expression 66
2.5.5 Implication of Shv in Future Stem Cell Studies 67
2.6 Acknowledgments 68
2.7 Supplementary Figures 69

CHAPTER 3: Activity-induced Synaptic Structural Modification by an Activator
of Integrin Signaling at the Drosophila Neuromuscular Junction 78
3.1 Abstract 78
3.2 Introduction 79
3.3 Materials and Methods 82
3.4 Results 87
3.4.1 Shriveled is a Presynaptically Secreted Protein that Modulates
Synaptic Growth 87
3.4.2 Shv Modulates Integrin Receptor Activation at the NMJ 94
3.4.3 Shv is Released during Intense Activity to Acutely Activates
Integrin Signaling 98
3.4.4 Activation by Shv during Intense Activity Triggers Synapse
Maturation 102
3.4.5 Shv is Required for Functional Plasticity 108
3.5 Discussion 111
3.5.1 Summary of Results 111
3.5.2 Secretion of Shv Acutely Activates Integrin and Induces
Synaptic Structural Modification 112
3.5.3 Release of Shv is Selectively Regulated by Synaptic Activity
to Modulate Synaptic Growth and Maturation 113
3.6 Acknowledgments 115
3.7 Supplementary Figures 116

CHAPTER 4: Summary and Future Directions 123
4.1 Summary 123
vi

4.2 Shv-dependent Integrin Activation Regulates E-cadherin Levels
to Preserve Niche Integrity 124
4.3 Shv is a Novel Integrin Activator in vivo and in vitro 125
4.4 Activity-dependent Release of Shv Induces Rapid Synaptic
Remodeling 126
4.5 Future Directions 129

REFERENCES 131

vii

LIST OF FIGURES
CHAPTER 1
1.1 The Drosophila male and female germline and somatic stem cells 5
1.2 Structure and signaling mechanisms in Drosophila male stem cell
niche 8
1.3 Cadherin and integrin-mediated stem cell niche adhesion 12
1.4 Learning-induced morphological changes in dendritic spines 16
1.5 Integrin engagement during LTP 20

CHAPTER 2
2.1 Shv is required for the maintenance of hub and GSCs during aging 38
2.2 Shv is present and required in multiple cell types 42
2.3 Shv genetically interacts with βPS integrin signaling 47
2.4 Shv is a secreted protein that activates with βPS integrin through
outside-in signaling 50
2.5 Shv activates βPS integrin signaling in vivo 55
2.6 Shv regulates DE-cadherin level through βPS integrin 59
2.7 Upregulation of Shv prevents loss of hub and GSCs during aging 62
S2.1 Hub cell and CySC quantification in differnet ages and altered
hub architecture seen in shv
1
mutants 69
S2.2 Shv sequence alignment, transcript levels, and antibody specificity 70
S2.3 shv alleles 71
S2.4 Shv distribution in testes 72
S2.5 Restoring Shv in CySCs recues GSC loss phenotype 73
S2.6 Extracellular Shv activates integrin signaling 74
S2.7 Secretion of Shv regulates DE-cadherin levels 75

CHAPTER 3
3.1 Shriveled is a presynaptically secreted protein 89
3.2 Shriveled modulates synaptic growth 92
3.3 Shv activates integrin receptor at NMJ 96
viii

3.4 Shv is secreted during intense synaptic stimulation to acutely
activate integrin signaling 100
3.5 Shv release during intense activity induces synaptic maturation 104
3.6 Extracellular incubation of Shv is sufficient to trigger synapse
maturation 106
3.7 Shv regulates functional plasticity 110
S3.1 Quantification of pFAK level and number of ghost boutons 116
S3.2 Pulsed high K
+
stimulation 119
S3.3 Synaptic stimulation induces synapse maturation 121
S3.4 Shv and Shv
LNV
isolation 122

LIST OF TABLES

CHAPTER 2
Table 2.1 Hub cell quantification 39

CHAPTER 3
Table S3.1 Table indicating the number of samples used to determine
relative pFAK levels in Figure 3.3.C and 3.3.F 118

LIST OF VIDEOS

CHAPTER 2
Video S2.1 3D presentation of Drosophila stem cell niche 76
Video S2.2 3D presentation of Drosophila stem cell niche 77

ix

ABSTRACT
Cell adhesion molecules (CAM) mediate adhesion between cellular components
and play pivotal roles during the development and throughout the adult life of an
organism. Integrin, a heterodimer of α and β subunits, modulates adhesion between
cells and the extracellular matrix (ECM) to support physical structure of the cells and
mediate intracellular signaling cascade that govern cell survival and differentiation. Cell
surface integrin receptors have been implicated in optimizing stem cell niche
environment, and synaptic plasticity by stabilizing synaptic strength. The goal of this
dissertation is to identify the roles of a novel DNAJ domain protein, Shriveled (Shv), in
maintaining cellular integrity through integrin signaling pathway. Over the course of my
research, I investigated the genetic and functional interactions between Shv and integrin
by utilizing two different Drosophila model systems: 1) germline stem cell niche, and 2)
neuromuscular junction (NMJ). My data reveal that Shv activates integrin signaling in
vivo to enhance stem cell survival and modulate synaptic growth; therefore, I propose
that Shv functions as a novel activator of integrin signaling.
At the apical tip of Drosophila testes, germline stem cells (GSCs) and somatic
stem cells (CySCs) share the niche called the hub. Establishment of proper architecture
of the niche ensures dynamic stem cell behaviors and their functions. Depletion of Shv
results in deterioration of hub structure and decline in GSC, whereas upregulation of
Shv preserves age-dependent loss of stem cells. I find that Shv is a secreted protein
that activates integrin signaling, which in turn enhances E-cadherin expression to
maintain a healthy niche structure. Chapter 2 describes a new integration mechanism in
x

which Shv-mediated crosstalk between integrin and E-cadherin in stem cells serves to
promote a healthy niche structure in the Drosophila testes.
As integrin plays a crucial role in stabilizing the synaptic structure in response to
activity, I further examined the role of Shv in activity-induced synaptic remodeling at the
Drosophila neuromuscular junction in Chapter 3. Loss of Shv protein results in an
overgrowth of the NMJ structure and increased the number of undifferentiated synapse.
I demonstrate a new stimulation condition in which synapses undergo rapid structural
modification to achieve synaptic maturation in the form of local bouton enlargement and
increase in glutamate receptor abundance. This intense neuronal stimulation paradigm
selectively enhances Shv release at the synapse, which then promotes integrin
activation to rapidly stabilize the synaptic structure, presumably by recruiting additional
glutamate receptors to the surface. Since mild neuronal stimulation, which has been
shown to trigger new synapse formation, fails to induce such changes, my results imply
that synapse utilizes distinct machineries to adapt to the different stimuli. Importantly,
Shv is also required for normal post-tetanic potentiation in the context of functional
plasticity. Together, my data suggests that Shv is a trans-synaptic signal with its release
regulated by neuronal activity, which then activates integrin signaling to facilitate
synaptic remodeling process through integrin signaling.

1
Chapter 1
Introduction

1.1 Shriveled, The Drosophila Homolog of DNAJB11/Erdj3
DNAJB11, also known as Erdj3, is a co-chaperone protein that binds to unfolded
protein client and delivers it to immunoglobulin protein (BiP), thereby inducing
conformational change of Hsp70 to stabilize the binding to its substrates (Yu et al.,
2000; Shen and Hendershot, 2005; Kampinga and Craig, 2010; Guo and Snapp, 2013).
In addition to its relation with co-chaperoning pathway, DNAJB11 has been shown to
play a role in extracellular proteostasis upon ER stress (Genereux et al., 2014). Protein
aggregation pathology in the brain is a hallmark of Alzheimer’s disease (AD) and
studies have shown that unfolded protein response (UPR) signaling is activated in AD
brain to alleviate toxic build up of misfolded proteins (Teixeira et al., 2006; Hoozemans
et al., 2012). Genereux et al. reported that DNAJB11 is a secretable chaperone protein
that inhibits extracellular Aβ aggregation and protects extracellular environment from
toxic proteins (Genereux et al., 2014). In mammalian cephalic neural crest cell (CNCC),
DNAJB11 is secreted and binds to protogenin, a member of immunoglobulin
superfamily, which in turn recruits downstream molecule to induce conformational
change of integrin thereby promoting cell migration during neurogenesis (Wong et al.,
2010; Wang et al., 2013). While these studies indicate DNAJB11 as a secreted co-
chaperone protein and a putative ligand of protogenin, in this dissertation, I present
evidence that DNAJB11 functions as a novel binding partner of integrin receptor to
2
maintain stem cell niche structure in Drosophila testes as well as modulate synaptic
remodeling at Drosophila neuromuscular junction.
Dr. Chang isolated a Drosophila mutant exhibiting shorter life span, which also
displays male specific sterility and age-dependent decrease in testes size. We named
the mutant shriveled (shv) after its strikingly shrunken testes phenotype. Molecular
cloning revealed that Shv protein shares 63% similarity in its amino acid sequence with
human DNAJB11 (Erdj3) (Lee et al., 2016). DNAJB11 contains a signal peptide at the
N-terminus, a putative nuclear localization signal, and a RGD motif found in integrin
binding proteins (Ohtsuka and Hata, 2000; Shen and Hendershot, 2005; Humphries,
2006; Ludvigsen et al., 2009).
The focus of this dissertation is to explore the roles of Shv in two different model
systems of Drosophila: germline stem cell niche and neuromuscular junction. Our study
revealed that Shv is a secreted protein that binds to and activates integrin, which in turn
modulates E-cadherin level to uphold a healthy stem cell niche during aging (Lee et al.,
2016). Interestingly, secreted Shv also acts at the Drosophila NMJ to induce activity-
dependent modification of synaptic structure. These findings suggest a novel function of
Shv (DNAJB11/Erdj3) as an activator of integrin besides involvement in co-chaperoning
pathway.

1.2 Stem Cell Niches
1.2.1 Stem Cell Niche Functions: Self-Renewal and Differentiation
Established characteristics of adult stem cells are their ability to undergo self-
renewal and generate differentiating daughter cells that contribute to tissue repair and
3
regeneration. To preserve their “stemness” identity, local microenvironment, or niche is
required (Schofield, 1978). Stem cell niches provide tightly regulated signals to the stem
cells to control their behavior; quiescence, maintenance and proliferation (Jones and
Wagers, 2008; Morrison and Spradling, 2008). Stem cells are present in many tissues
including brain, bone, skin, muscle, and blood to replace and replenish injured or
damaged cells throughout life (Morrison and Spradling, 2008; Bussolati, 2014).
Uncovering mechanisms by which stem cell niche precisely calibrates self-renewal and
differentiation could have profound impact on understanding of regenerative capacity of
tissues. Generally, stem cell niche are classified into two types by their contact partner:
1) stromal niche forms direct contact with stem cells, and 2) epithelial niche are devoid
of niche support cells and make contact with basal membrane (Song and Xie, 2002;
Song et al., 2002; Morrison and Spradling, 2008). Example of stromal niche includes
Drosophila germline stem cell niche, which is among the first studies to yield insight into
underlying molecular mechanism of stem cell niche dynamics due to it’s simple anatomy,
which permits single cell resolution of the stem cell niche structure (Fuller, 1998; Lin,
2002; Yamashita et al., 2005; Fuller and Spradling, 2007). In order to maintain tissue
homeostasis, stem cells undergo asymmetric cell division, which results in two daughter
cells having different cell fates. A cluster of stromal cells (called the hub cell) in
Drosophila gonads secretes inductive molecules such as Unpaired (Upd) and Bone
Morphogenic Protein (BMP) ligand to their neighboring germline stem cells (GSC) to
control stem cell behavior (Kiger, 2001; Tulina and Matunis, 2001; Kawase, 2004; Singh
et al., 2005; Issigonis et al., 2009). Moreover, proper orientation of mitotic spindle
ensures cell fate of stem cells where a stem cell with the spindle oriented toward niche
4
support cells generates one daughter cell with retained stem cell identity and the other
daughter cell displaced away from the niche undergoes differentiation as a
consequence of physical distance from the hub cells (Yamashita et al., 2003).
Mammalian hematopoietic stem cell (HSC) primarily reside in mammalian bone
marrow exhibits a unique characteristic of homing and migration through vasculature to
defined microenvironment in different tissues (Lapidot, 2005; Laird et al., 2008). Thus,
precise signals from specific environment are essential to control cell fate of
hematopoietic stem cell: chemokine CXCL12 regulates HSC maintenance (Sugiyama et
al., 2006), Angiopoietin-1 (Tie2) is to proposed to control quiescence (Arai et al., 2004),
and Notch signaling and Wnt promotes self-renewal (Willert et al., 2003; Fleming et al.,
2008). HSC niches are composed of distinct cell population including osteoblasts,
mesenchymal stem cell, vascular endothelia cells and Schwann cell (Wang and
Wagers, 2011). In order to succeed in long-term repopoulation of hematopoietic stem
cell, proper localization of HSC by engraftment and migration to their niche is imperative.
Cell adhesion to the niche or ECM-mediated cell adhesion is crucial in controlling stem
cell anchorage and homing, and also chemokine gradient is essential in directing
migration toward supportive stem cell niche (Sahin and Buitenhuis, 2012; Gattazzo et
al., 2014).

5

Figure 1.1 The Drosophila male and female germline and somatic stem cells. Post-
mitotic stromal hub cells (green) are surrounded by germline stem cells (GSC, red),
which is enveloped by two somatic stem cells (CySC, blue). Differentiated daughter cell
(gonialblast in male, cytoblast in female) undergoes transit-amplifying division to give
rise to 16 spermatogonia/cytocytes (Fuller and Spradling, 2007).

6
1.2.2 The Drosophila Testes as a Model for Niche Studies
The Drosophila germline stem cells in testes and ovary are among the well-
established model system of adult stem cells. At the apical tip of each reproductive
organ, anatomically simple stem cell niches house easily identifiable stem cells that give
rise to sperm and eggs (Fuller, 1998; Fuller and Spradling, 2007; Spradling et al., 2011).
In Drosophila testes, germline stem cells (GSC) and somatic stem cells (CySC) resides
in common niche formed by hub cells. Hub cells are composed of ~10 post-mitotic
somatic cells and surrounded by 6-10 GSCs and CySCs, which envelop the developing
GSCs (Hardy et al., 1979; Boyle et al., 2007). GSC and CySC maintain their stemness
through activated JAK-STAT signaling (Janus kinase and signal transducer and
activator of transcription) by ligand Unpaired (Upd) secreted from hub cell, while the cell
that lies outside the niche becomes gonialblast due to lack of exposure to Upd signal
and subsequent failure of JAK-STAT activation (Kiger, 2001; Tulina and Matunis, 2001).
In addition, Bone Morphogenic Protein (BMP) such as Glass bottom boat (Gbb) and
Decapentaplegic (Dpp) signaling are secreted from the niche and CySC thereby
repressing expression of differentiation factor, Bag of Marble (Bam), to promote GSC
self-renewal (Xie and Spradling, 1998; Kawase, 2004; Leatherman and DiNardo, 2010).
Similar to male stem cell niche, in Drosophila ovary, daughter cell that is only one-cell
distance away from the niche failed to receive BMP signal resulting in germ cell
differentiation into cytoblast (Xie and Spradling, 1998; Chen, 2003). A differentiated
gonialblasts undergo four rounds of transit amplifying mitotic division to produce a
cluster of 16 spermatogonial cells enclosed by two cyst cells (Fuller, 1998) (depicted in
Fig. 1.1). In addition to molecular signals from the niche, GSCs receives signals from
7
neighboring CySCs to prevent excessive proliferation of stem cell population by
activating epidermal growth factor receptor (EGFR) signaling pathway. Disruption of
EGFR signaling and downstream mediator Raf in CySCs results in accumulation of
undifferentiated GSCs suggesting that inputs from CySCs are also important to keep
balanced population of stem cells (Kiger et al., 2000; Tran et al., 2000). Sustained
activation of JAK-STAT in CySC results in activation of transcription repressor, Zfh-1,
which causes expanded pool of both early somatic and germ cells suggesting a
communication between GSC and CySC is essential in maintaining GSC self-renewal
(Leatherman and DiNardo, 2010). Interestingly, conditional knockdown of JAK-STAT
signaling using temperature sensitive allele stat92E induced dedifferentiation of
spermatogonia (transit-amplifying cells) into regaining stem cell identity thereby
replenishing GSC pools (Brawley, 2004). Moreover, Cheng et al. found that
dedifferentiated GSCs are functional stem cell capable of reentering cell cycle to
undergo asymmetric division (Cheng et al., 2008). These insightful findings suggest that
self-renewal of stem cell is not only orchestrated by the niche but also requires signals
from neighboring stem cells; therefore understanding how molecular cues that
coordinates communication between different stem cell population may contribute to
other stem cell systems.

8

Figure 1.2 Structure and signaling mechanism of Drosophila male stem cell niche.
The hub cell secretes Unpaired, which activates JAK-STAT signaling pathway in
adjacent GSC and CySC to promote self-renewal. Also, BMP-like ligands are secreted
from hub and CySC to repress Bam expression to maintain stem cell identity revealing
dynamic communication between stem cell to ensure tissue homeostasis (Hsu and
Fuchs, 2012).

9
1.2.3 Adhesion in the Stem Cell Niche: Integrin and Cadherin
As key instructive molecules secreted from stem cell niche are short-rage signals
acting on adjacent stem cells to regulate self-renewal, retaining intimate distance
between the niche and stem cells is integral to maintain stem cell self-renewal and
differentiation; therefore adhesive forces are indispensible for anchoring the niche in
desired location and stem cells to their niche (Xi, 2009; de Cuevas and Matunis, 2011;
Bulgakova et al., 2012). Cell adhesion molecules (CAM), particularly integrins and
cadherins are known to serve such functions in Drosophila testes (Wang et al., 2006; Xi,
2009; Bulgakova et al., 2012; Lee et al., 2016).
Cadherins mediate cell-cell adhesion through a homophilic interaction and
provides anchor sites for actin cytoskeleton via α-catenin to enhance adherent junction
(Jou et al., 1995). The Drosophila E-cadherin (Shotgun) and β-catenin (Armadillo) are
highly accumulated at the membrane of hub cells where GSC and CySC make physical
contact (Song et al., 2002). Moreover, E-cadherin loss of function results in GSC drifting
away from their niche and become differentiated into gonialblast indicating that E-
cadherin is necessary for maintenance of stem cell niche (Song and Xie, 2002; Song et
al., 2002; Voog et al., 2008). Rap-GEP (Gef26) Drosophila mutants display impaired E-
cadherin-mediated adherent junction at the hub-GSC, which results in detachment of
both GSC and CySC from the niche demonstrating that proper cell adhesion is
significant to maintain stem cell identity (Wang et al., 2006).
Integrins are cell surface receptors that mediate adhesion between cell and
extracellular matrix (ECM) by connecting ECM protein to intracellular actin cytoskeleton.
They are composed of α and β subunits, which form transmembrane heterodimers. In
10
mammalian testes and adult hematopoietic stem cell, β1 integrin serve as essential
adhesion molecules in promoting stem cell homing to their niche via interaction with
basal membrane (Potocnik et al., 2000; Kanatsu-Shinohara et al., 2008). Similarly, in
Drosophila testes, βPS integrin, encoded by myospheroid (mys) is necessary to anchor
the hub at the apex of testes through interaction with ECM protein (Tanentzapf et al.,
2007; Lee et al., 2008; 2016). Deletion of Mys during gonad morphogenesis results in
failure of anchoring hub to the ECM that eventually leads to a loss of the hub and stem
cell population underscoring the importance of integrin-mediated ECM assembly in
proper niche positioning (Tanentzapf et al., 2007). Integrin receptors are highly
concentrated at the interface between hub-CySC of male stem cell niche, and its
expression is thought to be regulated by Socs36E, an inhibitor of JAK-STAT pathway.
Elevated level of integrin at the CySC caused by absence of Socs36E appears to out-
compete GSC by pushing GSC out of the niche (Issigonis et al., 2009). Moreover, Hox
transcription factor, Abdominal-B (Abd) in spermatocytes of larval testes has shown to
regulate hub positioning and niche architecture by controlling localization of integrin
through tyrosine-kinase Sevenless (Sev) signaling pathway (Papagiannouli et al., 2014).
These studies imply that integrin plays a crucial role in preserving stem cell niche
structure and stem cell population.
Proper stem cell function requires correct stem cell niche architecture and
positioning to secure stem cell population, and adhesion molecules are essential in
holding stem cell within the niche to govern short distance signals that regulates
balance between stem cell self-renewal and proliferation. Therefore, identifying intrinsic
and extrinsic cues modulating cellular adhesion molecules activity is of great interest
11
and will provide insight into underlying mechanism of preserving a healthy stem cell
environment.

12

Figure 1.3 Cadherin and Integrin-mediated stem cell niche adhesion. Cadherin,
which mediates cell-cell adhesion, is indispensible in maintenance of the niche structure
by promoting adhesion of GSC to the hub. Integrin-mediated cell-ECM adhesion is
necessary for proper anchoring of the hub at the apical tip of the testes. (Chen et al.,
2012).

13
1.3 Synaptic Plasticity
1.3.1 Activity-dependent Synaptic Remodeling
A synapse is a fundamental structure that is highly dynamic that creates complex
wiring of the nervous system. The formation of neural circuits is a long-term process of
continuous refinement and modification of synaptic connections throughout lifetime.
Fundamentally, it requires a neuron’s ability to find the suitable target in order to form a
functional synaptic network that is amenable to the environment. These plastic
properties of synapse are coordinated by interplay of various events such as
neurogenesis, differentiation, migration and synaptogenesis to ensure proper
development and promote complex brain functions like learning and memory (Martin
and Morris, 2002; Chklovskii et al., 2004; Ming and Song, 2005). From invertebrates to
mammalian, studies have proved that persistence of learning is associated with the
induction of changes in synaptic weight, which leads to rearrangements of synaptic
structures (Xie et al., 2007; Holtmaat and Svoboda, 2009; Caroni et al., 2012; Takeuchi
et al., 2014). The concept of structural plasticity encompasses remodeling of pre-
existing synaptic structures, formation or elimination of synapses yielding in functional
consequences. This morphological variability is dependent on the pattern of activity that
they experience, and the size of dendritic spine heads directly reflects the synaptic
strength (Chicurel and Harris, 1992; Matsuzaki et al., 2001; Yuste and Bonhoeffer,
2001; Lamprecht and LeDoux, 2004; Caroni et al., 2012). Pathologies of dendritic spine
distribution and shapes have been observed in association with the number of
neurodevelopmental and psychiatric disorders (Multani et al., 1994; Kelley, 1997; Garey
et al., 1998; Jiang et al., 1998; Glantz and Lewis, 2000; Swann et al., 2000; Fiala et al.,
14
2002; Lamprecht and LeDoux, 2004; Xie et al., 2007; Lewis, 2009). For example, in
hippocampal and neocortical pyramidal neurons from human epilepsy tissue and
comparable animal models show major decrease in dendritic spine density and
complexity (Multani et al., 1994; Jiang et al., 1998; Swann et al., 2000; Fiala et al.,
2002). Similarly, aberrant dendritic spine density have been observed in postmortem
schizophrenic tissues (Kelley, 1997; Garey et al., 1998; Fiala et al., 2002), which
appears to involve a reduction of the excitatory connection in a subset of pyramidal
neurons (Kelley, 1997; Glantz and Lewis, 2000; Fiala et al., 2002; Lewis, 2009). Such
plastic changes of structure are mediated by synaptic machineries that govern gene
transcription and post-translational modification of synaptic molecules thereby
contributing stabilization of the neural network, or long-term potentiation (LTP)
(Berninger and Bi, 2002; Lamprecht and LeDoux, 2004; Je et al., 2006; Cohen and
Greenberg, 2008; Kim et al., 2015; Vallejo et al., 2016). In experiment with adult
mammalian hippocampal neurons, it was found that during LTP induction, synapse
undergoes rapid morphological changes by rearranging the cytoskeleton in less than 2
minutes to induce synapse enlargement or increase the number of spine heads to
accommodate additional insertion of glutamate receptor thereby consolidating
enhanced synaptic transmission (Van Harreveld and Fifkova, 1975; Malinow and
Malenka, 2002; Lamprecht and LeDoux, 2004; Matsuzaki et al., 2004; Kramár et al.,
2006). In addition, widening of spine neck affects magnitude of postsynaptic calcium
influx, which is necessary for the induction of LTP (Majewska et al., 2000; Graupner,
2010), and polyribosomes are selectively distributed to dendritic spine of the enlarged
synapse and facilitate local protein synthesis to sustain long-term potentiation (Ostroff et
15
al., 2002; Pfeiffer and Huber, 2006). Therefore, the morphological characteristics of
synapses accounts for revealing the functional efficacy and stability of synapse.
Among these cellular events occur during LTP, actin cytoskeleton plays pivotal
role in mediating the architecture of synapse and perturbation of actin polymerization
results in impaired LTP maintenance and modifying synaptic structure (Matsuzaki et al.,
2004; Okamoto et al., 2004; Bramham, 2008; Honkura et al., 2008). Postsynaptic actin
filaments (F-actin) are capable of undergoing rapid polymerization and LTP induction
leads to pronounced increase in polymerized F-actin in dendritic spine resulting in
expansion of the synaptic structure (Allison et al., 1998; Fischer et al., 1998; Kramar et
al., 2002; Lamprecht and LeDoux, 2004; Okamoto et al., 2004). Moreover, excitatory
AMPA receptors are clustered in the postsynaptic density via actin cytoskeleton, which
also controls receptor trafficking and insertion into the surface of synapse (Zhou et al.,
2001; Lamprecht and LeDoux, 2004; Okamoto et al., 2004). Actin dynamics is tightly
regulated by cohort of signaling proteins, and cell adhesion molecules (CAM) are one of
the candidates that regulates actin polymerization and adhesive forces between pre-
and postsynaptic terminal.

16

Figure 1.4 Learning-induced morphological changes in dendritic spines. Long-
term potentiation induces changes in spine head morphology accompanied by increase
in postsynaptic receptor distribution, calcium influx and ribosome within spines.
Rearrangement of spine head requires cytoskeletal regulation (Lamprecht and LeDoux,
2004).

17
1.3.2 Integrin Receptor Activation in Synaptic Plasticity
Learning-induced synapse is preferentially stabilized to consolidate the structural
changes (Lamprecht and LeDoux, 2004; Okamoto et al., 2004; Xu et al., 2009; Arikkath,
2010; Caroni et al., 2012). Stabilization of synapse involves several molecular
machineries including growth factors, dynactin and cell adhesion molecules (CAM)
(Beumer et al., 2002; Eaton et al., 2002; Eaton and Davis, 2005; Kramár et al., 2006;
Jia et al., 2008; Arikkath, 2010). For the synapse to properly transduce trans-synaptic
signaling, presynaptic machineries must be precisely aligned with apposing
postsynaptic receptors, and adhesive force between the pre- and postsynaptic terminal
are known to play a predominant role in maintaining structural integrity and stability of
the synapse (Sanes and Lichtman, 1999; Benson et al., 2000; Yamagata et al., 2003;
Sindi et al., 2014); therefore, adhesion molecules are essential component in ensuring
synaptic efficacy (Stäubli et al., 1998; Murase et al., 2002; Togashi et al., 2002). CAM
have been implicated in activity-dependent structural modification process in which they
create a physical link between extracellular cues and actin cytoskeleton thereby
activating intracellular signaling pathways to meet external synaptic demand (Dityatev
and Schachner, 2003; Yamagata et al., 2003; Arikkath, 2010). Integrins have been
shown to serve such functions in Drosophila and mammalian nervous system to
stabilize both structural and functional changes in response to the synaptic activity.
(Rohrbough et al., 2000; Beumer et al., 2002; Kramár et al., 2006; McGeachie et al.,
2011; Tsai et al., 2012a).
Integrins are αβ heterodimeric transmembrane receptors that create a bridge
between extracellular matrix (ECM) environment and the intracellular actin cytoskeleton
18
permitting local cell morphology alterations (Brakebusch and Fässler, 2003; Shattil et
al., 2010; Kim et al., 2011). Integrins can signal bidirectionally across the membrane to
mediate cell-ECM adhesion: 1) outside-in signaling occurs when RGD-containing ligand
binds to extracellular domain of integrins, which induces integrin clustering and
recruitment of downstream signaling kinases to its cytoplasmic domain (Qin et al., 2004;
Shattil et al., 2010; Kim et al., 2011); 2) an intracellular signaling event can also trigger
conformational change of integrin to a high affinity state to achieve inside-out signaling
(Qin et al., 2004; Harburger and Calderwood, 2009; Shattil et al., 2010).
The Drosophila has successfully been used to discover molecular mechanisms
underlying synaptic plasticity. In Drosophila glutamatergic neuromuscular junction
(NMJ), there are 5 alpha (αPS1-5) and 2 beta integrin subunits (βPS and βν) (Brown,
1993; Gotwals et al., 1994). Experimental evidences indicates that αPS3 integrin is
required for functional synaptic plasticity process as αPS3 (volado) mutant displayed
deficit in learning and memory (Grotewiel et al., 1998; Rohrbough et al., 2000).
Moreover, larval crawling activity induces laminin release, which then regulates
activation of βν integrin to promote overall NMJ growth (Tsai et al., 2012a). In
mammalian hippocampal neurons, blockade of integrin activation by ligand mimetic
peptide containing Arg-Gly-Asp (RGD) consensus binding sequence, which acts as
integrin antagonists reverses LTP when it is applied 10 min after LTP induction implying
that integrin contributes to the late stage of LTP consolidation rather than early phase
(Bahr et al., 1997; Stäubli et al., 1998). Moreover, function-blocking antibodies to β
1

family integrin have shown to interfere with LTP stabilization and actin polymerization
19
event, again underscoring the role of integrin in LTP consolidation steps where it
involves rapid stabilization of synaptic structure and function.
Therefore, the findings from these studies suggests disengagement of adhesion
molecules at the cleft to induce LTP-associated synapse enlargement (McGeachie et
al., 2011), and disassembly of F-actin actin network creates shorter actin filament to
reorganize the structure in order to accommodate insertion of additional postsynaptic
receptor (Malinow and Malenka, 2002; Kramár et al., 2006; Chen et al., 2015; Stefen et
al., 2016). Subsequent consolidation step engages integrin receptors where new
integrins are inserted into the membrane, and its activation regulates ECM reassembly
and intracellular actin polymerization to further stabilize LTP (Gall and Lynch, 2005; Lin
et al., 2005; McGeachie et al., 2011) (depicted in Fig. 1.5).

20

Figure 1.5 Integrin engagement during LTP. Immediately after LTP induction, integrin
changes its conformation into inactive status and subsequent disassembly of ECM-actin
cytoskeleton to accommodate physical expansion of the postsynaptic terminal.
Additional AMPA receptors are inserted to enlarged postsynaptic surface to consolidate,
thus enhancing synaptic transmission (McGeachie et al., 2011).

21
1.3.3 Drosophila Melanogaster as a Model System for Studying Synaptic
Remodeling
Drosophila larval neuromuscular junction has provided valuable insights into
molecular and cellular mechanisms involved in synaptic development, function, and
plasticity with its powerful genetics and well-conserved signaling pathway. The larval
NMJ continuously undergoes synaptic remodeling event by forming and stabilizing the
new synapse to ensure proper synaptic transmission, therefore it enables us to
investigate how synaptic activity induces structural and functional changes to meet
synaptic demands. The structure of Drosophila NMJ is stereotypic, which allows easy
accessible analysis of structural and functional properties during synaptic activity.
Atament et al. have shown that larval NMJ displays an undifferentiated structure called
“ghost bouton” characterized by lack of pre- and postsynaptic proteins (Ataman et al.,
2006; 2008). The appearance of ghost bouton is normal physiological process during
NMJ development, however Budnik group uncovered that synaptic activity induced by
external pulse of high K
+
depolarization causes greater increase in ghost bouton
budding and enhances Wingless (Wg) secretion to activate Dfrizzled-2 (DFz2) receptor
indicating that synapse undergoes rapid structural modification in Wg-dependent
fashion in response to patterned stimulation (Ataman et al., 2008). Interestingly, they
found that this transient ghost bouton eventually differentiates into a functional synapse
equipped with postsynaptic glutamate receptors and active zones (Ataman et al., 2008).
On the other hands, high frequency light stimulation of motorneurons enhanced
clearance of immature ghost bouton that failed to form stable postsynaptic contact
(Fuentes-Medel et al., 2009). Together, these findings suggest that larval NMJ exhibits
22
dynamic process of synaptic remodeling and serves as an excellent model system to
study structural modification in response to synaptic activity.
In the Drosophila glutamatergic NMJ, studies reported the number of integrin
ligands such as Tiggrin (Fogerty et al., 1994), Teneurin (Graner et al., 1998; Mosca,
2015), and laminin A (Tsai et al., 2012a). Laminin A is one of extracellular matrix (ECM)
components that activates integrin signaling transduction (Humphries, 2006; Takagi,
2007) and only known ligand that its secretion is regulated by activity (Tsai et al.,
2012a). Synaptic activity downregulates retrograde laminin secretion from the
postsynaptic muscle cell to accommodate synaptic growth by reducing integrin
activation (Tsai et al., 2012a). In this dissertation, I present evidences that Shv, an
novel activator of integrin (Lee et al., 2016), is acutely released from presynaptic
terminal in activity-dependent manner to promote rapid structural remodeling at the
Drosophila NMJ. I propose a stimulation paradigm that selectively triggers Shv release
to promote local synaptic bouton enlargement and increases in postsynaptic glutamate
receptor abundance at the synapse.

1.4 Summary
Cell adhesion molecules (CAM) are key players in maintaining virtually every
biological process to ensure efficient communication between cellular components.
Integrin is one of the core cell adhesion molecules involved in a wide range of
physiological processes by promoting structural integrity. Although studies have
revealed that integrin signaling is implicated in governing stem cell survival and
maintaining synaptic potentiation, the molecular cue that regulates integrin activation
23
still remains unsolved. In this dissertation, I present a novel protein, Shriveled (Shv), as
a positive regulator of integrin signaling in two different systems in Drosophila: germline
stem cell niche and neuromuscular junction.
The Drosophila germline stem cell niche serves as a great in vivo model system
for studying molecular pathways contributing stem cell behaviors in all animals. Using
this effective model system, I characterize the role of Shv in maintaining a healthy stem
cell environment that involves integrin activation. Shv activates integrin signaling at the
somatic stem cell (CySC) to assure proper anchoring of stem cell niche at the tip of the
testes. In addition, I demonstrate that Shv not only activates integrin signaling but also
regulates E-cadherin expression to prevent age-dependent loss of germline stem cell
(GSC). Integrin and E-cadherin is thought to work in separate pathways involving hub
anchoring and GSC attachment. Surprisingly, my research reveals a Shv-coordinated
crosstalk between integrin and E-cadherin in preserving structural maintenance of stem
cell niche.
Integrin is a transynaptic receptor that plays important roles in synaptic
development and synaptic plasticity by contributing to the structural refinement. Integrin
signaling is associated with consolidating synaptic potentiation by aiding structural
support through enhancing adhesive forces between the terminal and regulating actin
cytoskeletal dynamics thereby promoting synaptic efficacy. However, mysteries remain
as to the molecular cue that regulates integrin activation during synaptic activity. At the
Drosophila neuromuscular junction, I identify that Shv also functions as an activator of
integrin to modulate synaptic growth, and Shv is selective released during intense
synaptic activity to acutely activate integrin signaling, which in turn favorably stabilizes
24
the synapse in the form of local synapse enlargement and increase in glutamate
receptor accumulation at the terminal. The goal of my dissertation is to answer the
question of how Shv secretion modulates cellular structural integrity by regulating
integrin activation. Utilizing two different model systems of Drosophila, I propose a novel
role of Shv in maintaining stem cell niche structure, and activity-regulated Shv secretion
modulates synaptic structural modification by activating integrin signaling pathway.

25
Chapter 2
Maintenance of stem cell niche integrity by a novel activator of integrin signaling

Joo Yeun Lee, Jessica Y. Chen, Jillian L. Shaw and Karen T. Chang

2.1 Abstract
Stem cells depend critically on the surrounding microenvironment, or niche, for
their maintenance and self-renewal. While much is known about how the niche
regulates stem cell self-renewal and differentiation, mechanisms for how the niche is
maintained over time are not well understood. At the apical tip of the Drosophila testes,
germline stem cells (GSCs) and somatic stem cells share a common niche formed by
hub cells. Here we demonstrate that a novel protein named Shriveled (Shv) is
necessary for the maintenance of hub/niche integrity. Depletion of Shv protein results in
age-dependent deterioration of the hub structure and loss of GSCs, whereas
upregulation of Shv preserves the niche during aging. We find Shv is a secreted protein
that modulates DE-cadherin levels through extracellular activation of integrin signaling.
Our work identifies Shv as a novel activator of integrin signaling and suggests a new
integration model in which crosstalk between integrin and DE-cadherin in niche cells
promote their own preservation by maintaining the niche architecture.

26
2.2 Introduction
Adult stem cells have the unique capacity to undergo self-renewal for extended
periods of time and to generate differentiating daughter cells with the potential for tissue
repair and regeneration. Such features of adult stem cells depend critically on the
microenvironment, or niche (Schofield, 1978). The stem cell niche -- comprised of
various molecular factors such as extracellular matrix (ECM), secreted proteins,
adhesion molecules and support cells -- provides the key molecular cues necessary for
stem cell maintenance and tissue homeostasis during development, aging and changes
in environment (Schofield, 1978; Jones and Wagers, 2008; Morrison and Spradling,
2008; de Cuevas and Matunis, 2011; Losick et al., 2011).Despite a wealth of knowledge
on how the niche-stem cell interactions control their self-renewal and differentiation,
mechanisms for how the niche is maintained over time are not well understood.
The Drosophila germline stem cell system is an excellent model system for
investigating the biology of stem cells in vivo in the context of their niche, mainly due to
its simple anatomy and easily identifiable stem cell populations (Fuller, 1998; Yamashita
et al., 2005; Fuller and Spradling, 2007; Chen, 2008; Spradling et al., 2011). In
Drosophila testes, germline stem cells (GSCs) and cyst stem cells (CySCs) share a
common niche formed by hub cells. Each hub contains roughly 10 somatic hub cells
located at the apical tip of the testes and is surrounded by ~ 6-10 GSCs (Hardy et al.,
1979; Boyle et al., 2007; Issigonis et al., 2009). Each GSC is also enveloped by two
CySCs that are also in direct contact with the apical hub. Dynamic signaling between
hub cells, CySCs, and GSCs facilitate the self-renewal, differentiation, and survival of
the GSCs (Fuller, 1998; Fuller and Spradling, 2007; Lehmann, 2012). It was shown that
27
hub cells secrete molecules such as Unpaired and Bone morphogenic protein ligands to
neighboring stem cells to govern stem cell self-renewal and maintenance (Kiger et al.,
2000; Tulina and Matunis, 2001; Kawase, 2004; Singh et al., 2005; Li et al., 2007;
Issigonis et al., 2009; Leatherman and DiNardo, 2010; DiNardo et al., 2011; Michel et
al., 2011; Amoyel and Bach, 2012).
The molecular cues that regulate stem cell self-renewal and differentiation are
short-range signals acting on adjacent somatic and stem cells; therefore, adhesive
forces are necessary to anchor hub cells to the appropriate location in the testis, and
stem cells to the hub (Xi, 2009; de Cuevas and Matunis, 2011; Bulgakova et al., 2012).
Two types of cell adhesion molecules have been shown to serve such functions in the
Drosophila germline: integrins and cadherins (Song et al., 2002; Jenkins et al., 2003;
Wang et al., 2006; Tanentzapf et al., 2007; Lee et al., 2008; Voog et al., 2008;
Leatherman and DiNardo, 2010; Chen et al., 2012; Srinivasan et al., 2012). Integrins
are heterodimeric transmembrane receptors that can signal bi-directionally across the
plasma membrane to mediate cell-ECM adhesion (Hynes, 2002; Campbell and
Humphries, 2011); cadherins mediate cell-cell adhesions via homophilic interactions of
the extracellular domains (van Roy and Berx, 2008). In the Drosophila male germline
system, integrins are crucial for anchoring the somatic hub cells to the basal lamina at
the tip of the testis (Tanentzapf et al., 2007), whereas DE-cadherin is required for
attaching the GSCs and CySCs to the hub (Wang et al., 2006; Voog et al., 2008;
Leatherman and DiNardo, 2010; Tarayrah et al., 2013). Altered integrin signaling
affects niche positioning and leads to loss of both hub and stem cell populations in the
adult testes (Tanentzapf et al., 2007; Lee et al., 2008; Papagiannouli et al., 2014), thus
28
underscoring the importance of hub cell anchoring in the maintenance of its neighboring
stem cells. DE-cadherin and integrin also sustain the “competitiveness” of GSCs and
CySCs (Song et al., 2002; Jin et al., 2008; Issigonis et al., 2009; Inaba et al., 2010),
respectively, although the role of integrin in niche competition is less clear. In the fly
testes, expression of a dominant negative construct of DE-cadherin caused GSC loss
only if it was expressed in a subset of GSCs, but not if in all GSCs, demonstrating DE-
cadherin influences competition between GSCs (Inaba et al., 2010). It has also been
shown that CySCs with elevated levels of βPS integrin at the hub-CySC interface
caused by loss of Socs36E out-compete the GSCs and displace them away from the
niche (Issigonis et al., 2009). However, more recent data suggest that loss of Soc36E
does not elevate integrin levels but activates MAPK to increase competitiveness of
CySCs, and that clonal overexpression of integrin in CySCs does not cause niche
competition (Amoyel et al., 2014; 2016). Despite current controversial results on the role
of integrin in niche competition, previous work on mechanisms maintaining GSC and
stem cell niche have suggested that optimum integrin and DE-cadherin signaling are
crucial for a healthy stem cell system.
Here we identify a novel activator of integrin signaling named Shriveled (Shv) in
the maintenance of stem cell niche integrity in the Drosophila testes. We report that
Shv is secreted extracellularly by somatic cells and GSCs to activate βPS integrin in
vivo to ensure anchoring of the hub cells and maintenance of niche architecture.
Importantly, our results indicate Shv modulates DE-cadherin levels through an integrin-
dependent pathway, thus uncovering a new integration mechanism in which crosstalk
between integrin and DE-cadherin in hub cells serves to maintain niche architecture and
29
GSC numbers. Furthermore, our findings that upregulation of Shv preserves the stem
cell niche in aging Drosophila males further implies that enhancing Shv function may be
a valuable strategy to strengthen adhesion within the niche in order to delay the effects
of aging on tissue homeostasis.

2.3 Materials and Methods
Fly Stocks and Antibody Generation
Flies were cultured at 25 ̊C on standard cornmeal, yeast, sugar, and agar
medium unless indicated otherwise. White-eyed flies (w
1118
) were used as wildtype
throughout all experiments. The following fly lines were used with Bloomington Stock #
in parenthesis: mys
ts1
(# 3169), c833-gal4 (# 6988), nanos-gal4 (#4937), upd-gal4 (from
D. Harrison), c587-gal4, shg (#26885), shv
9803
(# 9803), shv
C00496
(from Exelixis at
Harvard Medical School), UAS-Shg-GFP (#58445), UAS- DEFL(II) (from Y. Yamashita),
c587-gal4 (from E. Matunis and A. Spradling), UAS-MYS (from R. Xi). shv
1
mutant was
generated using random hop with Drosophila lines carrying p{lacZ,w
+
} (Amoyel et al.,
2012). The site of P element insertion was determined by plasmid rescue (Hamilton et
al., 1991). shv
PJ
was generated by precise excision of the P-element allele. Full length
shv transgene construct was generated by subcloning the coding regions of CG4164
into the pINDY6 vector, and Shv with signal peptide deleted (NoSp-shv) was generated
by deleting the first 22 amino acids corresponding to the predicted signal peptide as
determined using SignalP 4.1. Transgenic flies were generated by standard
transformation method (Montell et al., 1985). Affinity purified rabbit polyclonal antibody
for Shv was generated against amino acids 243-256 of Shv (Sigma Genosys). All other
30
stocks and standard balancers were obtained from Bloomington Stock Center
(Bloomington, IN).

Immunocyochemistry
Testes were dissected in PBS and fixed in 4% paraformaldehyde for 25 min.
Fixed samples were washed with 0.1% triton X-100 in PBS (PBST) then blocked with
5% normal goat serum (NGS) in PBST. Primary antibodies were diluted in blocking
solution and used as following: rabbit anti-Shv, 1:400; rat anti-Vasa, 1:20 (DSHB); rabbit
anti-Vasa 1:5000 (gift from P. Lasko); mouse anti-Fasciclin III, 1:15 (7G10, DSHB); anti-
adducin, 1:35 (1B1, DSHB); rat anti-DE-cadherin, 1:20 (DSHB); mouse anti-integrin
βPS, 1:100 (DSHB); rat anti-DN-cadherin, 1:10 (DSHB); rabbit anti-phosphoFAK, 1:200
(Invitrogen). Secondary antibodies used were Alexa-488, 555 or 405 conjugated, 1:250
(Invitrogen). Extracellular labeling of Shv protein was adapted from Zheng et al. (Zheng
et al., 2011). Briefly, testes were dissected in cold Ringer’s solution, incubated with
anti-Shv antibody in cold Ringer’s solution containing 5% NGS for 2 to 3 hrs at 4 °C.
Testes were then washed 3 times with cold Ringer’s solution and processed for
standard immunostaining. Images were captured using Zeiss LSM5 confocal
microscope using a 63X 1.6NA oil immersion objective with a 1x or 2x zoom. When
comparing intensity across genotypes, the exposure time was kept constant for all
genotypes per experiment. The staining intensities of pFAK and DE-cadherin were
normalized to Fasciclin III staining that labels the hub cells. All values were normalized
to control done within the same experimental set.

31
Antibody preabsorption for Western and immunocytochemistry
Antibody-peptide absorption was achieved by incubating 0.2ug of Shv peptide
with 1 µg of Shv antibody for 1hr at RT. Western blots and standard
immunocytochemistry were carried out as described earlier.

Cell counting and hub positioning determination
The number of hub cells was determined by counting DAPI nuclei that were
positive for Fasciclin III marker. Germ cells contacting the hub that were adducin and
Vasa positive were counted as GSCs. Image J was used to measure the DE-cadherin
signal intensity as defined by Fasciclin III positive hub area. DE-cadherin level was
normalized to the hub area. Hub location was determined by measuring the distance
from the apical tip of the testes to the center of the hub area. In addition, we strictly
scored the hub as mislocalized when it is located more than two Vasa positive germ
cells apart, or 14 µm, from the apical tip of the testes. We scored the hub as “pulled”, or
with altered structure, when a cross-section image of testis stained with Fascicilin III is
not circular.

Fluorescence in situ hybridization
shv RNA detection was performed following protocol by Toledano et al., except
testes were incubated with PBS+0.1% triton for five minutes following fixation and prior
to primary antibody addition (D’Alterio et al., 2012). Probes for shv was generated by
PCR using primers against shv (F: ATGCAGCTTATCAAGTGCTT and R:
TCACAGTCCATTGTATATGC) and subcloned into pGEM-Teasy (Promega).
32
Western Blotting
To detect Shv levels in flies, protein extracts were obtained by homogenizing flies
in RIPA lysis buffer (50 mM Tris-HCl, pH7.5, 1% NP-40, 0.5% NaDoc, 150 mM NaCl,
0.1% SDS, 2 mM EDTA, 50 mM NaF, 1 mM Na
3
VO
4
, 250 nM cycloporin A, protease
inhibitor cocktail (Roche) and phosphatase inhibitor cocktail 1 (Sigma) using mortar and
pestle. 20 µg protein homogenate was separated by SDS-PAGE and transferred to
nitrocellulose membranes. For Western blots using S2 cell extracts, 2 µg of cell protein
extracts or 15 µl of media were loaded. Primary antibodies were diluted in blocking
solution as following: rabbit Shv, 1:500; anti-tubulin 1: 500 (7E10, DSHB); anti-βPS
integrin, 1:1500 (from R. Hynes); anti-V5, 1:5000 (Invitrogen).

Quantitative RT-PCR
One µg of total RNA from adult testes was isolated using TRIzol reagent
(Invitrogen), converted to cDNA using SuperScriptII reverse transcriptase (Invitrogen),
and then used for quantitative RT-PCR with SYBR Green reagent (Applied Biosystems).
Primers were designed from mRNA sequence to detect shv and normalized gapdh
transcripts (shv: F- CCATGGAGATCAAGCACCTT, R-TTTCTTGAGCGCTTCCTTGT;
GAPDH: F-TGGTACGACAACGAGTTTGGC, R-GTCTCACCCCATTCTACCGC; crq: F-
TTCTCATCACCGGCATCACG, R-GCTATCACAAACTGCAAGACG). Thermocycling
was conducted in The Light Cycler 480 Real-Time PCR system (Roche). The Light
Cycler Analysis Software 4.05. (Roche) was used to analyze amplification plots. The
relative quantity of amplified cDNA corresponding to each gene was calculated by using
the ΔΔCt method and normalized for expression of gapdh in each sample.
33
S2 cell culture
Drosophila S2 cells (Invitrogen) were cultured at 29°C in Schneider’s insect
medium (Sigma-Aldrich) supplemented with 10% FBS. Full length Shv and Shv with
signal peptide deleted (noSP-Shv) were subcloned into pAc5.1/V5-His vector
(Invitrogen). Shv-GFP is generated by inserting eGFP sequence to c-terminus of full-
length Shv and subcloned into the same pAc5.1/V5-His vector. Transfection was
performed using Calcium Phosphate Transfection Kit (Invitrogen). To test for secretion
of Shv, transfected cells and media were harvested 72 hours after transfection. For cell
spreading assay and pFAK determination, untransfected S2 cells were seeded in 24-
well plate containing 12mm glass coverslip pre-coated 50 µg/ml of poly-D-lysine
(Millipore) at 0.5X10
6
cells/well. Filtered media (0.2µm polyethersulfone membrane,
VWR) collected from transfected cells were added to each well, and cells were allowed
to spread for 1hr at 29°C. Amount of media applied was kept at 1:2 ratios to the seeded
volume. Following aspiration, cells were fixed and processed for staining as described
above. To remove Shv from the collected media (Shv pull down), collected media were
incubated with anti-V5-agarose beads (Sigma Adrich) at 4° for 2hr. For control,
collected media was incubated with agarose A/G beads (Santa Cruz Biotechnology) for
the same amount of time. Beads were washed with 1X PBS four times. Pull-down was
confirmed via western blots using antibody against V5. For colocalization experiment,
detergent was omitted in all solutions. Primary antibodies were used as following: rabbit
anti-phosphoFAK, 1:400 (Invitrogen); rabbit anti-GFP, 1:1000 (Acris); mouse anti-V5,
1:5000 (Invitrogen); rat anti-DCAD2 1:20 (DSHB); mouse anti-integrin βPS, 1:100
(DSHB). 100uM working stock of Actin-stain 488 fluorescent phalloidin (Cytoskeleton)
34
was used to stain actin filaments. Coverslips were mounted in Pro-long Gold Antifade
reagent with DAPI (Invitrogen). For RGD peptide treatment, 1mg/mL of RGD peptide
(Sigma Aldrich, A8052) was added for 15 min prior to application of Shv containing
media.

dsRNA construction
cDNA was synthesized from S2 cell RNA using TRIzol reagent followed by
SuperScriptII reverse transcriptase under the recommended manufacturer’s conditions.
511 bp fragments of myospheroid (mys) gene were amplified by PCR with Taq
polymerase (KapaBiosystem). Double-stranded RNA (dsRNA) primer sequences were
obtained from Drosophila RNAi Screening Center (DRSC) #18799 and tailed with T7
sequences (F: CCTCTTCGGTGGAGATGAA, R: GGATTTGGTCGCTTGTGG). dsRNA
was synthesized using MEGAscript T7 Kit (Ambion) according to the manufacturer’s
instructions. 2µg dsRNA was introduced in S2 cells using Effectene Transfection
Reagent (Qiagen) and cells were harvested after 72hrs.

Site-directed Mutagenesis
The KND sequence on Shv was mutated to LNV (Shv
LNV
) using QuickChange Kit
(Stratagene). Oligonucleotide primers used for site-directed mutagenesis were (with
mutated nucleotides underlined: F- CATCCGCGATTCCTGCGCCTGAATGTTGAT
CTGTACACGAACGT and R-ACGTTCGTGTACAGATCAACATTCAGGCGCAGGAAT
CGCGGATG. Point mutation was confirmed by sequencing (Genewiz).

35
3D Construction
Confocal z-stack images were volume rendered for 3D reconstruction using
Imaris 7.7 software (Bitplane). Additional surface rendering was performed toward the
red channel with Imaris 7.7 software.

Statistics
For paired samples, Student’s T-test was used. For multiple samples, One-way
ANOVA followed by post hoc analysis with Bonferroni’s multiple-comparison test was
used to determine statistical significance. All plots show mean ± SEM.

2.4 Results
2.4.1 Deterioration of hub architecture in shriveled mutants
We isolated a Drosophila mutant that displayed male sterility and age-dependent
decrease in testes size (Fig. 2.1.A). Because of the shrinking testes phenotype, we
named the mutant shriveled (shv) and the allele shv
1
. To understand the cause of the
reduced testes size, we stained them with a hub marker Fasciclin III (Fas III) and germ
cell marker Vasa. shv
1
mutants showed a striking age-dependent loss of the hub
structure (Figs. 2.1.B and 2.1.C). By 35 days of age, about 50% of the shv
1
testes
showed a complete loss of hub cells while all of the control and shv
1
/+ testes still
contained a hub. We also counted the average number of hub cells during aging (Table
2.1), and found that shv
1
mutant showed a slight decrease in the number of hub cells at
day 3 (control: 8.9 ± 0.18 vs. shv
1
: 6.4 ± 0.28; p < 0.05). By 35 days of age, the
average number of hub cells is 8.18 ± 0.26 for control, but 1.8 ± 0.46 for shv
1
(or 4.0 ±
36
0.4, n = 16 if only counting testes that contained positive hub marker staining). To
further understand if shv
1
is required for normal hub formation, we counted the number
of hub cells in 3
rd
instar larval testes. shv
1
larval testes showed a normal number of hub
cell compared to control (Fig. S2.1.A), suggesting that Shv is not essential for hub
formation, but rather required for hub maintenance. Closer examination of the hub
architecture of young flies revealed that even though shv
1
mutants contained a Fas III-
positive hub, 58.3% of hub cells appeared to be “pulled”, spread throughout different
image planes of the testes, and frequently did not localize at the apical tip (Fig. 2.1.D,
Figs. S2.1.B, S2.1.C, and S2.1 and S2.2 Video). The age-dependent loss of hub cells
prompted us to examine the expression level of DE-cadherin, a cell adhesion molecule
highly expressed in the hub and previously shown to decline during aging (Yamashita et
al., 2003; Boyle et al., 2007). shv
1
testes displayed a decrease in DE-cadherin levels in
both the hub and hub/GSC border. Both DE-cadherin and DN-cadherin have been
shown to be important for niche-GSC interaction (Le Bras and Van Doren, 2006), we
therefore also examined the levels of DN-cadherin in shv
1
. We found that shv
1
testes
also showed a decrease in the level of DN-cadherin in the hub cells (Fig. S2.1.D). To
further confirm that the decrease in cadherins is not due to a loss of hub cell integrity,
we examined the level of another hub selective marker, cactus (DiNardo et al., 2011).
Similar to Fas III staining, cactus staining remained intact in shv
1
, suggesting that
decrease in DE-cadherin and DN-cadherin levels is not simply due to a loss of hub cell
integrity (Fig. 2.1.E and Fig. S2.1.E).
Studies have shown the CySCs, together with hub cells, form part of the niche for
GSCs. We therefore determined if shv
1
mutants also have altered CySCs by counting
37
the number of Zfh1-positive, Eya-negative cells (Amoyel et al., 2012). Supplementary
Fig. 1F shows that there is also a slight but significant decrease in the number of
CySCs. Accordingly, shv
1
testes have reduced number of GSCs (Fig. 2.1.F). Together,
our results demonstrate that Shv is essential for the maintenance of niche integrity and
GSCs.

38

Figure 2.1. Shv is required for the maintenance of hub and GSCs during aging. (A)
Representative images of testes dissected from control and shv
1
flies at the indicated
age. Scale bar = 100 µm. (B) Magnified images of 3 and 35 days old testes from
control, shv
1
/+ and shv
1
flies immunostained with antibodies as indicated. Dots highlight
GSCs. (C) Quantification of percentage of testes with hub cells. Sample numbers are
indicated in Table 1. (D) Representative confocal images (from a single Z-axis plane)
demonstrating hub architecture of young control and shv
1
testes. Asterisks show the
apical tip of the testis and arrows highlight hub mislocalization. (E) Representative
images of the testis tip stained with the indicated antibodies in control and shv
1
. *
highlights the hub. For (B), (D), and (E) scale bar = 10 µm. (F) Quantification of
average number of GSCs per testis during aging. Sample numbers are indicated in the
bar graph. Age of flies examined is 3 days after eclosion unless noted otherwise. For
multiple samples, One-way ANOVA followed by post hoc analysis with Bonferroni’s
multiple-comparison test was used to determine statistical significance. * p < 0.05
compared to control. All values represent mean ± SEM.
Lee et al. Figure 1
A B
Control shv
1

14 days old 1 day old
Vasa Fas3 Hts DAPI
E
E-cad Fas3 DAPI E-cad
Control shv
1

f
*
*
*
*
E-cad
Control shv
1

E-cad Fas3 Vasa
F
0
2
4
6
8
10
12
# of GSCs per testis
Control
shv
1
/+
shv
1

3-day 7-day 14-day
*
*
*
*
*
*
35-day
57
21
22
17
73
31
21
20
17
14
12
30
Days after eclosion
C
0 5 10 15 20 25 30 35
20
40
60
80
100
0
Control
shv
1

shv
1
/+
% of testis with hub cells
Days after eclosion
3 days old 35 days old
shv
1

shv
1
/+

Control

Vasa Fas3 Hts DAPI

D
*
*
*
*
Control shv
1

Vasa Fas3 Hts DAPI
39

Table 2.1. Hub cell quantification. Table showing quantification of hub cell numbers
and number of testes quantified. Each column represents designated days after
eclosion. Sample numbers are in parentheses. * p < 0.05 compared to control in the
same age group. For multiple samples, One-way ANOVA followed by post hoc analysis
with Bonferroni’s multiple-comparison test was used to determine statistical
significance.

Lee et al. Table 1
A
Table 1. Quantification of hub cells in each genotype (n)
Genotype
shv
1
/+
Control
shv
1

shv
1
;UAS-shv/c833-GAL4
UAS-shv/c833-GAL4
upd-GAL4;shv;UAS-shv
UAS-shv/nanos-GAL4
shv
1
;UAS-shv/nanos-GAL4
upd-GAL4;UAS-shv
35 DAE
8.18 ± 0.26
8.2 ± 0.25
1.8 ± 0.46
6.0 ± 0.98
7.3 ± 0.27
0.0 ± 0.00
7.8 ± 0.33
3.5 ± 0.71
7.0 ± 0.44
(17)
(17)
(30)
(10)
(7)
(23)
(20)
(20)
(10)
14 DAE
8.1 ± 0.29
8.9 ± 0.61
5.1 ± 0.70
7.9 ± 0.51
7.5 ± 0.29
2.4 ± 1.56
8.4 ± 0.27
6.2 ± 0.82
7.1 ±0.30
(21)
(14)
(22)
(8)
(8)
(20)
(20)
(10)
(10)
7 DAE
8.0 ± 0.18
9.3 ±0.45
5.8 ± 0.63
8.4 ± 0.57
8.4 ± 0.40
2.9 ±1.17
8.4 ± 0.26
6.4 ± 0.70
7.8 ± 0.22
(20)
(12)
(31)
(15)
(9)
(20)
(12)
(14)
(12)
3 DAE
8.9 ± 0.18
8.8 ± 0.25
6.4 ± 0.28
9.8 ± 0.44
9.3 ± 0.24
2.9 ± 1.15
9.8 ± 0.24
7.4 ± 0.37
8.1 ± 0.36
(106)
(19)
(33)
(23)
(26)
(15)
(11)
(134)
(33)
* * * *
* * * *
* * *
*
*
Total (n)
164
76
217
44
39
89
75
77
51
40
2.4.2 Shriveled encodes a conserved protein
Molecular cloning identified that shv
1
contains P-element insertion within an
uncharacterized fly gene, CG4164, which shares 63% identity in amino acid sequence
with human DNAJB11 (Fig. S2.2.A). Human DNAJB11, also known as ERDJ3, is a
unique chaperone protein that contains a signal peptide sequence at the N-terminus, a
putative nuclear localization signal, two DnaJ domains and a RGD motif found in some
integrin binding proteins (Ohtsuka and Hata, 2000; Shen and Hendershot, 2005;
Ludvigsen et al., 2009). It has been shown to act as a co-chaperone in assisting proper
protein folding in the ER and in increasing the activity and affinity of Hsp70 BiP for
substrates (Yu et al., 2000; Shen and Hendershot, 2005; Guo and Snapp, 2013). It also
acts as a secreted protein that modulates integrin affinity (Wang et al., 2013) and in
modulating unfolded protein response (Genereux et al., 2014). However, roles of
DNAJB11 in stem cell maintenance and spermatogenesis have not been established.
Quantitative RT-PCR revealed that the shv
1
mutant is nearly a null allele with shv
transcript level of only about 0.025 ± 0.002 fold of the control, and an undetectable level
of Shv protein on Western blot using an antibody specific for Shv (Fig. 2.2.A, Figs. S.2.B
and S2.2.C). Quantitative RT-PCR also confirmed that shv
1
does not affect the
transcription level of crq, an adjacent gene oriented in the opposite direction (Figs.
S2.2.A and S2.2.B). To further confirm that mutation in Shv is responsible for the
observed phenotypes, we isolated a precise excision allele, shv
PJ
, which reverted the
shv transcript level back to wildtype, as well as the positioning and structure of the hub,
number of GSCs and DE-cadherin level to normal (Fig. S2.3). In addition, we obtained
other alleles of Shv with transposon insertion within CG4164, shv
9803
and shv
c00496
(Fig.
41
S2.2A). Note that we did not characterize shv
9803
homozygous mutant because it is a
strong semi-lethal allele. Nevertheless, similar to shv
1
, shv
9803
/shv
1
, shv
c00496
, and
shv
c00496
/shv
1
also showed abnormal hub architecture, DE-cadherin reduction and loss
of GSCs (Fig. S2.3), but did not show a complete loss of hub with aging. Quantitative
RT-PCR revealed that the difference is likely caused by shv
c00496
and shv
9803
being less
severe alleles of shv
1
(Fig. S2.3.A). Together, these data suggest that reduction in Shv
level is responsible for the hub phenotypes and reduction in GSC number.

42

Figure 2.2. Shv is present and required in multiple cell types. (A) Representative
western blot showing Shv levels for the indicated genotypes. β-tubulin is used as a
loading control. (B) Testes immunostained with the indicated antibodies. Arrows point to
Shv staining often seen at the hub/GSC, GSC/CySC, and germ/cyst interface. (C) shv
1

mutant displays a significant decrease in Shv level. Antibody pre-absorbed with Shv
peptide confirms Shv antibody specificity. Arrows point to Shv present at hub/GSC
interface. (D) Control testes taken at lower magnification shows abundant Shv protein in
the nucleus of spermatocytes (arrowhead). Scale bar = 50 µm. For (B), (C), and (D),
Images of testes are from 3 day old males. (E), (G), and (H) Quantification of
percentage of testes with hub cells for the indicated genotypes. (F) Images of testes
from 14 day old flies showing rescue of the hub phenotype in flies overexpressing shv
using c833-GAL4 driver in shv
1
mutant background. In (B), (C), and (F), scale bar = 10
µm and sample number per age group is listed in Table 2.1.
G
0 5 10 15 20 25 30 35
20
40
60
80
100
0
Control
upd-GAL4;shv
1
;UAS-shv
shv
1

% of testis with hub cells
Days after eclosion
upd-GAL4;UAS-shv
Days after eclosion
E
0 5 10 15 20 25 30 35
20
40
60
80
100
0
Control
shv
1
;UAS-shv/c883-GAL4
shv
1

% of testis with hub cells
UAS-shv/c883-GAL4
H
Days after eclosion
0 5 10 15 20 25 30 35
20
40
60
80
100
0
Control
shv
1
;UAS-shv/nanos-GAL4
shv
1

% of testis with hub cells
UAS-shv/nanos-GAL4
D
Vasa Shv Fas3 Hts
Control
A
Control
shv
1
/+
shv
1

43
34
55
Shv
β-tubulin
B
UAS-GFP/c833-GAL4
Shv GFP Fas3
C
Control shv
1
Pre-absorption Control
Shv Vasa Fas3
F
Control shv
1
shv
1
;UAS-shv/c833-GAL4 UAS-shv/c833-GAL4
Vasa Fas3 Hts DAPI
Lee et al. Figure 2
43
To elucidate how Shv maintains the stem cell niche, we examined its distribution
in the fly testes. Fluorescent in situ hybridization revealed that shv RNA is ubiquitously
expressed in different cell types: low levels in hub cells, CySCs and GSCs; high levels
in spermatocytes and cyst cells; below the level of detection in shv
1
mutant (Fig.
S2.4.A). Immunostaining with the Shv antibody further showed low levels of Shv protein
in hub cells and CySCs at the apical tip of the wildtype testes, but barely detectable in
shv
1
mutants or not present if the antibody had been pre-absorbed with the Shv
immunizing peptide (Fig. 2.2.B, 2.2.C, and Fig. S.2.2.C). These results demonstrate the
specificity of the Shv antibody and further confirm that shv
1
is close to a null allele. Note
that because Shv staining often appeared punctate at the apical tip, we performed
additional colocalization studies using specific organelle markers. Supplementary Fig.
2.4.B and 2.4.C show that Shv displayed partial colocalization with the ER, but not with
spectrosome, peroxisome, lysosome, mitochondria, or golgi markers in cyst cells.
Instead, Shv is frequently seen at the GSC/hub interface and cyst/germ cell border (Fig.
2.2.B and 2.2.C; arrows), thus making it difficult to specifically locate in a specific cell
type. These staining patterns are characteristics of a secreted protein, and the
presence of a signal peptide sequence at the N-terminus of Shv further suggests that
Shv may be a secreted protein. We also noted that despite shv RNA being present in
the GSCs, Shv protein is usually below the level of detection in GSCs. In addition,
consistent with the presence of a predicted nuclear localization signal in the Shv protein
(Fig. S.2.2.A), Shv was detected in the nucleus of primary spermatocytes, but not in
shv
1
mutant (Fig. 2.2.D and Fig. S2.4.D). The presence of Shv in different cell types
suggests that Shv protein may play multiple roles in spermatogenesis.
44
To identify if Shv is required in specific cell types to maintain hub integrity and
preserve GSC number, we knocked down Shv in selective cells using different driver
and UAS-RNAi lines, but were not able to detect any phenotype likely because we were
not able to sufficiently reduce Shv level. We therefore restored Shv protein in shv
1

testes using the UAS/GAL4 system and independent driver lines specific for expression
in hub cells (upd-GAL4) (Le Bras and Van Doren, 2006), germ cells (nanos-GAL4)
(Doren et al., 1998), and the hub + cyst cells (c833-GAL4) (Hrdlicka et al., 2002;
Papagiannouli and Mechler, 2009). Expression of Shv using c833-GAL4 rescued both
hub integrity and GSC number (Fig. 2.2.E, 2.2.F, and Fig. S2.5.A). Note that all early
somatic drivers we tested show low expression in the hub, including the commonly used
c587-GAL4, ptc-GAL4, Tj-GAL4 and esg-GAL4 (Schulz et al., 2002; Joti et al., 2011;
D’Alterio et al., 2012). To confirm that this rescue is not driver specific, we also
performed the rescue experiment with C587-GAL4 driver. Similarly, expressing Shv in
shv
1
mutant using C587-GAL4 restored the number of hub cells (Fig. S2.5.B) and hub
mislocalization phenotype (0% hub mislocalization for c587-GAL4; shv
1
; UAS-Shv).
Expression of full-length Shv in hub cells of shv
1
mutants not only failed to rescue the
age-dependent loss of hub, but also exacerbated the phenotype (Fig. 2.2.G and Table
2.1). It is possible that abnormally high level of Shv locally during development may
interfere with hub formation, since the rescue experiment using upd-GAL4 driver had a
dominant effect over shv
1
mutants. Notably, even though Shv is below the level of
detection in GSCs by immunostaining, expression of full-length Shv in shv
1
germ cells
using nanos-GAL4 also partially preserved the hub and protected against decreases in
GSC number (Fig. 2.2.H, Table 2.1, and Fig. S2.5.C). Together, our results indicate
45
that expression of Shv in either somatic cells or GSCs could maintain hub integrity and
GSC health via both cell-autonomous and non-cell-autonomous mechanisms.

2.4.3 Shriveled interacts with integrin to control hub anchoring
Integrin is an essential cell adhesion molecule found in somatic cells of
Drosophila testes. Loss of integrin signaling causes the hub to drift away from the
apical tip, and in severe cases, leads to loss of hub cells as seen in talin-RNAi lines
(Tanentzapf et al., 2007; Lee et al., 2008; Papagiannouli et al., 2014). The hub
mislocalization and loss of hub phenotypes seen in shv
1
mutants are similar to those
described for βPS integrin mutants; we therefore tested the possibility that Shv interacts
with integrin signaling pathway. We first assayed for genetic interaction between Shv
and βPS integrin, myospheroid (mys) in Drosophila (Bunch and Brower, 1992), by
measuring the hub to testes tip distance. Since null alleles of mys are embryonic lethal
due to muscle detachment (Leptin et al., 1989), we used a viable mys hypomorphic
allele (mys
ts1
) that had been shown to decrease levels of βPS integrin when raised at
25°C (Beumer et al., 1999). mys
ts1
testes indeed showed hub mislocalization that was
rescued by mys overexpression in somatic cells. Similarly, hub in shv
1
was
mislocalized but was rescued by Shv upregulation (Figs. 2.3.A and 2.3.B), indicating
Shv is responsible for the phenotype. Double mutants of mys
ts1
; shv
1
showed the same
hub mislocalization phenotype as either mutant alone, suggesting that Shv and Mys act
in the same pathway to ensure normal anchoring of the hub. Upregulation of Mys in
shv
1
mutant background partially restored the hub mislocalization phenotype (Fig. 2.3.A
46
and 2.3.B), confirming genetic interaction between the two proteins. However, the weak
rescue further raises the possibility that Shv is required for effective integrin activation.

47

Figure 2.3. Shv genetically interacts with βPS integrin signaling. (A)
Representative images of testes dissected from 3 day old flies reared at 25°C.
Asterisks show the apical tip of the testis and arrows highlight distally located hub. (B)
Quantification of hub position relative to the apical tip of the testis. * p < 0.05 compare to
control and ** p < 0.05 compared to the indicated genotypes. All values represent mean
± SEM and sample numbers are indicated in the bar graph. (C) Testes of mys
ts1
raised
at 29°C show similar hub phenotype as seen in shv
1
. Arrowheads highlight the “pulling”
of hub cells and asterisks indicate the apical tip. Note that DE-cadherin staining is also
reduced in mys
ts1
raised at 29°C. Age of flies examined is 3 days after eclosion unless
noted otherwise. For multiple samples, One-way ANOVA followed by post hoc analysis
with Bonferroni’s multiple-comparison test was used to determine statistical
significance. Scale bar in (A) and (C) = 10 µm.

C
A
mys
ts1
E-cad Integrin βPS VASA
29°C
Control

* *
Lee et al. Figure 3
Vasa
Fas3 Hts DAPI
Control shv
1
shv
1
/+ mys
ts1
*
*
mys
ts1
;shv
1
/+

*
mys
ts1
;shv
1
*
*
*
shv
1
;UAS-shv/
c833-GAL4
shv
1
;UAS-mys/
c833-GAL4
mys
ts1
;UAS-mys/
c833-GAL4

UAS-mys/
c833-GAL4
*
* * *
*
UAS-shv/
c833-GAL4
Vasa
Fas3 Hts DAPI
C833-GAL4

I
**

0
5
10
15
20
25
30
Distance from
the tip to hub (µm)
I I I I I I I I I I
110
67
81
118
94
20
37
56
27
38
*
*
*
*
*
**
**
I
40
B
48
Curiously, even though mys
ts1
exhibited hub mislocalization, the overall hub
structure was not altered. This suggests that either there is still sufficient level of
functional integrin receptors, or that Shv acts through integrin-independent pathways to
influence hub architecture. To further reduce integrin, we reared the mys
ts1
flies at
29°C, which resulted in semi-lethality. However, in addition to showing hub
mislocalization, hub cells from the testes of surviving myst
s1
flies often exhibited a
similar mis-aggregation phenotype of hub as seen in shv
1
mutants (Fig. 2.3.C; 75% of
testes examined). These results suggest that integrin, in addition to anchoring the hub
at the tip, is required for the maintenance of hub architecture.

2.4.4 Shriveled is a secreted protein that activates integrin signaling in vitro
Having established that Shv genetically interacts with integrin to maintain stem
cell niche integrity, we tested whether Shv activates integrin signaling. Based on the
presence of a signal peptide sequence, we propose Shv is secreted to activate integrin
pathway extracellularly. To examine secretion of Shv, we transfected Drosophila
Schneider’s (S2) cells with Shv tagged with V5 and assayed for the presence of Shv in
the media. Shv was detected in the media of transfected cells, but not if the signal
peptide was removed from Shv (NoSP-Shv; Fig. 2.4.A), indicating Shv is a secreted
protein. To understand whether Shv activates integrin via an outside-in signaling
mechanism, we tested the ability of extracellularly applied Shv to activate integrin by
measuring the extent of focal adhesion kinase (FAK) phosphorylation. Previous studies
have indicated a strong correlation between integrin activation and autophosphorylation
of FAK at Tyr397 site (Mitra et al., 2005; Harburger and Calderwood, 2009; Campbell
49
and Humphries, 2011). Figure 2.4.B shows that addition of Shv containing media to
untransfected S2 cells was sufficient to increase pFAK levels and induce cell spreading
that is indicative of cell adhesion activation as detected by phalloidin staining. However,
treatment of cells with media collected from cells transfected with NoSP-Shv failed to
induce any change in cell shape or pFAK levels. Preabsorption of Shv (Shv pull down)
from the media also prevented both effects, indicating extracellular Shv is responsible
for the changes (Figs. 2.4.B, 2.4.C, and Fig S2.6.A). Furthermore, reducing βPS
integrin levels using mys-RNAi abolished the effects of Shv on cell spreading and FAK
phosphorylation (Figs. 2.4.D, 2.4.E, and Fig. S.6.B), suggesting that extracellular Shv
induces cell spreading through integrin activation. To rule out the possibility that Shv
modulates integrin signaling via an inside-out manner, we examined the levels of FAK
phosphorylation in mock, Shv, and NoSP-Shv expressing cells incubated with fresh
media. Despite the presence of intracellular Shv, no change in cell shape or FAK
phosphorylation was detected when Shv was removed from the media (Fig. 2.4.F and
Fig. S2.6.C). Together, these data support the claim that extracellular Shv is both
necessary and sufficient for integrin activation.

50

Figure 2.4. Shv is a secreted protein that activates βPS integrin through outside-
in signaling. (A) Western blot showing presence of Shv-V5 protein in the media of
transfected cells but not if the signal peptide was truncated. C: transfected cell extract;
M: media collected from the transfected cells. (B) Untransfected S2 cells after treatment
with the indicated media. Phalloidin (green) staining shows that Shv containing media
caused spreading or changes in cell shape and increase in pFAK staining intensity. (C)
Quantification of pFAK levels after media treatment. * p < 0.05 compared to cells
incubated with fresh media. (D) Images of control and mys-RNAi transfected cells
following treatment with Shv containing media. Shv containing media failed to induce
cell spreading and phosphorylation of FAK when integrin is knocked down. (E)
Quantification of relative pFAK intensity. * p < 0.05 compared to control. (F) S2 cells
transfected with the indicated constructs but incubated with fresh S2 media did not
show changes in pFAK staining. (G) Shv-GFP partially colocalizes with βPS integrin on
the S2 cell surface. S2 cells were incubated with either Shv-GFP containing media or
fresh S2 media only and processed for staining without permeabilization. (H) Images of
F
G I
I I I I
I I I I
0.0
0.5
1.0
Media
Relative pFAK intensity
≈
I
4.0
4.5
5.0
D
Shv media treated
pFAK DAPI
Phalloidin
control mys-RNAi
pFAK
Fresh Shv-GFP
GFP DAPI
Integrin
GFP
DAPI
B A
55
40
C M
S2
Shv-V5
NoSP-Shv-V5
C M C M
C
Shv Fresh
pFAK DAPI
Phalloidin
Shv pull down NoSP-Shv
Media treated
pFAK
J
E
pFAK
DAPI V5
pFAK
Shv Mock NoSP-Shv
Transfected cells
*
I
I

*
I
5
I
*
I I I I
I I I I I I I I
0
1
2
3
4
Media treated
Relative
pFAK intensity
99
178
34
91
69
I I
I I I I
I I I I I
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Relative
pFAK intensity
Shv media - +
*
17
41
46
52
H
GFP DAPI RGD -
+
Shv Media + +

91
99
178
Lee et al. Figure 4
pFAK DAPI
Phalloidin
pFAK
Shv media Shv
LNV
media
51
S2 cells pre-incubated with RGD peptide followed by exogenous application of Shv-
GFP protein. (I) Representative images of cell spreading and phospho-FAK levels in S2
cell treated with the indicated media. (J) Quantification of the relative phospho-FAK
levels in S2 cells conditioned with the indicated media. For multiple samples, One-way
ANOVA followed by post hoc analysis with Bonferroni’s multiple-comparison test was
used to determine statistical significance. * p < 0.05 compared to control. All values
represent mean ± SEM. All scale bars = 5 µm.

52
We next examined whether Shv colocalizes with βPS integrin receptors by
generating Shv with GFP tagged at the C-terminus. We found Shv-GFP is secreted into
the media and can activate integrin signaling as indicated by increased FAK
phosphorylation (Figs. S2.6.D and S.2.6.E). Extracellularly applied Shv-GFP also
colocalized with βPS integrin receptors on the S2 cell surface (Fig. 2.4.G).
Furthermore, we found that pre-incubation of S2 cells with the RGD peptide competed
against the ability of Shv-GFP to adhere to the S2 cell surface (Fig. 2.4.H and Fig.
S2.6.F), suggesting that Shv interacts and binds to integrin receptors. Integrin
receptors have been shown to bind to a wide variety of ligands, including, but not
exclusively, to those that contain the RGD sequence (Humphries, 2006). Sequence
alignment shows that even though the RGD sequence in human DNAJB11 is not
conserved in fly, KND sequence in Shv does contain sequence similarity to RGD
(highlighted in Fig. S.2.2.A). Furthermore, a similar sequence, KGD, had previously
been shown to be sufficient for integrin activation (Barja-Fidalgo et al., 2005). To test
whether KND sequence belonging to Shv is required for activation of integrin, we
mutated the charged residues of KND to hydrophobic amino acids, LNV (Shv
LNV
). While
Shv
LNV
is still secreted by S2 cells (Fig. S2.6.G), it diminished the ability of Shv to
activate integrin signaling as depicted by the absence of cell spreading and comparable
pFAK levels to control (Figs. 2.4.I and 2.4.J). Together, our results suggest that Shv can
bind to and interact with integrin directly to modulate integrin activation.

53
2.4.5 Secretion of Shriveled activates integrin signaling and modulates E-
cadherin levels in vivo
Having demonstrated that Shv is a secreted protein that activates integrin
signaling in vitro, we next tested if Shv is present extracellularly in fly testes. Shv
antibody staining of the fly testes without detergent confirmed the extracellular presence
of Shv (Fig. 2.5.A and 2.5.B). Shv signal was observed at the tip region, present as
puncta at the hub and GSC/CySC interface regions of the control testes but absent in
shv
1
. Next, we determined if Shv can indeed activate integrin signaling in stem cell
niche in vivo by examining 1) integrin clustering, and 2) FAK phosphorylation, both of
which strongly correlate with integrin activation (Mitra et al., 2005; Harburger and
Calderwood, 2009; Campbell and Humphries, 2011). Testes from control adult flies
showed strong integrin staining at the hub/CySC interface (Fig. 2.5.C), consistent with
previous reports (Issigonis et al., 2009; Joti et al., 2011; Papagiannouli et al., 2014).
Strong phosphorylated FAK (pFAK) signals were also seen at the hub/CySC interface
(Fig. 2.5.D). mys
ts1
mutants showed reduced integrin and pFAK levels, confirming
pFAK is indeed sensitive to integrin signaling (Fig. 2.5.D, 2.5.E). shv
1
mutants showed
a virtual absence of integrin clustering and reduced pFAK staining at the hub/CySC
border that is markedly enhanced by upregulation of full-length Shv in mutant
background. To further confirm that secretion of Shv is necessary for integrin activation
in vivo, we generated transgenic flies containing NoSP-Shv under UAS control.
Expression of NoSp-Shv in shv
1
mutant background did not restore integrin clustering
and pFAK levels (Figs. 2.5.C-E), supporting the claim that integrin activation and
clustering is achieved by extracellular Shv. Furthermore, upregulation of Shv alone was
54
sufficient to increase integrin clustering and pFAK staining at the hub/CySC border, but
these were dampened in mys
ts1
mutant background. This result is consistent with Shv
being an activator of integrin in vivo. Interestingly, elevation in localized integrin
signaling at the hub/CySC border in shv overexpression testes is accompanied by a
reduced number of GSCs (Fig. S2.5.A), consistent with previously reported integrin-
dependent niche competition between GSCs and CySCs (Issigonis et al., 2009).
shv
1
mutants also showed a dramatically reduced DE-cadherin level in the hub
cells. One plausible mechanism is that Shv regulates DE-cadherin level through
integrin. We first tested this hypothesis in vitro using S2 cells. Incubation of S2 cells
with Shv containing media elevated DE-cadherin signal compared to cells treated with
control media. Knockdown of βPS integrin using mys-RNAi blocked the effects of Shv
in triggering DE-cadherin elevation (S7A and S7B Fig). These results not only suggest
that Shv can induce DE-cadherin expression but confirm that Shv regulates DE-
cadherin level through integrin activation.

55

Figure 2.5. Shv activates βPS integrin signaling in vivo. (A) Staining of Shv antibody
without detergent shows extracellular Shv accumulation (arrowhead), whereas punctate
extracellular Shv is absent in shv
1
. (B) Arrows point to extracellular Shv labeling in hub
and GSC/CySC interface. (C) Integrin clustering revealed by staining with βPS integrin
antibody staining (yellow arrowheads). shv
1
mutant show localized decrease in integrin
clustering. (D) Levels of FAK phosphorylation (pFAK) at the hub/CySCs interface in the
Lee et al. Figure 5
C D
shv
1
;
UAS-mys
Control UAS-mys
Control UAS-mys
shv
1
;
UAS-mys
* *
*
*
* *
Control
shv
1
;
UAS-shv
UAS-shv shv
1

Integrin βPS
E-cad
Integrin βPS
mys
ts1

mys
ts1
;
UAS-shv
shv
1
;
UAS-NoSP-shv/
*
UAS-shv shv
1
Control
Vasa pFAK
Fas3
pFAK
*
*
*
*
mys
ts1

mys
ts1
;
UAS-shv
*
*
*
*
*
*
* *
*
*
shv
1
;
UAS-shv
shv
1
;
UAS-NoSP-shv
A
B
GFP Fas3
Shv (Extracellular)
Shv (Extracellular)
UAS-GFPS65/
C833-GAL4
Control shv
1

Fas3
Shv (Extracellular)
Shv (Extracellular)
23
I I I
*

0.0
0.2
0.4
0.6
0.8
1.0
1.2
Relative pFAK
immunoreactivity
C833-GAL4

I I I I I I I
36
41
40
12
5
16
26
9
**

I I I I I I I I
1.4
*
*
*
**
**
*
E
56
indicated genotypes. Yellow asterisks highlight the hub. (E) Quantification of the
relative pFAK intensity normalized to Fas III levels across genotypes. * p < 0.05
compared to control and ** p < 0.05 compared to indicated genotypes. All values
represent mean ± SEM and n is indicated in the bar graph. Age of flies examined is 3
days after eclosion. Age of flies examined is 3 days after eclosion. For multiple samples,
One-way ANOVA followed by post hoc analysis with Bonferroni’s multiple-comparison
test was used to determine statistical significance. All scale bars = 10 µm.

57
Next, we took a genetic approach to test in vivo that DE-cadherin expression can
indeed be regulated by integrin activation. We focused on DE-cadherin levels since it
has previously been shown to be essential for hub compaction, GSC and CySCs
maintenance (Jenkins et al., 2003; Voog et al., 2008). We found that reducing βPS
integrin in the testes led to lower DE-cadherin staining in the hub cells that is restored
by expressing mys in mys
ts1
using c833-GAL4 driver (Fig 2.6.A). To further confirm that
this decrease in DE-cadherin intensity is specific and not due to a change in the number
of hub cells, we plotted the relative DE-cadherin intensity normalized to Fas III intensity
(Fig. 2.6.B). Again, mys
ts1
showed reduced level of DE-cadherin to Fas III ratio,
consistent with the claim that integrin regulates DE-cadherin expression in the hub cells.
Expression of full-length, but not NoSP-Shv, rescued the shv
1
mutant phenotype, again
verifying that secretion of Shv is important for normal DE-cadherin level. Expression of
NoSP-Shv in shv
1
using the nanos-GAL4 driver also resulted in the similar findings for
DE-cadherin expression (Figs. S2.7.C and S2.7.D). Furthermore, double mutants
mys
ts1
;shv
1
showed a similar decrease in DE-cadherin level as single mutant (Figs.
2.6.A and 2.6.B), supporting the notion that Mys and Shv is in the same pathway to
regulate DE-cadherin level. We found overexpression of mys in shv
1
partially rescued
the decrease in DE-cadherin level (Figs. 2.6.A and 2.6.B). This weak rescue is
consistent with our findings that Shv needs to be present for efficient integrin activation
and thus DE-cadherin expression.
Because our data indicates that integrin alters DE-cadherin levels in hub cells,
we next tested whether this reduction in DE-cadherin level contributes to hub
mislocalization. Figure 6C shows that upregulating DE-cadherin in mys
ts1
mutants
58
sufficiently restored hub location. Furthermore, while heterozygous mutants of shv
1

(shv
1
/+) and DE-cadherin (shotgun in Drosophila; shg/+) did not show hub
mislocalization, shv
1
/shg mutants have hub cells distantly located away from the tip
(Fig. 2.6.D and Fig. S2.7.E; 85% of testes examined). Previous reports demonstrated
that loss of DE-cadherin alone is not sufficient to cause hub mislocalization (Lee et al.,
2008); however, our results imply that although the primary role of DE-cadherin in hub
cells is not to anchor the hub, it does contribute to hub localization.
We next asked if reduction in DE-cadherin level contributes to the shv
1
mutant
phenotypes. To this end, we restored DE-cadherin level in shv
1
using the c833-GAL4
driver (Figs. 2.6.E and 2.6.F). We found that elevating DE-cadherin in shv
1
significantly
rescued the hub “pulling” and hub mislocalization phenotype (Figs. 2.6.E, 2.6.F and
2.6.G). Moreover, GSC loss of shv
1
was prevented by DE-cadherin upregulation
(average GSC number in shv
1
;UAS-shg-GFP/c833-GAL4 is 8.45 ± 0.23 vs. 6.27 ± 0.58
in shv
1
, p < 0.05

). However, it did not rescue the number of hub cells of shv
1
to control
levels, suggesting that restoring DE-cadherin alone is not sufficient, presumably
because activation of integrin signaling by Shv affects multiple downstream
pathways/targets in addition to DE-cadherin, such as N-cadherin.

59

Figure 2.6. Shv regulates DE-cadherin level through βPS integrin. (A) Images of the
apical tip of testes stained with DE-cadherin (E-cad) and indicated antibodies. Lower
panels show pseudo-colored images of DE-cadherin staining intensity. (B)
Quantification of the relative DE-cadherin intensity normalized to Fas III levels across
genotypes. (C) Upregulation of DE-cadherin in mys
ts1
background rescued hub
Lee et al. Figure 6
D
shv
1
/+ shg/+ shv
1
/shg
E-cad Vasa
βPS integrin
*
*
*
A
Low

High

E-cad
Fas3 Vasa
Fas3 E-cad
Control shv
1
/+ shv
1
mys
ts1
/Y mys
ts1
;shv
1
/+ mys
ts1
;shv
1
shv
1
;UAS-mys mys
ts1
;UAS-mys UAS-mys
c833-GAL4

shv
1
;UAS-shv shv
1
;UAS-NoSP-shv
C
mys
ts1
;UAS-shg;
c833-GAL4
E-cad Vasa Fas3
mys
ts1

*
*
E-cad Vasa Fas3
mys
ts1
;UAS-shg;
Nanos-GAL4
Control
B
5
**
I I I
*

0.0
0.2
0.4
0.6
0.8
1.0
1.2
E-cad/Fas3 immunoreactivity
**
*
* *
C833-GAL4

*
I I I I I I I
37
15
36
23
28
11
27
8
20
I
19
**
*
**
E-cad
Fas3 Vasa
E-cad
Control shv
1
shv
1
;UAS-shg
/C833-GAL4
* *
*
E
F
G
0.0
0.2
0.4
0.6
0.8
1.0
Ecad/Fas3
Immunoreactivity
1.2
*
21
7
36
Percentage of altered hub structure (“Pulled”) and mislocalized hub cells
Genotype
shv
1

Control
shv
1
;UAS-shg/C833-GAL4
% of pulled hub cells
0
58.3
5.66
% of mislocalized hub cells
0
59.4
1.89
60
mislocalization. (D) Reducing Shv and DE-cadherin (shg) caused hub mislocalization.
Asterisks highlight the apical tip of the testes and arrows point the observed hub
position. (E) Images of testes stained with DE-cadherin and indicated antibodies. (F)
Quantification of the relative DE-cadherin intensity normalized to Fas III levels across
genotypes. (G) Table showing percentage of altered hub structure and mislocalized hub
cells. * p < 0.05 compared to control and ** p < 0.05 compared to indicated genotypes.
All values represent mean ± SEM and n is indicated in the bar graph. Age of flies
examined is 3 days after eclosion. For multiple samples, One-way ANOVA followed by
post hoc analysis with Bonferroni’s multiple-comparison test was used to determine
statistical significance. All Scale bars = 10 µm.

61
2.4.6 Upregulation of Shv preserves stem cell niche during aging
Having demonstrated that loss of Shv results in deterioration of hub architecture
and stem cell niche integrity, we next tested whether upregulation of Shv could prevent
decline in hub cell number and DE-cadherin expression in older animals. Interestingly,
upregulation of Shv in hub cells, hub+cyst cells, or GSCs preserved hub cell number
and DE-cadherin levels in 50 days old testes when compared to age matched control
testes. Only expression of Shv in hub cells or GSCs (but not hub+cyst cells using c833-
GAL4) preserved the number of GSCs during aging (Figs. 2.7.A and 2.7.B). Again,
these results are consistent with integrin-dependent competition for niche space by
somatic cells (Issigonis et al., 2009). We also determined if Shv level in the testes
changes with age. Immunostaining of testes dissected from young and older flies show
that Shv level is reduced in 30 days old flies (Fig. S2.7.F). This observation is
consistent with our model that Shv is required for the maintenance of hub structure
since testes from older flies are known to have reduced DE-cadherin levels and reduced
number of hub cells (Boyle et al., 2007). Together our results indicate that upregulation
of Shv has the ability to preserve stem cell niche during aging.

62

Figure 2.7. Upregulation of Shv prevents loss of niche and GSCs during aging.
(A) Staining of testes dissected from 50 days old males for the indicated genotypes.
Upregulation of Shv preserved DE-cadherin intensity during aging. Scale bar = 10 µm.
(B) Quantification of DE-cadherin intensity, average hub cell number per testis, and
average GSC number per testis in 50 days old flies. Sample numbers are indicated in
the graph. For multiple samples, One-way ANOVA followed by post hoc analysis with
Bonferroni’s multiple-comparison test was used to determine statistical significance. All
values represent mean ± SEM and * p < 0.05 compared to control. (C) Model for how
Shv maintains niche integrity in the Drosophila testes. In control testis (top), Shv
activates integrin receptors, promoting hub anchoring at the tip. Shv-dependent integrin
A
E-cad
Fas3 Vasa
Control
E-cad
UAS-shv/
c833-GAL4
UAS-shv/nanos-
GAL4
50 days old
upd-GAL4;
UAS-shv
Low

High

C
FAK
P
E-cadherin
FAK
Control
Shriveled
Active integrin
Inactive integrin
E-cad
Shv
Active integrin
E-cad
Inactive integrin
E-cad
Hub cells
GSC
Hub cells
GSC
shv
1

Lee et al. Figure 7
B
*
# of hub cells
*
*

0
2
4
6
8
15
31
15
11
10
12
*
*
*

0
2
4
6
8
15
31
15
11
# of GSCs cells
*

Control
UAS-shv/c833-GAL4
UAS-shv/nanos-GAL4
upd-GAL4;UAS-shv
*
*
0.0
0.5
1.0
1.5
2.0
22
31
39
11
E-cad/Fas3
immunoreactivity
*
2.5
63
activation also increases DE-cadherin expression, thus enhancing hub-hub and hub-
GSC cell adhesion to maintain hub architecture, niche integrity, and GSC health. In the
absence of Shv (shv
1
mutant; bottom), loss of integrin activation causes the hub cells to
drift away from the tip, and leads to disrupted cell-cell adhesion. This results in altered
niche structure, reduced number of GSCs, and eventually loss of the hub as observed
in shv
1
mutant.

64
2.5 Discussion
2.5.1 Summary of Results
In this study, we identify Shriveled as a key factor in preserving niche integrity
and GSC number in the Drosophila testes in vivo. We show that Shv is a secreted
protein that activates integrin signaling and in turn controls DE-cadherin levels. While
previous experiments suggest that integrin and DE-cadherin work independently in
steps involving hub anchoring and GSC attachment to the hub (Wang et al., 2006;
Tanentzapf et al., 2007; Voog et al., 2008; Leatherman and DiNardo, 2010; Tarayrah et
al., 2013), respectively, our results reveal a tight link between integrin activation and
DE-cadherin levels in hub cells. Cooperation between integrin and DE-cadherin
signaling may serve to ensure maintenance of the 3-dimensional structure of the hub by
promoting hub-hub cell interaction and GSC-hub adhesion following hub anchoring.
Furthermore, secretion of Shv by somatic cells and GSCs may act as a feedback signal
to sustain optimum integrin activation, DE-cadherin expression, and a healthy stem cell
niche during aging (modeled in Fig 2.7.C).

2.5.2 Maintenance of Stem Cell Niche Structure by Extracellular Presence of Shv
Our immunostaining data revealed that Shv is found in multiple cell types and in
distinct subcellular locations in the Drosophila testes. Due to the striking hub
deterioration phenotype seen in shv
1
mutants, we focused our studies at the apical tip.
We found that despite the presence of shv RNA in multiple cell types including the
GSCs, Shv protein was not detected inside the GSCs but often seen at the hub/GSCs
or germ/cyst cell borders. Together with our in vitro data showing release of Shv
extracellularly, we conclude that Shv is secreted efficiently in the testes by GSCs.
65
Consistent with this interpretation, expressing Shv in GSCs rescued the shv
1

phenotype, but not if the signal peptide had been deleted. Hub and CySCs also likely
secrete Shv, as it is also present at hub-hub or hub-CySC cell interface and expression
of Shv using hub+cyst cell driver rescued the mutant phenotype. Together, extracellular
Shv may allow multidirectional communication between different cell types for niche
maintenance. Note that based on reports showing hub mislocalization does not cause
sterility (Tanentzapf et al., 2007; Lee et al., 2008), we do not think that altered hub
structure and localization seen in shv
1
directly contributed to sterility in young flies.
Given that Shv is also found in the nucleus spermatocytes, it is likely that Shv plays
multiple roles during spermatogenesis. Future studies examining Shv function in the
nucleus and factors regulating Shv subcellular distribution will lead to better
understanding of its multiple roles in Drosophila testes and spermatogenesis.

2.5.3 Shv Interacts and Activates Integrin Signaling
Upon release, Shv activates integrin signaling. This is supported by 1) genetic
data demonstrating interaction between Shv and βPS integrin, and 2) in vitro data
showing extracellular Shv application triggers integrin-dependent changes in pFAK, and
3) mutating Shv at potential integrin-interaction sites diminished its ability to activate
integrin signaling. Based on our in vitro data that pre-incubation of RGD peptide can
block the ability of Shv to bind to integrin, we believe that Shv can bind to Position
Specific 2 (PSs) integrin group, which mediates binding through RGD peptide. In
Drosophila, it is thought that αPS3, αPS4, αPS5 and one βPS subunit (mys) are
expressed in the gonads (Brown et al., 2000; Tanentzapf et al., 2007). It is thus
66
possible that a single cell expresses multiple integrin receptors comprised of different
αPS subunit together with βPS, thus allowing it to bind to different ligands to promote
adhesion in a temporal and spatial manner. Furthermore, integrin receptors have
different affinity for different ligands. We thus envision that Shv acts to serve a
modulatory role, and not to out compete the binding of normal ligand. Altogether, our
results demonstrate that Shv is a novel ligand that interacts and activates integrin
extracellularly.

2.5.4 Shv-dependent Signaling Modulates DE-cadherin Expression
Our finding that Shv-dependent integrin signaling modulates DE-cadherin
expression in hub cells is surprising, since these two adhesion molecules are thought to
act in separate pathways involving hub anchoring and GSC attachment. Nevertheless,
the effects of integrin activation on DE-cadherin levels in the hub cells were not
examined previously, and while reducing DE-cadherin levels alone did not alter hub
positioning (Tanentzapf et al., 2007), contribution of DE-cadherin to hub anchoring
could not be ruled out. Indeed, we showed that DE-cadherin contributes to hub
anchoring, apparent only when integrin function is compromised. Furthermore, DE-
cadherin expression was increased upon extracellular application of Shv, but not if
integrin receptors were silenced, revealing direct relationship between DE-cadherin and
integrin signaling pathway. Shv-dependent integrin activation may lead to transcriptional
and/or translational increase of DE-cadherin rather than re-localization of DE-cadherin
to the cell surface. This is supported by observations in S2 cells showing an overall
elevation of DE-cadherin intensity rather than membrane redistribution following Shv
67
incubation. In addition, consistent with data that loss of DE-cadherin disrupts hub
compaction and reducing DE-cadherin in hub and CySCs leads to hub cell loss (Jenkins
et al., 2003; Voog et al., 2008), shv
1
mutants showed a hub compaction phenotype in
which the hub cells appear pulled away from the apical tip and a gradual loss of hub
cells during aging. Indeed, upregulation of DE-cadherin in shv
1
testes successfully
restored hub mislocalization as well as pulled hub structure, supporting the claim that
DE-cadherin serves downstream of Shv to maintain healthy stem cell niche structures.
Taken together, it is likely that Shv-dependent integrin activation enhances cadherin
expressions, which ensures proper cell-cell adhesion during aging. This may result in a
physical barrier that passively contributes to hub cell anchoring by decreasing the ease
by which hub cells could move away from the tip. Note that although integrin activation
modulates cadherin expression, other intrinsic and extrinsic factors likely regulate
cadherin expression independently, especially during early embryogenesis. This may
explain why mys null mutants still showed normal GSC formation around the mis-
positioned hub cells during embryonic stage (Tanentzapf et al., 2007). However, severe
reduction in integrin signaling in adults did lead to an age-dependent loss of hub
(Tanentzapf et al., 2007), similar to what was observed in shv
1
mutants.

2.5.5 Implication of Shv in Future Stem Cell Therapy
The identification of a novel activator of integrin signaling may have broad
implications since integrin is involved in a wide range of biological processes ranging
from development to cancer (Desgrosellier and Cheresh, 2010). Interestingly, the
human homolog of Shv, DNAJB11, has recently been identified in secretome profiling
68
as a protein upregulated in oral cavity squamous cell carcinoma (Hsu et al., 2014), as
well as secreted during unfold protein response activation (Genereux et al., 2014). In
addition, DNAJB11 has been shown to be secreted in mice and influences integrin
signaling (Wong et al., 2010; Wang et al., 2013), suggesting that the functions of Shv
may be evolutionarily conserved. Strikingly, we found that upregulation of Shv was
sufficient to preserve the number of hub cells and GSCs that normally decline during
aging. An understanding of how Shv functions in niche maintenance may thus
contribute to future stem cell therapy, as activation of integrin by Shv could potentially
enhance our ability to optimize niches and promote survival of in vitro derived stem cells
after transplantation.

2.6 Acknowledgements
We thank Drs. S. Dinardo (University of Pennsylvania), D. Harrison (University of
Kentucky), R. Hynes (Massachusetts Institute of Technology), P. Lasko (McGill
University), E. Matunis (Johns Hopkins University), A. Spradling (Howard Hughes
Medical Institute and Carnegie Institute for Science), R. Xi (National Institute of
Biological Science, Beijing), and Y. Yamashita (University of Michigan) for prompt and
generous sharing of fly stocks and antibodies. We are grateful to The Developmental
Hybridoma Bank (Iowa, USA) for multiple antibodies. We also thank Dr. K.-T. Min for
critical reading of the manuscript and members of the Chang laboratory for discussions.

69
2.7 Supplementary Figures

Figure S2.1. Hub cell and CySC quantification in different ages and altered hub
architecture seen in shv
1
mutants. (A) Quantification of control and shv
1
larval hub
and GSC. (B) Montage showing individual confocal z-stack images of control and shv
1

mutant testes stained with the indicated antibodies. Note that the overall hub
architecture is disrupted in shv
1
with hub cells spread throughout different z focal planes
and is not clustered at the tip. (C) Table showing percentage of altered hub structure.
(D) Representative images of the testes tip stained DN-cadherin antibody. (E)
Characterization of hub cells using anti-cactus antibody. (F) Quantification of CySCs by
counting Zfh-1(+), Eya(-) cells. Scale bar in (B), (D) and (E) = 10 µm.

Vasa Fas3 1B1 DAPI
Control shv
1

A
Control shv
1

Vasa Cactus DAPI
D
Percentage of altered hub structure (“Pulled”)
Genotype
shv
1
/+
Control
shv
1

Pulled
0
0
85
Total
110
57
141
% of pulled hub cells
0
0
58.3
C
E
Quantification of CySCs
Genotype
shv
1
/+
Control
shv
1

Zfh-1 (+) Eya (-)
36.4 ± 1.114
35.8 ± 0.892
27.8 ± 1.747
Total
20
10
22
B
0
2
4
6
8
10
12
Control shv
Number of cells
per larval testes
Hub cell
GSC
20
24
20
24
Lee et al. Figure S1
N-cad
Fas3
Control
N-cad
shv
1
/+ shv
1

*
*
*
F
70

Figure S2.2. Shv sequence alignment, transcript levels, and antibody specificity.
(A) Diagram depicting genomic region and P-element insertions within CG4164, which
we have renamed shriveled (shv). Orange boxes indicate coding region of shv and gray
boxes show untranslated mRNA. An adjacent gene, crq, is oriented in the opposite
direction. Green boxes indicate crq coding region and darker gray boxes show
untranslated mRNA. Blue inverse triangle indicates P-element insertions within the 5’-
UTR of shv. SP represents signal peptide. A predicted nuclear localization signal
(NLS) is also highlighted. Sequence alignment between human DNAJB11 and Shv are
shown below. Red residues highlight the RGD sequence and the similar residues in
fly. (B) Quantification of the relative shv and crq transcripts in control and shv
1
. * p <
0.05 compared to control. All values represent mean ± SEM. n = 4 independent
experiments. For paired samples, Student’s T-test was used. (C) Shv antibody is
specific for Shv. Western blot performed using fly extract detected with Shv antibody
(control) and antibody pre-absorbed with Shv peptide.
DNAJB11_HUMAN MAPQNLSTFCLLLLYLIGAVIAGRDFYKILGVPRSASIKDIKKAYRKLAL 50
CG4164 MQLIKCLVIIQLSLLLVEESFAGRDFYKILNVKKNANTNEVKKAYRRLAK 50
* : .: * * *: :*********.* :.*. :::*****:**
DNAJB11_HUMAN QLHPDRNPDDPQAQEKFQDLGAAYEVLSDSEKRKQYDTYGEEGLKDGHQS 100
CG4164 ELHPDKNKDDPDASTKFQDLGAAYEVLSNPDKRKTYDRCGEECLKKEGMM 100
:****:* ***:*. *************:.:*** ** *** **.
DNAJB11_HUMAN SHG-DIFSHFFGDFGFMFGGTPRQQDRNIPRGSDIIVDLEVTLEEVYAGN 149
CG4164 DHGGDPFSSFFGDFGFHFGGDGQQQD--APRGADIVMDLYVSLEELYSGN 148
.** * ** ******* *** :*** ***:**::** *:***:*:**
DNAJB11_HUMAN FVEVVRNKPVARQAPGKRKCNCRQEMRTTQLGPGRFQMTQEVVCDECPNV 199
CG4164 FVEIVRNKPVTKPASGTRKCNCRQEMVTRNLGPGRFQMIQQTVCDECPNV 198
***:******:: *.*.********* * :******** *:.********
DNAJB11_HUMAN KLVNEERTLEVEIEPGVRDGMEYPFIGEGEPHVDGEPGDLRFRIKVVKHP 249
CG4164 KLVNEERTLEIEVEQGMVDGQETRFVAEGEPHIDGEPGDLIVRVQQMPHP 248
**********:*:* *: ** * *:.*****:******* .*:: : **
DNAJB11_HUMAN IFERRGDDLYTNVTISLVESLVGFEMDITHLDGHKVHISRDKITRPGAKL 299
CG4164 RFLRKNDDLYTNVTISLQDALVGFSMEIKHLDGHLVPVTREKVTWPGARI 298
* *:.*********** ::****.*:*.***** * ::*:*:* ***::
DNAJB11_HUMAN WKKGEGLPNFDNNNIKGSLIITFDVDFPKEQLTEEAREGIKQLLKQGSVQ 349
CG4164 RKKGEGMPNFENNNLTGNLYITFDVEFPKKDLTEEDKEALKKILDQSSIN 348
*****:***:***:.*.* *****:***::**** :*.:*::*.*.*::
DNAJB11_HUMAN KVYNGLQGY 358
CG4164 RIYNGL--- 354
::****
A
B C
72
55
40
32
Shv
*

0.0
0.2
0.4
0.6
0.8
1.0
1.2
Relative
transcript level
shv
crq
CG4164/
Shv protein
DnaJ DnaJ
SP
NLS
shv
1
shv
c00496
shv
9803
crq CG4164 Genomic region
Lee et al. Figure S2
71

Figure S2.3. shv alleles. (A) Quantification of the relative shv transcripts. * p < 0.05
compared to control. All values represent mean ± SEM. n = 4 independent experiments.
(B) Table showing percentages of testes with altered hub structure. (C) Representative
images of testes dissected from 3-day old flies. Asterisks show the apical tip of the
testis and arrows highlight distally located hub. (D) Testes stained with DE-cadherin (E-
cad) and indicated antibodies. Lower panels show pseudo-colored images of DE-
cadherin staining intensity. (E) Quantification of GSCs per testis dissected from 3-day
old flies. * p < 0.05 compared to control. For multiple samples, One-way ANOVA
followed by post hoc analysis with Bonferroni’s multiple-comparison test was used to
determine statistical significance. Scale bars in (C) and (D) = 10 µm

I I I I I I
I I I I I I
0.0
0.2
0.4
0.6
0.8
1.0
Relative shv
transcript level
I
1.2
*
*
*
*
A
C
Percentage of altered hub cell structures (“Pulled”)

Genotype
shv
1

Control
shv
c00496

shv
PJ

shv
c00496
/shv
1

shv
9803
/shv
1

%
0.0
58.3
20.0
0.0
15.4
58.3
B
E-cad
Fas3 Vasa
E-cad
Control shv
1
shv
c00496
shv
c00496
/shv
1
shv
9803
/shv
1
shv
PJ

Low

High

D
Lee et al. Figure S3
Vasa
Fas3 E-cad
Control shv
1
shv
c00496
shv
c00496
/shv
1
shv
9803
/shv
1
shv
PJ

* * * * *
*
I
E
I I I I I I
I I I I I I
0
2
4
6
8
10
Average number
of GSCs per testes
12
*
*
* *
I
14
57
30
10
13
12
5
72

Figure S2.4. Shv distribution in testes. Dual fluorescent shv RNA and protein
detection to mark different cell types. (A) Control and shv
1
testes taken at lower
magnification demonstrating ubiquitously expressed shv RNA at the apical tip of the
testis. Higher magnification images representing the presence of shv RNA in the
spermatocytes. shv RNA is seen in the hub, CySCs, germ and cyst cells of the control
testes, but barely detectable in shv
1
mutant. Similarly, shv RNA is seen at high level in
control spermatocytes and cyst cells, but not in shv
1
mutant. (B) Control testes stained
with Shv, fusome and indicated antibody to see where Shv is located. Scale bar = 10
µm (C) Shv subcellular distribution is further investigated by staining testes expressing
fluorescently-tagged organelle markers with Shv antibody. Peroxisome marker (SKL-
GFP); lysosme marker (SPIN-myc-eGFP); golgi marker (Golgi-GFP); mitochondria
marker (mito-GFP); ER marker (RFP-KDEL). Scale bar = 10 µm. (D) Abundant Shv
protein is detected in the nucleus of control spermatocytes but is significantly reduced in
shv
1
(arrowhead). Scale bar = 50 µm. Age of flies examined is 3 days after eclosion.
Lee et al. Figure S4
A
B
D
C
Shv Laminin
Control shv
1

Control shv
1

Shv Vasa Fas3 Hts Shv
Shv Vasa Fas3 Hts Shv
Control shv
1

Control
Fas3 Fusome Shv Vasa
RFP-KDEL
SKL-GFP SPIN-myc-eGFP
Mito-GFP
GFP Shv Fas3
Golgi-GFP
GFP Shv Fas3 Shv RFP
73

Figure S2.5. Restoring Shv in CySCs rescues GSC loss phenotype. (A)
Quantification of the average number of GSCs per testes for the indicated genotypes.
(B) Quantification of the average number of hub and GSCs per testes for the indicated
genotypes. (C) Quantification of the average number of GSCs per testes for the
indicated genotypes. * p < 0.05 compared to control. For multiple samples, One-way
ANOVA followed by post hoc analysis with Bonferroni’s multiple-comparison test was
used to determine statistical significance.

Lee et al. Figure S5
A B
C
0
2
4
6
8
10
12
# of GSCs per testes
3-day 7-day 14-day 35-day
Control
shv
1

shv
1
;UAS-shv/c833-GAL4
*
29
*
*
29
13
12
20
23
19
23
20
UAS-shv /c833-GAL4
*
*
*
*
*
57
30
12
21
22
14
17
17
Days after eclosion
*
*
*
# of GSCs per testes
I I I I I I I
0
2
4
6
8
10
12
14
3-day 7-day 14-day
*
35-day
11
10
16
Days after eclosion
Control
shv
1

shv
1
;UAS-shv/nanos-GAL4
I
11
9
11
10
10
UAS-shv/nanos-GAL4
57
30
12
21
22
14
17
17
*
*
*
*
0.0
2.0
4.0
6.0
8.0
10.0
12.0
Number of cells
per testes
Hub Cell
GSC
74

Figure S2.6. Extracellular Shv activates integrin signaling. (A) Western blot
depicting Shv levels in the media for the indicated conditions. Shv pull down efficiently
removed Shv proteins from the media. (B) Western blot demonstrating efficiency of
mys-RNAi. (C) Quantification of pFAK intensity in cells transfected with the indicated
constructs. Intracellular expression of Shv did not alter pFAK levels when Shv was
removed extracellularly. Number of cells examined is indicated in the bar graph. (D)
Representative images of cell spreading and pFAK levels in S2 cells treated with the
indicated media. (E) Western blot depicting the normal secretion of Shv protein tagged
with GFP. (F) Quantification of GFP levels on the S2 cell surface normalized to the
amount of GFP without RGD peptide incubation. * p < 0.05 compared to control. (G)
Western blot demonstrating the presence of Shv
LNV
extracellular in the media. For
paired samples, Student’s T-test was used. For multiple samples, One-way ANOVA
followed by post hoc analysis with Bonferroni’s multiple-comparison test was used to
determine statistical significance. Number of cells examined is indicated in the bar
graph. All values represent mean ± SEM. Scale bar = 5 µm.

Lee et al. Figure S6
A
55
40
Shv
Shv pull down
Control pull down
media
V5
B
mock
mys-RNAi
130
55 β-tubulin
βPS integrin
C
0.0
0.2
0.4
0.6
1.2
Relative pFAK intensity
0.8
1.0
1.4
Transfected cells
60
48
51
49
Shv
LNV
55
40
Shv
Media
V5
control
F
D

0.0
0.4
0.8
1.2
Normalized
GFP intensity
* 41
52
0.2
0.6
1.0
1.4
-
+
+ +
RGD
Shv Media
E
G
pFAK DAPI
Fresh
Shv-GFP
media
GFP media
72
Shv-GFP

Mock
Media
GFP
55
75

Figure S2.7. Secretion of Shv regulates DE-cadherin levels. (A) Representative
images of cell spreading and E-cad levels in S2 and mys-RNAi cells treated with the
indicated media. Scale bar = 5 µm. (B) Quantification of E-cad intensity in control and
mys-RNAi transfected cells following media treatment. (C) Pseudo-colored images of
DE-cadherin in 3-day old testes. Asterisks indicate the hub. (D) Quantification of relative
DE-cadherin intensity across genotypes. * p < 0.05 compared to control. ** p < 0.05
between the indicated genotypes. All values represent mean ± SEM. For multiple
samples, One-way ANOVA followed by post hoc analysis with Bonferroni’s multiple-
comparison test was used to determine statistical significance. n is indicated in the bar
graph. (E) Percentage of mislocalized hub cells observed across genotype. (F) 3 and 30
days old testes labeled with Shv and indicated antibody. Scale bar in (C) and (F) = 10
µm.

Lee et al. Figure S7
*
Percentage of mislocalized hub cells
Genotype
shg/+
shv
1
/+
shv
1
/shg
% of mislocalized hub
0
10
85
n
20
10
20
E
C
Low

High

Control shv
1

shv
1
;UAS-
NoSP-shv
E-cad
shv
1
;UAS-shv
*
*
nanos-GAL4

E-cad
I

0.0
0.2
0.4
0.6
0.8
1.0
1.2
Relative
DE-cadherin intensity
*
nanos-GAL4

I I I
12
14
12
5
I
*
*
D
Vasa Shv Fas3 Shv Fas3
3 days old 30 days old
Control
F
A
B
0.0
2.0
4.0
6.0
8.0
10.0
Relative
DE-cadherin intensity
22
40
45
*
Media treated
mys-RNAi
I I I I I
S2 cell
I
E-cad DAPI
Fresh Shv Shv
Media treated
Control mys-RNAi
76

Video S2.1. 3D presentation of Drosophila testes stem cell niche. 3D projection
rendered from confocal z-stack images of control testis stained with Vasa (green), FasIII
and 1B1 antibodies (red). Arrows point to the hub as visualized by Fas III staining
clustered at the apical tip.

Lee et al. Video S1
77

Video S2.2. 3D presentation of Drosophila testes stem cell niche. 3D projection
rendered from confocal z-stack images of shv mutant testis stained with Vasa (green),
FasIII and 1B1 antibodies (red). Arrows point to the “pulled” hub stained by Fas III.

Lee et al. Video S2
78
Chapter 3.
Activity-induced synaptic structural modifications by an activator of integrin
signaling at the Drosophila neuromuscular junction
Joo Yeun Lee, Junhua Geng, Juhyun Lee, Andrew R. Wang, and Karen T. Chang

3.1 Abstract
Activity-induced synaptic structural modification is crucial for neural development
and synaptic plasticity, but the molecular players involved in this process are not well
defined. Here, we report that a protein named Shriveled, Shv, regulates synaptic
growth and activity-dependent synapse maturation at the Drosophila neuromuscular
junction. Depletion of Shv causes synaptic overgrowth and an accumulation of
immature boutons. We find that Shv physically and genetically interacts with βPS
integrin. Furthermore, Shv is secreted during intense, but not mild, neuronal activity to
acutely activate integrin signaling, induce synaptic bouton enlargement, and increase
postsynaptic glutamate receptor abundance. Consequently, loss of Shv prevents
activity-induced synapse maturation and abolishes post-tetanic potentiation, a form of
synaptic plasticity. Our data identifies Shv as a novel trans-synaptic signal secreted
upon intense neuronal activity to promote synapse remodeling through integrin receptor
signaling.

79
3.2 Introduction
The synapse is a highly dynamic structure that undergoes rapid changes in
morphology and strength in response to synaptic activity (Berninger and Bi, 2002; Fiala
et al., 2002; Chklovskii et al., 2004; Bourne and Harris, 2008; Holtmaat and Svoboda,
2009). Such activity-induced structural and functional plasticity underlies complex brain
functions such as learning and memory and is associated with neurodevelopmental and
psychiatric disorders (Fiala et al., 2002; Trachtenberg et al., 2002; Lamprecht and
LeDoux, 2004; Xie et al., 2007). Multiple synaptic machineries that govern gene
transcription and post-translational modification of synaptic molecules have been shown
to play an important role in activity-induced synaptic plasticity by coordinating efficient
synaptic transmission between pre- and postsynaptic compartments (Berninger and Bi,
2002; Lamprecht and LeDoux, 2004; Je et al., 2006; Cohen and Greenberg, 2008; Kim
et al., 2015; Vallejo et al., 2016). However, despite a wealth of discoveries on molecules
that play major roles in functional plasticity, the signaling mechanism underlying activity-
induced structural modifications is not well understood.
The synaptically located extracellular matrix (ECM) environment and cell
adhesion molecules (CAMs), which modulate adhesive forces between the pre- and
postsynaptic terminals, are thought to coordinate communication across the synaptic
cleft to regulate synapse growth, remodeling, and stabilization (Dityatev and Schachner,
2003; Yamagata et al., 2003; Rushton et al., 2009; Arikkath, 2010; Dityatev et al., 2010;
Broadie et al., 2011). In the Drosophila glutamatergic neuromuscular junction (NMJ),
several secreted molecules that establish the synaptic cleft ECM environment have
been discovered. For example, laminin, heparan sulfate proteoglycans, Hikaru Genki,
80
Mind-the-Gap (Mtg) are secreted extracellular molecules that regulate synaptic
development (Hoshino et al., 1996; 1999; Johnson et al., 2006; Inoue and Hayashi,
2007; Rohrbough et al., 2007; Rushton et al., 2009; Tsai et al., 2012a). In addition, the
Drosophila Wingless (Wg) is a secreted signal that acts via Frizzled (Dfz2) receptors to
modulate activity-dependent synaptic growth (Ataman et al., 2008). Among CAMs,
integrin receptors are heterodimeric transmembrane receptors that enable intercellular
signaling by bridging the ECM to the intracellular actin cytoskeleton (Campbell and
Humphries, 2011). Emerging data suggest that integrin receptors are important for
coordinating activity-dependent structural and functional modifications in Drosophila and
mammalian nervous system (Beumer et al., 1999; Rohrbough et al., 2000; Chavis and
Westbrook, 2001; Beumer et al., 2002; McGeachie et al., 2011; Dani et al., 2014).
Studies indicate that integrins contribute to synaptic stabilization by promoting new
AMPA receptor insertions into the postsynaptic membrane in hippocampal neurons
(Kramár et al., 2006; Cingolani et al., 2008; McGeachie et al., 2011), and that integrins
have been shown to increase its surface expression to stabilize long-term potentiation
(LTP) (Lin et al., 2005). Consistent with these observations, functional blockade of
integrin-ECM ligand interaction have been shown to significantly reduce consolidation of
LTP in mammalian hippocampal neurons (Grooms and Jones, 1997; Stäubli et al.,
1998; Chun et al., 2001; Chan et al., 2006; Huang et al., 2006) and activity-dependent
synaptic plasticity in Drosophila (Rohrbough et al., 2000; Dani et al., 2014).
While several lines of evidence suggest that integrin activation is essential for
synaptic plasticity (Bahr et al., 1997; Beumer et al., 1999; Rohrbough et al., 2000;
Chavis and Westbrook, 2001; Chun et al., 2001; Kramár et al., 2006; McGeachie et al.,
81
2011; Dani et al., 2014), very little is known about the molecular cues that contribute to
integrin activation during activity-induced synaptic plasticity. The Drosophila
glutamatergic NMJ is an excellent model to identify molecular players modulating
activity-dependent synaptic plasticity due to its powerful genetics and well conserved
signaling pathways. There are 5 α integrin subunits (αPS1-5) and 2 β integrin subunits
(βPS and βν) in Drosophila (Brown, 1993; Gotwals et al., 1994). Several integrin ligand
such as Tiggrin (Fogerty et al., 1994), laminin A (Inoue and Hayashi, 2007), Teneurin
(Graner et al., 1998; Mosca, 2015), have been reported. However, to date, laminin is
the only known integrin ligand with its release regulated in an activity-dependent
manner at the fly NMJ (Tsai et al., 2012a). Synaptic activity has been shown to down
regulate laminin release from the postsynaptic muscle cells, thereby permitting synaptic
growth and expansion through reduced integrin activation (Tsai et al., 2012a). Here, we
present evidence that an activator of integrin signaling, Shriveled (Shv) (Lee et al.,
2016), is released presynaptically in an activity-dependent manner at the Drosophila
NMJ. Shv is a fly homolog of human DNAJB11/ERDJ3 protein, which is thought to be a
secretable chaperone protein that modulates unfolded protein response and integrin
affinity (Wang et al., 2013; Genereux et al., 2014). We demonstrate that Shv regulates
proper synaptic growth at the Drosophila NMJ and plays an important role in activity-
dependent synaptic plasticity via integrin signaling pathway. We find that Shv is acutely
secreted during intense neuronal activity, but not during mild activity, to promote
synaptic bouton enlargement and increase postsynaptic glutamate receptor abundance.
We further demonstrate that Shv release is required for synaptic plasticity. Together,
our data suggest that intense neuronal activity triggers Shv release to contribute to
82
activity-dependent synapse maturation through integrin signaling at the synapse, and
further imply that neurons utilize distinct molecular cues to modulate synapse growth
and plasticity in response to different synaptic stimulation strength.

3.3. Materials and Methods
Drosophila Genetics
Flies were cultured at 25 °C on standard cornmeal, yeast, sugar, and agar
medium under a 12-hour light and 12-hour dark cycle unless noted otherwise. White-
eyes flies (w
1118
) were used as wildtype throughout all experiments. The following fly
lines were used with Bloomington Stock # in parenthesis: mys
ts1
(#3169), 24B-GAL4
(#1767), UAS-mys (from R. Xi), shv
1
(Lee et al, 2016), UAS-NoSP-shv (Lee et al, 2016).
Transgenic RNAi flies UAS-shv-RNAi (#108576) and UAS- β
ν
Integrin-RNAi (#40895)
were obtained from Vienna Drosophila RNAi center. UAS-shv-GFP and UAS-NoSP-
shv-GFP were generated by inserting eGFP sequence to c-terminus of full-length Shv
and Shv with deleted signal peptide and subcloned into the pINDY6 vector, respectively.
Transgenic files were generated by standard transformation method. All other stocks
and standard balancers were obtained from Bloomington Stock Center (Bloomington,
IN).

Immunochemistry
Third-instar larvae were dissected in Ca
2+
free dissection buffer: NaCl 128mM,
KCl 2mM, MgCl
2
4.1mM, sucrose 35.5mM, HEPES 5mM, EGTA 1mM. Motor nerves
were cut and dissected preparations were fixed in 4% paraformaldehyde solution for 25
83
min at room temperature (except Bouin’s fixative was used for GluRIII staining). Fixed
samples were then washed with 0.1% triton X-100 in PBS (PBST) or with PBS for
detergent-free condition. Samples were blocked with 5% normal goat serum in PBST or
PBS as indicated. Primary antibodies were diluted in blocking solution and used as
following: rabbit anti-Shv, 1:400 (Lee et al, 2016); mouse anti-Dlg, 1:500 (4F3, DSHB);
rabbit anti-phosphoFAK, 1:50 (Abcam); rabbit anti-GluRIII, 1:1000 (from DiAntonio);
Cy3-conjugated anti-HRP, 1:100 (Jackson ImmunoResearch). Secondary antibodies
used were Alexa-488 or 405 conjugated, 1:250 (Invitrogen).

Synapse Activity Stimulation

Third-instar larval body wall were dissected in normal HL-3 solution without Ca
2+

(NaCl 110mM, KCl 5mM, MgCl
2
10mM, sucrose 30mM, HEPES 5mM, EGTA 1mM,
trehalose 5mM, NaO
3
10mM, pH 7.2), leaving the brain and peripheral nerved intact.
High K
+
stimulation was achieved by applying solution with 90mM KCl in HL-3 solution
with Ca
2+
(NaCl 25mM, KCl 90mM, MgCl
2
10mM, CaCl
2
1.5mM, sucrose 30mM, HEPES
5mM, trehalose 5mM, NaHCO
3
10mM, pH 7.2) for 10min and replaced with resting
solution (normal HL-3 solution without EGTA) for 2 min followed by 25 min fixation. For
the experiment with exogenous Shv application, dissected preps were incubated with 1
µg/ml of purified Shv-HA or Shv
LNV
-HA in HL-3 for 10 minutes. 3X spaced high K
+
depolarization paradigm was adapted from Vasin et al. (Vasin et al, 2014) except the
CNS and nerve kept intact in our preparation. 5X spaced high KCl stimulation paradigm
was adapted from Ataman et al. (Ataman et al, 2008). For inhibition of transcription or
translation, dissected NMJs were preincubated with either 5µM actinomycin-D (Biotium)
84
or 200µM cycloheximide (Amresco) for 20 min in HL-3, and the inhibitor was also
included in normal/high K
+
solutions, and applied throughout stimulation protocol (Frank
et al, 2006; Koon et al, 2011). For electrical stimulation, the severed nerve was
stimulated by suction electrode at either 1Hz or 10Hz for 5min in normal

HL-3 solution.

Image Quantification
Images of synaptic terminal from NMJ 6/7 in A2 and A3 were captured using
Zeiss LSM5 confocal microscope using a 63X 1.6NA oil immersion objective with a 0.7x
or 1x zoom. The NMJ at muscle 4 in A3 to A5 were imaged and used for quantifying
axonal branching. An axonal projection was defined as a single branch only when two
or more synaptic boutons were observed. Average of bouton size was determined by
outlining individual bouton using image J. Number of boutons were calculated by
counting HRP-labelled boutons then normalized to the surface are of muscle 6/7 taken
with a 10x objective. Synaptic boutons without Dlg, NC-82 or GluRIII staining were
counted as a ghost bouton. Ghost bouton numbers were then normalized to the total
number of synaptic terminal in muscle 6/7. When comparing intensity across genotypes,
the exposure time was kept constant for all genotypes per experiment. Staining
intensities were measured by normalizing the fluorescence intensity to bouton area
outlined by either HRP or disc-large using Image J. All values were normalized to
control done within the same experimental set.

85
Electrophysiology
Third-instar larvae were dissected and then bathed in a modified HL-3 (NaCl
70mM, KCl 5mM, MgCl
2
10mM, sucrose 115mM, HEPES 5mM, trehalose 5mM,
NaHCO
3
10mM, pH 7.1) with 0.2 or 0.5mM CaCl
2
as indicated. Current-clamp
recordings were performed on muscles 6 in abdominal segments A2 or A3, and severed
ventral nerves were stimulated with suction electrodes at 0.3 msec stimulus duration.
The recording electrode with resistance between 15-40MΩ was filled with 3M KCl and
data with resting potential more hyperpolarized than -60mV were analyzed. Data sets
were rejected if resting potential were deviated by >10% during the recording and EPSP
amplitude dropped abruptly indicating stimulated nerves did not fully function throughout
the recording. Data was acquired using an Axopatch 200B amplifier, digitized using a
Digidata 1440A, and controlled using pClamp 10.3 software (Molecular Devices,
Sunnyvale, CA). Data was analyzed using MiniAnalysis (Synaptosoft), Clampfit
(Molecular Devices), and Microsoft Excel. Average EPSP was corrected using nonlinear
summation.

Western Blotting
To detect Shv levels in flies, protein extracts were obtained by homogenizing flies
in RIPA lysis buffer (50 mM Tris-HCl, pH7.5, 1% NP-40, 0.5% NaDoc, 150 mM NaCl,
0.1% SDS, 2 mM EDTA, 50 mM NaF, 1 mM Na
3
VO
4
, 250 nM cycloporin A, protease
inhibitor cocktail (Roche) and phosphatase inhibitor cocktail 1 (Sigma) using mortar and
pestle. 20 µg protein homogenate was separated by SDS-PAGE and transferred to
nitrocellulose membranes. Primary antibodies were diluted in blocking solution as
86
following: rabbit Shv, 1:500; anti-tubulin 1:500 (7E10, DSHB); anti-beta actin,1:2500
(AC-15, Abcam); anti- βPS integrin, 1:1500 (from R. Hynes), anti-HA, 1:200 (Santa
Cruz).

Immunoprecipitation
Fly heads (100) were collected on dry ice and homogenized in lysis buffer (10
mM HEPES, 100 mM NaCl, 10 mM EDTA, 1% NP-40, 1 mM Na
3
VO
4
, 50 mM NaF, 250
nM cyclosporin A) supplemented with complete EDTA-free protease inhibitor cocktail
tablets (Roche). Anti-HA-Agarose beads (Sigma) (20 µl) were added to the extracts and
rotated at 4°C overnight for immunoprecipitation. After washing 3 times with lysis buffer,
the immunocomplexes were eluted with SDS sample buffer, and all of the eluates were
used for Western blotting.

Protein Purification and pull-down experiments
Protein purification was conducted as previously described (Chen et al, 2014).
Briefly, His and HA doubly tagged shv or shv mutant (His-shv-HA or His-shv
LNV
-HA)
were obtained by cloning the sequences of shv or shv
LNV
into pET15b vector (Novagen).
BL21 strain containing the expression plasmids were grown at 37 °C until A600 of the
culture reached 0.6–0.8. Expression of the proteins was induced by 1.0 mM IPTG for
4 hrs at 30 °C, bacteria were harvested and stored at −80 °C for the future using. Ni-
NTA Purification System (Invitrogen) was used to purify His-tagged proteins. Eluted
His-Shv-HA and His-Shv
LNV
-HA were dialyzed into PBS using Slide-A-Lyzer Dialysis
(ThermoScientific) and concentrated down using Vivaspin centrifugal concentrator
87
(Vivaproducts). For the pull-down experiments, His-Shv-HA or His-Shv
LNV
-HA
containing bacterial cell lysates were incubated with anti-HA-agarose beads (Sigma
Aldrich) at 4 °C overnight and washed with lysis buffer 4 times. Fly protein (2.5 mg from
wildtype flies) were pre-cleared with anti-HA-agarose beads for 1 Hr, then incubated
with anti-HA-agarose beads coupled with Shv or Shv
LNV
at 4 °C for 3 hrs, washed with
lysis buffer for 4 times, and eluted with SDS-PAGE sample buffer. All of the eluates
were used for Western blotting.

Statistics
For paired samples, Student’s T-test was used. For multiple samples, One-way
ANOVA followed by post hoc analysis with Bonferroni’s multiple-comparison test was
used to determine statistical significance. All plots show mean ± SEM.

3.4 Results
3.4.1 Shriveled is a presynaptically secreted protein that modulates synaptic
growth
We previously characterized a sterile fly mutant which we named shriveled (shv)
due to the appearance of shriveled testes (Lee et al., 2016). Our results revealed that
Shv is a secreted protein that activates βPS integrin in the fly testes to maintain niche
architecture (Lee et al., 2016). We also found that Shv protein is expressed in the
Drosophila nervous system and present in the larval motor neurons (Figs. 3.1.A and B).
We thus investigated if Shv is present locally at the NMJ. Immunostaining using non-
permeabilizing conditions confirmed that low levels of Shv can be detected at the NMJ
of wildtype larvae but not in shv
1
mutant (Fig. 3.1.C), a virtually null allele with only
88
about 3% of the shv transcript as the wildtype (Lee et al., 2016). Shv protein appeared
punctate and could only be observed in the NMJ using detergent-free conditions,
suggesting that Shv is present extracellularly as a secreted protein. To further confirm
that Shv is released extracellularly, we generated full-length Shv protein tagged with
GFP (Shv-GFP) as well as Shv with truncated signal peptide tagged with GFP (NoSP-
Shv-GFP). Figure 3.1.D shows that neuronal expression of Shv-GFP leads to positive
extracellular GFP signal outside of synaptic boutons but not if the signal peptide is
deleted, confirming Shv-GFP is a secreted protein at the NMJ.

89

Figure 3.1. Shriveled is a presynaptically secreted protein. (A) Representative
Western blot showing Shv protein is expressed in Drosophila nervous system and shv
1

is virtually a null allele. (B) Immunostaining of Drosophila larval brain of control and shv
1

with indicated antibodies. Boxed region is enlarged representing Shv expression in the
ventral ganglion and motor neurons. Scale bar = 20 µm. (C) Representative images of
the NMJs stained with Shv antibody in detergent-free condition. (D) Images of synaptic
terminal highlighting the presence of Shv-GFP outside of the boutons but not if the
signal peptide has been deleted. Scale bar = 5 µm.

B
Shv HRP Shv
1b
1s
1b
1s
1b
1s
Control shv
1

A
Figure 1
C D
UAS-shv-GFP UAS-NoSP-shv-GFP
nSynb-GAL4
HRP Shv-GFP
Control
shv
1

Head
Head
43
34
Shv
Body
Body
β-actin 42
Control shv
1

Elav Shv
90
Immunostaining of the NMJ with HRP revealed that Shv is required to restrict
synaptic growth. shv
1
mutant showed a synapse over-proliferation phenotype with an
increase in the number of synaptic boutons and synapse complexity in the form of
branching (Figs. 3.2.A-D). shv
1
mutant synapses also displayed an elevated number of
“ghost boutons”, or undifferentiated boutons that are immature and have not yet been
stabilized (Figs. 3.2.E and F). These ghost boutons contain synaptic vesicles and
express the neuronal membrane marker recognized by anti-HRP antibody, but lack
active zones or postsynaptic Dlg (Ataman et al., 2008; Vasin et al., 2014). The
observation that there are more ghost boutons as well as an overall increase in synaptic
complexity further suggest that the presence of Shv is required to restrict synaptic
growth and promote synapse maturation.
We next asked whether presynaptic and/or postsynaptic release of Shv is
important for modulating synaptic growth. To this end, we knocked down Shv either
pre- or postsynaptically using RNAi and tissue specific drivers (Figs. 3.2.A-F). We
found that driving shv-RNAi using a pan-neuronal driver, nSynb-Gal4, caused a
synaptic over- growth phenotype and an accumulation of immature ghost boutons, but
not when shv-RNAi was expressed in muscles using the 24B-Gal4 driver (Figs. 3.2.A-
F). In addition, neuronal expression of full-length Shv sufficiently rescued the synaptic
phenotypes of shv
1
but not when Shv was expressed in muscle. Deleting the signal
peptide of Shv abolished the ability of neuronal Shv to rescue shv
1
phenotype.
Together, these results indicate that release of Shv from neurons is necessary for
normal synaptic growth, perhaps because neurons have the proper machineries to
process Shv and control its release. We also found that overexpression of shv alone
91
did not affect synaptic growth and complexity (Figs. 3.2.A-F), suggesting that even
though the presence of Shv is required to restrict synaptic growth, it does not inhibit
synaptic growth.

92

Figure 3.2. Shriveled modulate synaptic growth. (A) Muscle 6/7 NMJ at A2 stained
by HRP for the indicated genotypes. Scale bar = 10 µm. (B) Number of boutons
normalized to muscle surface area (MSA). (C) Quantification of synaptic branch
Figure 2
shv
1
;
UAS-shv
UAS-shv
24B-GAL4
Control shv
1

shv
1
;
UAS-shv
UAS-shv
nSynb-GAL4
shv
1
;
UAS-NoSP shv
UAS-
shv-RNAi
UAS-
shv-RNAi
A
B
*
*
*
*
0.0
0.5
1.0
1.5
2.0
Normalized
bouton #/MSA (µm
2
)

nSynb-GAL4 24B-GAL4
24
28
11
20
11
21
16
27
8
C
*
* *
0.0
1.0
2.0
3.0
4.0
5.0
6.0
Branch Numbers
*
nSynb-GAL4 24B-GAL4
22
20
11
12
23
10
13
18
9
24B-GAL4
nSynb-GAL4
DLG HRP
Control
shv
1

shv
1
;UAS-shv
shv
1
;UAS-shv
UAS-shv
shv
1
;
UAS-NoSP-shv
UAS-shv-RNAi
UAS-shv UAS-shv-RNAi
DLG HRP
D
F
*
*
*
*
0.0
2.0
4.0
6.0
8.0
10.0
Normalized
Ghost bouton#/NMJ
nSynb-GAL4 24B-GAL4
E
30
29
10
7
12
11
8
15
8
Control shv
1

shv1;
UAS-shv
UAS-shv
nSynb-GAL4
shv1;
UAS-shv
shv
1
;
UAS-NoSP-
shv
24B-GAL4
UAS-
shv-RNAi
UAS-
shv-RNAi
UAS-shv
DLG HRP
93
numbers in muscle 4. (D) Representative images of NMJs at muscle 4 stained with HRP
and Dlg across genotypes. Scale bar = 10 µm. (E) Number of ghost bouton
appearances per NMJ across genotypes. (F) Images of NMJs labelled with the
indicated antibodies. Arrows points to ghost boutons that are recognized by HRP
labelling while lacking Dlg immunostaining. Scale bar = 5 µm. * p < 0.05 compared to
control. All values represent mean ± SEM.

94
3.4.2 Shv modulates integrin receptor activation at the NMJ
Our previous study in fly testes and S2 cells demonstrated that Shv can activate
βPS integrin signaling (Lee et al., 2016), we therefore tested if Shv acts as a novel
regulator of integrin signaling at the NMJ. First, we confirmed that Shv can physically
interact with βPS integrin receptor via immunoprecipitation (Fig. 3.3.A). Next, we asked
if (1) Shv indeed modulates integrin signaling at the NMJ, and (2) if Shv genetically
interacts with βPS integrin receptor to regulate synaptic growth. As a measure of
integrin activation, we determined the levels of phosphorylated focal adhesion kinase
phosphorylation (pFAK), since its level strongly correlates with integrin activation (Mitra
et al., 2005; Tsai et al., 2008; Harburger and Calderwood, 2009; Campbell and
Humphries, 2011). As βPS integrin receptors are present pre- and postsynaptically
(Beumer et al., 1999), we monitored the levels of pFAK in the presynaptic terminal
(within boundary of neuronal membrane marker HRP) and the total levels at the
synapse (within the boundary of postsynaptic marker Dlg) (Fig. S3.1.A). We found that
consistent with Shv being an activator of integrin signaling, depleting Shv in shv
1
or
neuronal knockdown of Shv using RNAi both led to decreased pFAK levels
presynaptically, as well as the total levels of pFAK at the NMJ (Figs. 3.3.B and C; Table
S3.1). Neuronal overexpression of shv also elevated both presynaptic and total pFAK
levels at the NMJ compared to control, but not if shv is overexpressed in muscles using
the 24B-Gal4 driver. These results indicate that release of Shv by neurons activates
integrin signaling presynaptically and postsynaptically, in an autocrine and paracrine
fashion. In support of this, neuronal expression of the full-length Shv restored pFAK
level of shv
1
both pre- and postsynaptically, whereas the non-secretable Shv (NoSP-
95
Shv) did not (Figs. 3.3.B and C). This suggests that Shv activates integrin signaling via
an outside-in mechanism, consistent with previous experiments done using S2 cultures
(Lee et al., 2016).

96

Figure 3.3. Shv activates integrin receptor at NMJ. (A) Immunoprecipitation (IP)
assay followed by Western blots with the indicated antibodies demonstrate interaction
between Shv and βPS integrin. (B) and (G) Representative images of NMJs stained
with HRP and pFAK across genotypes. (C) and (F) Quantification of presynaptic pFAK
and total pFAK levels at synaptic terminal by outlining HRP or Dlg respectively. (D)
Representative images of NMJs stained with HRP for the indicated genotypes. (E)
Quantification of boutons numbers normalized to muscle surface area (MSA). * p < 0.05
compared to control and ** p < 0.05 compared to indicated genotypes. *** Represents p
< 0.05 when comparing value of total pFAK between the indicated genotypes. All
values represent mean ± SEM.
130 kD 100 kD 40 kD input IP:HA HA βPS
Integrin Control UAS-shv/
nSynb-GAL4 Figure 3
B
nSynb-GAL4
*
24B-GAL4
*
*
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Normalized pFAK intensity
Total pFAK
Pre pFAK
*
*
*
*
*
*
*
A C
Control shv
1
mys
ts1
mys
ts1
;
shv
1
shv
1
;
UAS-mys

mys
ts1
;
UAS-shv

nSynb-GAL4
shv
1
;
UAS-mys

UAS-mys

24B-GAL4
UAS-mys

D
*
*
*
*
*
**
nSynb-GAL4
0.0
0.5
1.0
1.5
2.0
Normalized
bouton #/MSA (µm
2
)

24B-GAL4
11
28
24
5
6
7
13
13
15
E
G F
UAS-shv-RNAi
shv
1
;UAS-shv
shv
1
;UAS-NoSP-shv
UAS-shv
nSynb-GAL4
HRP pFAK
Control
shv
1

UAS-shv-RNAi
shv
1
;UAS-shv
24B-GAL4
nSynb-GAL4
24B-GAL4
HRP pFAK
Control shv
1
mys
ts1
mys
ts1
;
shv
1
shv
1
;
UAS-mys

mys
ts1
;
UAS-shv

shv
1
;
UAS-mys
UAS-
mys

UAS-mys

HRP pFAK
*
nSynb-GAL4
24B-GAL4
*
*
*
*
*
*
*
*
*
*
*
*
*
**
0.0
0.5
1.0
1.5
2.0
Normalized pFAK intensity
Total pFAK
Pre pFAK
*
*
***
97
Next, we investigated if Shv interacts with βPS integrin to modulate integrin
signaling and synaptic growth. We found that βPS hypomorphic mutant, mys
ts1
,
displayed a similar increase in bouton number, synaptic complexity, and ghost bouton
numbers as shv
1
(Figs 3.3.D and E; Fig. S3.1.B and C). Like shv
1
, we also saw a
corresponding decrease in the levels of presynaptic and total pFAK in the mys
ts1

mutant. This result confirms reduced integrin activation both pre- and postsynaptically
in mys
ts1
mutant, consistent with the known distribution of βPS integrin at the NMJ.
Note that because mys null allele is embryonic lethal, we used mys
ts1
, a strong
hypomorphic allele of mys that shows substantially reduced Mys protein level at 25°C
(Beumer et al., 1999). To further confirm genetic interaction between Shv and βPS
integrin, we examined double mutants of mys
ts1
and shv
1
. We found that mys
ts1
; shv
1

double mutants displayed the same phenotypes as either mutant alone in synaptic
morphology and pFAK levels (Figs. 3.3.D-G; Figs. S3.1.B, C, and Table S3.1),
confirming they act in the same genetic pathway. In addition, mys
ts1
hypomorphic allele
dampened the effect of shv overexpression on pFAK, suggesting that integrin acts
downstream of Shv. Upregulating Shv in mys
ts1
hypomorphic mutant also partially
alleviated the synaptic overgrowth phenotype of mys
ts1
(Figs. 3.3.D and E; Figs. S3.1.B
and C), consistent with restoration of pFAK levels compared to mys
ts1
(Figs. 3.3.F and
G). We also attempted to restore integrin signaling by overexpressing mys (UAS-mys)
in shv
1
neuron or muscle. However, both failed to restore the synaptic overgrowth
phenotype of shv
1
(Figs. 3.3.D and E), likely because the presence of Shv is required to
activate integrin signaling. Indeed, pFAK staining revealed that upregulation of the
integrin receptors alone failed to restore integrin signaling in the absence of an
98
activator, Shv. However, in the presence of Shv (wildtype background), overexpression
of mys in neurons and muscles increased the corresponding level of presynaptic pFAK
and total pFAK, respectively, confirming that the presence of Shv is necessary for
efficient integrin activation. Together, these results indicate that activation of integrin
receptors by extracellular Shv restricts synaptic growth.

3.4.3 Shv is released during intense activity to acutely activate integrin signaling.
In addition to modulating synaptic growth during development, integrin receptors
also contribute to activity-dependent synaptic plasticity (Rohrbough et al., 2000; Tsai et
al., 2012a; Dani et al., 2014). Having demonstrated that Shv is a secreted protein that
activates integrin signaling, we next asked if Shv release is regulated by activity. First,
we used an established spaced depolarization protocol with 3 pulses of high KCl that
has been shown to induce new bouton formation (Vasin et al, 2014). However, there
was no detectable change in Shv level at the NMJ, and pFAK level was significantly
reduced (Fig. S3.2.A-C). We also tested another spaced depolarization protocol that
can more robustly induce new bouton formation (5X high KCl; (Ataman et al., 2008)).
However, this 5X spaced stimulation protocol also failed to elevate Shv level and
resulted in reduced pFAK at the synapse (Fig. S3.2.D-F). These results are consistent
with previous findings that activity-induced synaptic growth and elongation down
regulates integrin signaling, presumably to minimize the opposing adhesive forces
generated by integrin activation at the pre- and postsynaptic terminals (Tsai et al.,
2012a; Dani et al., 2014). Interestingly, wildtype larval prep stimulated with a persistent
pulse of high KCl (for 10 minutes) led to extensive upregulation of Shv signal at the NMJ
99
detected using non-permeabilizing conditions that was not observed in shv
1
mutant (Fig.
3.4.A), suggesting that Shv is released extracellularly at the synapse. Electrical
stimulation at high frequency (10 Hz), but not at low frequency (1 Hz), also led to
substantial Shv signal at the NMJ (Fig. 3.4.B), revealing that Shv is selectively released
by intense synaptic activity. Enhanced Shv release by prolonged high K
+
is
accompanied by greater integrin activation as measured by increase in either
presynaptic or total pFAK levels, but not in shv
1
mutant or mys
ts1
mutants (Figs. 3.4.C
and D). High frequency stimulation, but not low frequency nerve stimulation, also
elevated pFAK levels in control larvae. Again, such change in pFAK was not detected
in shv
1
or mys
ts1
following high frequency nerve stimulation (Figs. 3.4.E and F), implying
that only intense neuronal activity triggers Shv release and activation of integrin
signaling.

100

*
*
*
*
*
N.S N.S
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Control shv
1
mys
ts1

13
26
8
11
6
9
Mock
10min Stimulation
A B
C
H
G
0.0
0.5
1.0
1.5
2.0
2.5
Normalized presynaptic
pFAK intensity
Mock
10min Stimulation
*
*
*
*
N.S
*
9
12
8
11
4
6
D
E
F
*
* *
0.0
0.5
1.0
1.5
2.0
2.5
Normalized pFAK intensity
Mock
1Hz 5min
10Hz 5min
Control shv
1
mys
ts1

N.S
*
*
* *
N.S
6
13
11
6
7
9
6
6
8
Mock 10min K
+
Stimulation
Control mys
ts1

UAS-βν-
RNAi/
nSynb-
GAL4
HRP pFAK
Mock 5min @ 10Hz
Shv
HRP
Control
5min @ 1Hz
Shv
Shv
HRP
Shv
shv
1

Mock 10min K
+
Stimulation
pFAK
HRP
pFAK
pFAK
HRP
pFAK
Control shv
1

pFAK
HRP
pFAK
mys
ts1

5min @ 1Hz
pFAK
HRP
pFAK
Control shv
1

5min @ 10Hz
pFAK
HRP
pFAK
pFAK
HRP
pFAK
mys
ts1

Mock
HRP Shv Shv
Mock
10min
K
+
Stimulation
HRP Shv Shv
Control shv
1

0.0
0.5
1.0
1.5
2.0
Mock
10min Stimulation
*
*
*
* *
N.S
N.S
Control shv
1
mys
ts1
Normalized presynaptic pFAK
Normalized total pFAK
101
Figure 3.4. Shv is secreted during intense synaptic stimulation to acutely activate
integrin signaling. (A) Representative images of NMJ stimulated with high K
+
for
10min followed by detergent-free Shv antibody labelling. Scale bar = 5 µm. (B) Images
representing Shv release after electrical stimulation at 10Hz for 5min but not by
stimulation at 1 Hz for 5 mins. Extracellular Shv was stained using detergent-free
condition. (C) Quantification of presynaptic and total pFAK level after 10 min high K
+

stimulation. (D) Levels of pFAK immunostaining within NMJs treated with persistent
pulse of high K
+
stimulation across genotypes. Scale bar = 10 µm. (E) Images
representing elevated pFAK level following high frequency electrical stimulation but not
in low frequency or shv
1
and mys
ts1
(F) Quantification of normalized total pFAK level
upon high frequency electrical stimulation. (G) Representative images of UAS-
β
ν
RNAi/nSynb-GAL4 showing elevated pFAK staining after high K
+
stimulation for 10
min. (H) Quantification of presynaptic pFAK levels upon high K
+
stimulation. Scale bar =
5 µm. * p < 0.05 compared to control. All values represent mean ± SEM.

102
We also tested the involvement of another integrin subunit, β
ν
integrin, which is
present presynaptically at the Drosophila NMJ and has been shown to affect pFAK
levels and synaptic growth via retrograde laminin signaling (Tsai et al., 2008; 2012a).
We found that consistent with a previous report, knockdown of β
ν
integrin in neurons
generated synaptic overgrowth (Fig. S3.2.G) and decreased presynaptic pFAK level
(Figs. 3.4.G and H), phenotypes similar to shv
1
and mys
ts1
. However, unlike shv
1
or
mys
ts1
mutants, intense activity still triggered presynaptic increase in pFAK levels in
NMJ with neuronal β
ν
integrin knockdown despite lower initial pFAK level, further
suggesting that strong activity mainly triggers βPS integrin activation independent of β
ν

integrin receptors and retrograde laminin signaling (Figs. 3.4.G and H). Together, these
results support the claim that activity-dependent Shv secretion acts to acutely activate
βPS integrin signaling.

3.4.4 Activation by Shv during intense activity triggers synapse maturation
What is the physiological consequence of activity-dependent Shv secretion and
activation of integrin receptors? We hypothesized that while mild or patterned synaptic
activity triggers synaptic growth, intense neuronal activity leads to Shv release to
acutely activate integrin and promote synaptic bouton maturation. Similar to spaced K
+

stimulation, 10 minutes stimulation paradigm triggered an increase in the number of
ghost boutons in control larvae (Figs. 3.5.A and B). Unstimulated shv
1
and mys
ts1
both
started with higher number of ghost boutons comparable to stimulated control, and the
number of ghost boutons increased slightly (albeit not significantly) upon stimulation,
perhaps because bouton budding reached a threshold. These results imply that Shv is
103
not required for new bouton formation, instead it may serve a modulatory function in
stabilizing the newly formed synapse. Interestingly, this stimulation paradigm also
acutely increased the size of synaptic boutons (Figs. 3.5.A, C, and D), implying that
prolonged stimulation triggered structural modification to achieve synaptic stabilization.
In contrast, prolonged stimulation of shv
1
did not lead to bouton enlargement, confirming
Shv plays a crucial role in activity-dependent synaptic remodeling. Similarly, mys
ts1

mutant did not expand its bouton size upon persistent stimulation (Figs. 3.5.A, C, and D)
whereas neuronal knockdown of β
ν
integrin still exhibited increase in synaptic bouton
size, consistent with Shv acting through a pathway independent of β
ν
integrin (Figs.
3.5.C and D). Furthermore, in accord with enhanced Shv release by high frequency
nerve stimulation, we found that intense neuronal activity selectively increased the size
of synaptic boutons (Fig. S3.4.A and B). Such increase in bouton size was not
observed in control larvae under mild stimulation (1 Hz), or in shv
1
and mys
ts1
mutants
under intense stimulation. Lastly, in agreement with hypotheses that different synaptic
demand activates distinct synaptic remodeling program and that Shv is a key player in
activity-dependent synaptic remodeling, we found that the pulsed potassium stimulation
paradigm, which did not trigger Shv release or elevate pFAK, was not sufficient to
increase synaptic bouton size (Fig. S3.3.C and D).

104

Figure 3.5. Shv release during intense activity induces synaptic maturation. (A)
Representative images of ghost bouton before and after intense stimulation with high K
+

application (highlighted with arrowhead). (B) Number of ghost bouton present upon
10min high K
+
depolarization. (C) Images representing increase in individual synaptic
bouton size after high K
+
stimulation. (D) Quantification of average size of type 1b
bouton following 10min high K
+
application. (E) Images representing staining of GluRIII
at the NMJ with or without 10min high K
+
stimulation. (F) Quantification of relative
GluRIII staining intensity at the NMJ treated with 10min high K
+
depolarization. Scale
bar = 5 µm. * p < 0.05 compared to control; ** p < 0.05 when comparing the indicated
conditions. All values represent mean ± SEM.
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
Normalized
ghost bouton #/NMJ
Mock
10min Stimulation
*
*
*
*
N.S
N.S
*
Control shv
1
mys
ts1

25
42
12
18
13
17
Figure 5
A B
C
10min K
+
Stimulation
HRP GluRIII
Mock
Control shv
1
mys
ts1

D
Control shv
1
mys
ts1

Mock 10min K
+
Stimulation
HRP
UAS-βν-
RNAi/
nSynb-
GAL4
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
Bouton size (µm
2
)
Mock
10min Stimulation
*
*
*
N.S
N.S
26
13
8
12
8
13
4
6
*
*
*
**
E
F
0.0
0.5
1.0
1.5
2.0
2.5
Normalized GluRIII intensity
Mock
10min Stimulation
*
*
*
*
N.S
N.S
Control shv
1
mys
ts1

13
13
7
12
6
10
*
Control shv
1
mys
ts1

Mock 10min Stimulation
HRP
GluRIII
GluRIII
HRP
GluRIII
GluRIII
HRP
GluRIII
GluRIII
105
Next, to further confirm that changes in the size of the synaptic boutons
represents activity-induced synapse maturation, we measured the levels of postsynaptic
glutamate receptors. The level of GluRIII receptor, which is an essential subunit of the
Drosophila glutamate receptors at the NMJ, was monitored before and after stimulation.
Following high KCl stimulation and high frequency nerve stimulation, we found a
significant increase in glutamate receptor abundance (GluRIII) in control NMJs (Figs.
3.5.E and F; Figs. S3.3.E and F). Interestingly, we noticed that shv
1
and mys
ts1
showed
lower levels of glutamate receptor staining intensity prior to stimulation as compared to
control, and glutamate receptor abundance was not enhanced by neuronal activity
(Figs. 3.5.E and F; Fig. S3.3.E and F). Altogether, our results demonstrate that Shv and
integrin activation is essential for synapse maturation during development and for
activity-induced synapse remodeling and maturation.
To further determine if transcription or synthesis of new proteins contribute to this
activity-dependent synaptic remodeling, we treated the NMJ with transcriptional inhibitor
actinomycin or translational inhibitor cycloheximide. While inhibition of transcription or
protein synthesis abolished ghost bouton formation following prolonged stimulation, it
did not block bouton enlargement or increase in glutamate receptor staining intensity
(Fig. 3.6.A-E). These results are consistent with previous findings that new bouton
formation requires transcription and new protein synthesis (Ataman et al., 2008), and
further demonstrate that synapse maturation acts through a pathway separate from one
required for new bouton formation, likely dependent on the local release of Shv as we
found that cycloheximide did not block Shv secretion following prolonged depolarization
(Fig. S3.4.A).
106

Figure 3.6. Extracellular incubation of Shv is sufficient to trigger synapse
maturation. (A) Representative images of HRP and GluRIII immunoreactivity at
synaptic boutons of control NMJ subjected to (A) 5X spaced depolarization, (B) 10 min
high K
+
depolarization in the presence of transcriptional inhibitor actinomycin or
translational inhibitor cycloheximde. Ghost bouton highlighted by white arrowhead.
Quantifications of (C) ghost bouton number per NMJ, (D) normalized GluRIII levels, (E)
Figure 6
A B
C
J
Shv treated Shv
LNV
treated
HRP
pFAK
Control shv
1

HRP
pFAK
pFAK pFAK
Mock
HRP
GluRIII
GluRIII
HRP
GluRIII
GluRIII
Shv
LNV
treated Shv treated
shv
1
Control
Mock
*
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Normalized pFAK intensity
Control shv
1

*
9
9
4
10
Shv
LNV
Shv

**
**
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
Bouton size (µm
2
)
*
Control shv
1

*
8
9
4
10
Shv
LNV
Shv

*
**
**
0.0
0.5
1.0
1.5
2.0
2.5
Normalized GluRIII intensity
Control shv
1

*
*
6
11
7
6
Shv
LNV
Shv

*
**
**
Mock 5X K
+
spaced
5X K
+
spaced
Actinomycin
5X K
+
spaced
Cycloheximide
HRP
GluRIII
GluRIII
F
D
G
H
I
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Normalized GluRIII intensity
*
High K
+
Cycloheximide

Mock

High K
+
High K
+
Actinomycin

*
*
*
*
9
12
7
6
10
11
5
6
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
Normalized
ghost bouton #/NMJ
10min
stimulation
5X spaced
stimulation
Mock

High K
+
High K
+
Actinomycin

High K
+
Cycloheximide

*
*
9
12
7
6
10
11
5
6
*
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
Bouton size (µm
2
)
Mock

High K
+
High K
+
Actinomycin

High K
+
Cycloheximide

*
*
*
9
12
7
6
10
11
5
6
Mock
10min
Stimulation
10min
Stimulation
Actinomycin
10min
Stimulation
Cycloheximide
HRP
GluRIII
GluRIII
E
10min
stimulation
5X spaced
stimulation
10min
stimulation
5X spaced
stimulation
107
individual bouton size upon 5X spaced or 10min high K
+
stimulation. * p < 0.05 when
compared to respective mock treated control. (F) Representative images of pFAK level
changes upon incubation of purified Shv or mutated Shv (Shv
LNV
) protein. (G)
Quantification of total pFAK levels at the NMJs after Shv or Shv
LNV
application
normalized to mock treated control. (H) Enlargement of individual bouton size upon
addition of purified Shv but not Shv
LNV
. (I) Quantification of GluRIII intensity at the NMJ
following Shv or Shv
LNV
protein incubation normalized to mock treated control. (J)
Images representing elevation of GluRIII level upon Shv treatment. Scale bar = 5 µm. *
p < 0.05 compared to mock treated control; ** p < 0.05 when comparing the indicated
conditions. All values represent mean ± SEM.

108
Thus, we next asked if the presence of Shv is sufficient to induce synapse
remodeling, or that other factors released during intense synaptic activity are also
required. To this end, we incubated dissected NMJ with purified His-tagged Shv protein
expressed in bacteria, and determined the effects of purified Shv on synaptic
morphology and maturation. In support of the hypothesis that Shv release is crucial for
synapse maturation, incubating the dissected NMJ with wildtype Shv in the absence of
synaptic stimulation was sufficient to elevate pFAK levels, augment the size of synaptic
boutons, and boost glutamate receptor intensity of both control and shv
1
mutant (Figs.
3.6.F-J; Figs. S3.4.B and C). The fact that we were able to observe these changes
even in shv
1
further imply that developmental problem was not the underlying cause of
lack of activity-dependent structural remodeling in shv
1
, but rather the presence of
extracellular Shv was necessary. In contrast, we found that incubating the NMJ with a
mutant Shv protein, Shv
LNV
, which does not activate integrin signaling due to mutation in
the putative integrin binding site (Lee et al., 2016), failed to trigger these changes (Fig.
3.6). Indeed, we found that Shv
LNV
did not interact with integrin in our pull down
experiment (Fig. S3.4.D). These results further support the claim that activation of
integrin signaling by Shv is sufficient to trigger synapse remodeling.

3.4.5 Shv required for functional plasticity
Having established that Shv modulates structural plasticity at the Drosophila
NMJ, we asked if Shv also alters functional plasticity. First, we measured the baseline
synaptic transmission in shv
1
mutant. Shv depletion reduced the amplitude of the
miniature excitatory postsynaptic potential (mEPSP), whereas neuronal expression of
109
Shv (but not muscle expression) rescued the phenotype of shv
1
mutant (Fig. 3.7.A).
This decrease in mEPSP amplitude is consistent with reduced postsynaptic glutamate
receptor abundance as observed by immunostaining (Figs. 3.5.E and F). Furthermore,
despite the decrease in mEPSP amplitude in shv
1
, the evoked EPSP amplitude
remained normal (Fig. 3.7.B), suggesting that homeostatic mechanism modulating
synaptic response is not affected. We next examined the effects of Shv depletion on
synaptic augmentation and post-tetanic potentiation (PTP), a form of synaptic plasticity.
In control larvae, high frequency stimulation at 10 Hz in low extracellular Ca
2+
concentration normally elicits synaptic augmentation that is followed by a potentiation of
the response that lasts for minutes. We found that shv
1
showed severely reduced
synaptic augmentation and impaired PTP that were rescued by neuronal expression of
Shv (Fig. 3.7.C), whereas neuronal expression of Shv in shv
1
mutant background
rescued synaptic plasticity. These results confirm that Shv is essential for functional
plasticity. Interestingly, overexpression of shv alone also abolished synaptic
augmentation and PTP, further implying that functional plasticity requires dynamic
change in integrin activation rather than persistent activation of integrin and downstream
signaling pathways.

110

Figure 3.7. Shv regulates functional plasticity. (A) Representative mEPSP and
average mEPSP amplitude recorded using HL-3 containing 0.5mM Ca
2+
. (B)
Representative evoked EPSP and average evoked EPSP amplitude recording in HL3
containing 0.5mM Ca
2+
. (C) Normalized EPSP amplitude upon 10min tetanus
stimulation shows synaptic augmentation and post-tetanic potentiation in control NMJ
whereas shv
1
failed to response to the activity. Upregulation of Shv in shv
1
mutant
restored synaptic augmentation and PTP but overexpression of shv alone inhibited
augmentation and PTP. Control n = 8, shv
1
n = 11, shv
1
;UAS-shv/nSynb-GAL4 n = 10,
UAS-shv/nSynb-GAL4 n = 4. * p < 0.05 compared to control. All values represent mean
± SEM.

Figure 7
B
C
A
*
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
mEPSP Amplitude (mV)
18
12
9
11
6
0
10
20
30
40
50
60
EPSP Amplitude (mV)
8
9
6
10
6
2 min @ 10 Hz
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Normalized EPSP amplitdue (mV)

I I I I
0

2

4

6

shv
1
Control

shv
1
;UAS-shv/nSynb-GAL4

UAS-shv/nSynb-GAL4

Time (min)
0.2mM Ca
2+
HL-3
*
*
*
*
* *
* * * * * * * * *
Control

shv
1
Control

shv
1
111
3.5 Discussion
3.5.1 Summary of Results
In the present study, we show that Shv is a novel protein secreted at the
Drosophila NMJ. We report that Shv is required during synaptic development to
maintain normal synaptic growth and is released upon intense neuronal stimulation to
acutely promote synapse maturation. We further demonstrate that activity-induced
release of Shv by neurons activates integrin signaling, induces bouton enlargement and
increases postsynaptic glutamate receptor abundance. Moreover, Shv is required to
achieve synaptic augmentation and maintain potentiation following tetanic stimulation.
These data thus suggest that trans-synaptic Shv released in response to strong
neuronal activity acts as a molecular cue that triggers activity-induced synaptic
structural modifications and synapse maturation through integrin activation.
Our data reveals that presynaptically secreted Shv plays an important
modulatory role in maintaining normal synaptic growth during development. We found
that Shv is specifically targeted to the synapse by neurons and released at low levels in
the vicinity of the synaptic terminal as shown by both immunostaining with Shv antibody
and Shv-GFP fusion protein puncta (Figure 1). Furthermore, secretion of Shv from the
synapse is essential in restraining synaptic growth and promoting synapse maturation,
since only the full length Shv rescued the synaptic overgrowth and ghost bouton
phenotypes of shv
1
, whereas deleting the signal peptide of Shv did not (Figure 2).
Previous studies have suggested that ghost boutons are newly formed, undifferentiated
boutons in the process of maturing into functional boutons with postsynaptic glutamate
receptors (Ataman et al., 2008; Menon et al., 2013). The observation that shv
1
mutants
112
have more ghost boutons suggest that presynaptically released Shv is not required for
bouton formation, but rather is important for bouton maturation. Consistent with this,
shv
1
mutant also showed reduced glutamate receptor abundance and a decrease in
mEPSP amplitude.
The low amounts of Shv present extracellularly likely activate βPS integrin
signaling both pre- and postsynpatically to modulate synaptic growth. This is supported
by our previous work showing that activation of βPS integrin receptors by Shv in S2
cells (Lee et al., 2016), data that Shv can biochemically and genetically interact with
βPS integrin to regulate synaptic growth, and that presynaptic knockdown of Shv
significantly reduced pFAK levels in both presynaptic terminals and postsynaptic muscle
(Fig. 3.3). We thus envision that Shv, once released at presynaptic terminals, is a
trans-synaptic molecule that acts on βPS integrin receptors bidirectionally to affect
synaptic growth through signaling pathways downstream of βPS integrin receptors. As
there are multiple integrin ligands shown to affect synaptic growth and different integrin
receptors at the NMJ (Broadie et al., 2011), it is likely that Shv acts in concert with other
integrin ligands to coordinate and optimize integrin activation and synaptic growth
during development.

3.5.2 Secretion of Shv Acutely Activates Integrin and Induces Synaptic Structural
Modification
Although the involvement of integrin in synaptic growth and plasticity has been
studied extensively (Beumer et al., 1999; Rohrbough et al., 2000; Chavis and
Westbrook, 2001; Beumer et al., 2002; Kramár et al., 2006; Tsai et al., 2008; 2012a;
113
2012b), the identity of an extracellular ligand that modulate integrin signaling in
response to synaptic activity remains to be elucidated. Excitingly, we found that
persistent synaptic activity or high frequency stimulation induced release of Shv, which
led to a robust increase in pFAK level and synaptic bouton enlargement, a process
normally associated with synaptic maturation. Furthermore, incubation of purified Shv
protein in dissected larval NMJ elevated the levels of pFAK and enlarged the size of
synaptic boutons, while the mutant Shv
LNV
that cannot interact with βPS failed to elicit
such changes (Fig. 3.6.F-J). These results suggest that the extracellular presence of
Shv is an instructive cue that is sufficient to acutely activate integrin signaling and
induce synaptic structural modification. Moreover, our finding that the absence of Shv
failed to elicit multiple phases of activity-dependent synaptic plasticity including initial
augmentation during tetanus and PTP support the claim that Shv required for synaptic
plasticity.

3.5.3 Release of Shv is Selectively Regulated by Synaptic Activity to Modulate
Synaptic Growth and Maturation
To date, aside from Shv, laminin A is the only known integrin ligand with its
release regulated in an activity-dependent manner at the Drosophila NMJ. However,
laminin release by postsynaptic muscles is instead down-regulated by synaptic activity
and crawling, resulting in reduced pFAK through its retrograde action on presynaptically
expressed β
ν
integrin receptors (Tsai et al., 2012a). Here we find that Shv’s release is
stimulated by strong synaptic activity and acts independently of β
ν
integrin receptors
(Figs. 3.4.G and H; Figs. 3.5.C and D). Interestingly, we also found that spaced
114
potassium depolarization paradigm, which has been shown to induce new bouton
formation and synaptic growth through release of wingless (Wg) (Ataman et al., 2008),
did not trigger Shv release or elevate pFAK at the NMJ. Furthermore, we found that
while new bouton formation were blocked by transcription or translational inhibitors
similar to previous reports (Fig. 3.6.A-C) (Ataman et al., 2008), bouton maturation in the
form of bouton enlargement and increase in glutamate receptor abundance were not
affected (Figs. 3.6.B, D, and E). Together, these data support a model in which
neurons activate different programs to differentially modulate synaptic growth and
maturation in response to different synaptic demand.
We propose that mild or patterned neuronal activity triggers release of factors
such as Wingless (Wg) and down regulates laminin release from muscles to initially
promote synaptic growth and allow synaptic expansion (increase the number of
boutons); persistent or intense synaptic activity then leads to release of Shv from
neurons to activate βPS integrin signaling bi-directionally to promote local synapse
maturation. Activation of βPS integrin signaling may lead to local synaptic enlargement
in bouton size by promoting actin branching (Levi et al., 2006; Legate et al., 2009),
which has been associated with growth in the size of boutons (Collins and DiAntonio,
2004). The exact mechanism for how activation of βPS integrin leads to increase in
glutamate receptor abundance at the Drosophila NMJ is not clear, but mutations that
reduce the levels of βPS integrin has also been shown to diminish glutamate receptor
abundance (Liebl and Featherstone, 2005). Secreted extracellular protein such as
mind-the-gap, which has been suggested to recruit integrin receptor to the synaptic
cleft, also affects synaptic localization of glutamate receptor clusters (Rohrbough et al.,
115
2007; Rushton et al., 2009). Furthermore, PS integrin receptors have been reported to
associate with disc large, Fasciclin II cell adhesion molecule, and CaMKII known to
affect synaptic glutamate receptor assembly and anchoring (Beumer et al., 1999; Albin
and Davis, 2004; Chen and Featherstone, 2005; Chen et al., 2005). Understanding the
downstream mechanism by which Shv-dependent integrin activation modulates
localization of glutamate receptor to promote stability of synaptic structure during
synaptic plasticity remains an interesting area to study in the future. It will also be
particularly interesting to elucidate how neuronal activity triggers Shv release in the
future. As a robust neuronal circuitry depends on the ability of neurons to dynamically
adjust synaptic strength and modify synaptic structure in response to synaptic demand,
elucidating how Shv functions in activity-induced synaptic plasticity has implications for
understanding mechanisms underlying cognition and psychiatric disorders.

3.6 Acknowledgement
We thank Drs. R. Xi (National Institute of Biological Science, Beijing), A. DiAntonio
(Washington University, St Louis, MO) for prompt and generous sharing of fly stocks
and antibodies. We are grateful to The Developmental Hybridoma Bank (Iowa, USA) for
multiple antibodies. We also thank members of the Chang laboratory for critical reading
of the manuscript and discussions.

116
3.7 Supplementary Figures

Figure S3.1. Quantification of pFAK level and number of ghost boutons. (A)
Presynaptic pFAK signal was calculated by outlining the presynaptic bouton using HRP
Figure 3 – figure supplement 1
A
C
Control shv
1

HRP
pFAK
Dlg
HRP pFAK
pFAK
Dlg
Dlg
Control shv
1
mys
ts1
mys
ts1
;shv
1
mys
ts1
;UAS-shv

shv
1
;UAS-mys

nSynb-GAL4
UAS-mys

shv
1
;UAS-mys

UAS-mys

24B-GAL4
C
B
DLG HRP
DLG HRP
0
1
2
3
4
5
Normalized
Ghost bouton#/NMJ
*
*
*
*
*
**
nSynb-GAL4
24B-GAL4
30
29
15
5
6
7
13
14
13
*
117
channel, whereas total pFAK signal was determined by outlining the postsynaptic area
using Dlg staining. (B) Images of NMJs labelled with HRP and Dlg. Arrows points to
ghost boutons that are HRP positive but Dlg negative. Scale bar = 5 µm. (C)
Quantification of normalized ghost bouton number. * p < 0.05 compared to control. ** p
< 0.05 compared to indicated genotypes. All values represent mean ± SEM.

118

Table S3.1. Table indicating the number of samples used to determine relative pFAK
levels in Figure 3.3.C and 3.3.F.

Quantification of sample number used in Figure 3C and 3F
Genotype Pre pFAK Total pFAK
Control 66 50
shv
1
23 9
UAS-shv-RNAi/Synb-GAL4 7 6
UAS-shv//Synb-GAL4 8 7
UAS-shv-RNAi/24B-GAL4 8 8
shv
1
;UAS-shv/Synb-GAL4 10 9
mys
ts1
7 6
shv
1
;UAS-shv/24B-GAL4 11 12
shv
1
;UAS-NoSP-shv/Synb-GAL4 11 12
mys
ts1
;shv
1
7 7
mys
ts1
;UAS-shv/Synb-GAL4 8 7
shv
1
;UAS-mys/Synb-GAL4 10 10
shv
1
;UAS-mys/24B-GAL4 17 13
UAS-mys/24B-GAL4 10 10
UAS-mys/Synb-GAL4 6 6
Figure 3 – figure supplement 2
119

Figure S3.2. Pulsed high K
+
stimulation. (A) A schematic diagram of 3X spaced
stimulation protocol. Representative images of levels of Shv at the NMJs with or without
3X spaced high K
+
stimulation stained using detergent-free condition. (B) Image of
NMJs stained with pFAK antibodies with and without 3 pulses high K
+
stimulation. Scale
bar = 5 µm. (C) Quantification of pFAK levels upon 3X spaced K
+
stimulation compared
to mock treatment (without high K
+
). * p < 0.05 when compared to mock treated control.
Figure 4 – figure supplement 1
A B
C
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Normalized pFAK intensity
Mock
3X Stimulation
**
**
*
Control shv
1

8
5
4
4
D
UAS-βνRNAi/nSynb-GAL4
Control
E F
HRP Shv
Control shv
1

Mock
3X spaced
K
+
Stimulation
HRP Shv Shv Shv
pFAK
HRP
Control
pFAK
Mock
3X spaced
K
+
Stimulation
shv
1

pFAK
HRP
pFAK
HRP
pFAK
pFAK
0X K
+

5X spaced
K
+
stimulation
Control
G
HRP Shv Shv
0X K
+

5X spaced
K
+
stimulation
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Normalized pFAK intensity
*
0X K
+

5X K
+
spaced
stimulation

6
6
120
** p < 0.05 when comparing mock treated or 3X stimulated. (D) A schematic drawing of
5 pulses of high K
+
stimulation. Representative images of Shv staining with and without
5X spaced high KCl depolarization performed using detergent-free condition. (E) Levels
of pFAK staining within NMJ treated with and without 5X spaced high K
+
stimulation. (F)
Quantification of pFAK levels upon 5X spaced K
+
depolarization. * p < 0.05 when
compared to mock treated control. All values represent mean ± SEM. (G)
Representative images showing that presynaptic knockdown of β
ν
integrin results in a
synaptic overgrowth phenotype. Scale bar = 10 µm.

121

Figure S3.3. Synaptic stimulation induces synapse maturation. Representative
images of NMJ stained with HRP staining showing size of synaptic boutons (A)
following nerve-evoked stimulation using the indicated conditions (C) with and without
pulses of high K
+
incubation as marked. (B) and (D) Quantification of bouton size. (E)
Representative images of GluRIII staining upon low (1Hz) or high (10Hz) frequency
electrical stimulation. (F) Quantification of relative GluRIII intensity at the synaptic
terminal after the stimulation. Scale bar = 5 µm. * p < 0.05 compared to control. All
values represent mean ± SEM.

Figure 5 – figure supplement 1
A
C
E
0.0
0.5
1.0
1.5
2.0
2.5
Normalized GluRIII intensity
Mock
1Hz 5min
10Hz 5min
Control shv
1
mys
ts1

*
N.S
* * *
N.S
* * *
9
10
9
7
7
7
7
7
8
Control shv
1
mys
ts1

Mock 5min @ 1Hz 5min @ 10Hz
0.0
1.0
2.0
3.0
4.0
5.0
6.0
Bouton Size (µm
2
)
Mock
1Hz 5min
10Hz 5min
Control shv
1
mys
ts1

*
N.S
*
*
*
N.S
6
8
8
7
10
9
6
7
8
B
F
D
Mock 5min @1Hz 5min @ 10Hz
Control shv
1
mys
ts1

HRP GluRIII
Control shv
1

*
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Bouton size (µm
2
)
Mock

3X K
+
stimulation
5X K
+
stimulation
8
5
4
4
N.S
N.S
*
12
4
Control shv
1

Mock 3X K
+
Stimulation 5X K
+
Stimulation
122

Figure S3.4. Shv and Shv
LNV
isolation. (A) Representative images of Shv release at
the synapse upon prolonged high K
+
stimulation with and without translational inhibitor
cycloheximide. Detergent-free staining condition was used. (B) Coomassie gel showing
relative amounts of Shv or Shv
LNV
following purification. Arrowhead points to the
position of the expected protein size. (C) Western blot confirming expression and
isolation of Shv and Shv
LNV
proteins. Western blot detected with HA antibody. (D) Pull-
down experiment using HA fusion proteins and fly protein lysates reveals that Shv
LNV

does not interaction with integrin. Representative blots are shown, and results were
confirmed in 3 independent experiments.

Figure 6 – figure supplement 1
B C
40
35
Shv
Shv
LNV
130 100 55 40 Input
Beads
Shv
Shv
LNV

IP:HA HA
βPS Integrin D
A
Mock 10min Stimulation
10min Stimulation
Cycloheximide
HRP
Shv
Shv
Shv
Shv
LNV
- Control
40
35
HA
123
Chapter 4.
Summary and Future Directions

4.1 Summary
Utilizing the model organism Drosophila melanogaster, I have proposed a novel
role of DNAJB11/Shv as an activator of integrin signaling pathway in maintaining stem
cell niche structure and synaptic remodeling in response to neural activity. In Drosophila
male stem cell niche, deletion of shv results in age-dependent deterioration of hub
architecture and loss of stem cells, whereas upregulation of Shv preserve the niche
during aging. Shv activate integrin signaling from extracellular (outside-in) in vivo and in
vitro as putative integrin binding motif is found in Shv. Surprisingly, Shv-dependent
integrin activation further modulates E-cadherin level in hub cell revealing cooperation
between the two adhesion molecules in maintaining niche integrity.
Integrin is an important cell adhesion molecule that functions in synaptic growth,
transmission and plasticity; therefore, I explored whether Shv also acts as a positive
regulator of integrin in stabilizing synaptic structure during activity. I find that while
intense neuronal stimulation triggers Shv secretion that acutely activate integrin
signaling, induce local bouton enlargement, and increase in postsynaptic glutamate
receptor abundance, mild patterned stimulation fail to elicit such changes implying that
synapse triggers distinct synaptic remodeling program in response to different synaptic
stimuli. The finding that extracellular presence of Shv in the absence of neuronal
stimulation is sufficient to trigger synapse maturation claims Shv as a key player in
inducing activity-dependent synaptic remodeling. In addition, post-tetanic potentiation is
124
impaired in shv mutant elucidating a role of Shv in modulating functional plasticity. I
propose Shv as a trans-synaptic molecule with its release regulated by neuronal activity
to elicit activity-dependent structural modification through integrin signaling.

4.2 Shv-dependent Integrin Activation Regulates E-cadherin Levels to Preserve
Niche Integrity
Studies have established that cell adhesion molecules are fundamental for
maintaining a healthy niche structure by enhancing cell-ECM or cell-cell adhesion. In
Drosophila germline stem cell niche, integrin and E-cadherin are major adhesion
mediators governing proper hub anchoring and GSC adhesion to the hub, respectively
(Song and Xie, 2002; Tanentzapf et al., 2007; Voog et al., 2008; Lee et al., 2016). In
this dissertation, I illustrate a novel secreted protein, Shv, as a positive regulator of
integrin receptor, which in turn modulates E-cadherin expression in hub to preserve
niche integrity. Although in the context of Drosophila stem cell niche, integrin and E-
cadherin are thought to regulate hub anchoring and GSC attachment through
independent pathway, in Chapter 2 I demonstrate the interesting findings that Shv-
dependent integrin activation modulates E-cadherin level in the hub cells. Extracellular
application of Shv-conditioned media in Drosophila Schneider’s (S2) cells elevates E-
cadherin expression, but not if integrin function is compromised revealing the crosstalk
between E-cadherin and integrin signaling pathway. While studies found that E-cadherin
is dispensable for proper hub localization at the apical tip of the testes, here, I find that
E-cadherin contributes to the hub anchoring when integrin expression is impaired.
Upregulation of E-cadherin rescues hub mislocalization and deterioration of hub
125
architecture seen in shv
1
mutant testes indicating that E-cadherin acts downstream of
Shv to preserve stem cell niche architecture. Moreover, upregulation of Shv delays age-
dependent loss of hub cell and GSC, probably via enhancing E-cadherin level mediated
cell-cell adhesion. Taken together, Shv maintains ideal stem cell niche environment
during aging through integrin-dependent regulation of E-cadherin.

4.3 Shv is a Novel Activator of Integrin in vivo and in vitro
Integrins transmit bidirectional signaling across the plasma membrane to
modulate actin dynamics to achieve cell migration, branching, and protrusion. They
regulate their ligand-binding affinity through binding of cytoplasmic β-subunit tail to
intracellular adapter protein, such as talin and kindlin to induce conformational change
into the active state (referred as inside-out signaling) (Calderwood, 1999; Calderwood
and Ginsberg, 2003; Montanez et al., 2008). Outside-in signaling is mediated by binding
of ligand in the ECM that leads to clustering of integrin, which creates focal adhesion, a
complex of several adaptor proteins such as paxillin, Src and FAK, resulting in
coordination of downstream signaling pathway and actin reorganization (Hynes, 2002;
Harburger and Calderwood, 2009). FAK is the first tyrosine kinase to be recruited to
focal adhesion and autophosphorylated at the Tyr397 site upon integrin conformational
change, therefore serving as an reliable readout for integrin activation (Mitra et al.,
2005; Harburger and Calderwood, 2009; Campbell and Humphries, 2011). In this
dissertation, I highlight that secreted Shv directly interacts and binds to integrin receptor
in vivo and in vitro as an extracellular signaling molecule. Chapter 2 illustrates Shv as a
secreted protein confirmed by the presence of Shv in Drosophila Schneider’s (S2) cell
126
media and in vivo extracellular labeling (Fig. 2.4.A and 2.5.B). Exogneous application of
Shv enhances FAK phosphorylation and cell spreading of S2 cells, while such changes
are absent in integrin knockdown cells indicating that extracellular Shv is both
necessary and sufficient to activate integrin signaling (Fig. 2.4). As RGD motifs are
reported as a binding site for integrin ligand, mutation of KND sequence found in Shv,
which shares similarity with human RGD peptide results in abolishing the effect of Shv
secretion on integrin receptor suggesting that Shv can directly bind to and interact with
integrin to modulate integrin activation (Fig. 2.4.I and J). Similarly, extracellularly
present Shv is required for proper integrin clustering and activation in Drosophila male
testes. shv
1
mutant shows severely impaired integrin expression and FAK
phosphorylation while upregulation of Shv in mutant background rescues the phenotype
suggesting that Shv is responsible for integrin activation in germline stem cell niche to
maintain intimate contact with the niche (Fig. 2.5). Moreover, truncation of signal
peptide (NoSP-Shv) fails to rescue the phenotypes supporting the claim that integrin
activation is achieved by extracellular presence of Shv. In agreement with these
findings, Chapter 3 proposes that Shv also acts as an activator of integrin signaling at
the NMJ to control normal synaptic growth and that secretion of Shv is regulated by
synaptic activity.

4.4 Activity-dependent Release of Shv Induces Rapid Synaptic Remodeling
Diverse synaptic proteins are recruited to govern proper synaptic structure and
functions during development. Integrin is an essential synaptic adhesion molecule and
its role has previously been examined in the context of regulating synaptic structures
127
and functions (Beumer et al., 1999; Rohrbough et al., 2000; Beumer et al., 2002; Tsai et
al., 2008; 2012a; 2012b). However, there is a gap remained in understanding of the
molecular cues that regulate integrin activation during synaptic activity. In this
dissertation, I demonstrate in Chapter 3 that Shv is released from synaptic bouton in an
activity-dependent manner and that Shv functions as a novel ligand of integrin receptor
to induce structural remodeling. Shv is required to maintain normal synaptic growth
during development by acting as a trans-synaptic secreted molecule to activate integrin
receptor in an autocrine and paracrine fashion (Fig. 3.2). Surprisingly, Shv secretion is
enhanced upon intense neuronal activity induced by persistent pulse of K
+
solution and
high frequency electrical stimulation, which led to greater integrin activation and local
bouton enlargement, a process normally associated with synaptic maturation (Fig. 3.4
and 3.5). Prolonged stimulation also triggers robust expression of glutamate receptor
supporting the claim that activity-dependent secretion of Shv regulates activity-
dependent synaptic remodeling and maturation. In addition, extracellular presence of
purified Shv protein without neuronal stimulation elicits elevated integrin activation and
synaptic maturation suggesting that extracellular Shv is sufficient and necessary to
trigger synaptic remodeling (Fig. 3.6.F-J). Together, this research presents the first in
vivo regulator of βPS integrin activator that modulates synaptic remodeling in response
to activity.
Another research team proposed laminin A as an integrin ligand that its released
is regulated by activity. Crawling activity induced by high temperature and food
starvation inhibits laminin A secretion from postsynaptic muscle resulting in reduced
activation of presynaptically expressed βν integrin receptor (Tsai et al., 2012a). I
128
demonstrate that Shv release upon intense neuronal stimulation acts independently of
βν integrin in promoting synaptic maturation (Fig. 3.4.G-H and 3.5.C-D). Wingless (wg)
signal is thought to be secreted during mild patterned synaptic activity to triggers new
synaptic bouton formation (Ataman et al., 2008); however, this mild activity had no
effect in Shv-dependent integrin activation and subsequent synaptic maturation (Fig.
S3.2.A-E). The observations that synaptic maturation still occurred in the presence of
transcription and translation inhibitors, the process responsible for new synapse
budding (Ataman et al., 2008), and occurrence of ghost bouton in shv depleted mutant
upon strong neuronal stimulation indicate that Shv is not required for a new synapse
formation rather it may serve a modulatory function in stabilizing the synapse (Fig.
3.6.A-E and 3.5.A-B). Therefore, it implies that synapse has ability to selectively activate
distinct molecular signaling pathways to the different synaptic demands to promote
synaptic growth and maturation.
The coupling of integrin receptors to actin cytoskeleton regulates local actin
polymerization and broad dynamic rearrangement of actin filaments. FAK has been
shown to directly interact and recruit the Arp2/3 complex, which organizes branched
actin network and its actin nucleation function is activated by Wiskott-Aldrich Syndrome
(WASP) protein (Serrels et al., 2007; Postel et al., 2008; Legate et al., 2009; Padrick et
al., 2011; Swaminathan et al., 2016). Thus, local bouton enlargement observed in
stimulated synapse may be resulted from integrin-induced actin branching instead of
protrusion (Fig. 3.5.C-D). Mutation in βPS integrin has been shown to cause impairment
in expression/localization of glutamate receptor (Liebl and Featherstone, 2005);
moreover, integrin is thought to interact with disc large and CaMKII that are known to
129
regulate localization of postsynaptic glutamate receptors (Beumer et al., 2002; Kazama
et al., 2003; Albin and Davis, 2004; Chen and Featherstone, 2005). In future work, it
would be interesting to investigate the downstream mechanism how Shv-dependent
integrin activation regulate glutamate receptor localization at the synapse to stabilize the
synapse following structural modification.

4.5 Future Directions
While numerous groups have provided insights into the role of integrin in
structural plasticity, identification of molecular cues that activates integrin in an activity-
dependent manner remains a mystery. During neuronal activity, various molecules are
recruited or disengaged to meet synaptic demand by enhancing release of
neurotransmitter and modifying synaptic structures. My data suggest that during period
of high activity, Shv is released to activate integrin signaling thereby promoting synaptic
maturation shown by increased surface expression of postsynaptic glutamate receptors.
Elucidating mechanism how Shv is released during synaptic activity is intriguing area to
explore in future. It would be interesting to determine whether Shv release is dependent
on Ca
2+
, packaged into synaptic vesicle or exosomal release. Moreover, as we
observed synaptic maturation in the form of increase in glutamate receptor abundance
and local bouton enlargement, it will be particularly interesting to investigate underlying
mechanism by which integrin activation enhances glutamate receptor anchoring.
Integrin is known to associate with disc large and fascilin II-adhesion molecule via
activation of CaMKII pathway, which has been shown to affect localization of glutamate
receptors (Koh et al., 1999; Lisman et al., 2002; Morimoto et al., 2010). Therefore, it
130
would be interesting to test if CaMKII acts as a downstream of Shv-dependent integrin
activation thereby enhancing glutamate receptor accumulation. In addition, as we ruled
out the possibility of changes in gene transcription/translation of glutamate receptor
during stimulation (Fig. 3.6.A), investigating how Shv secretion facilitates glutamate
receptor trafficking to the cell surface is an interesting future direction. Furthermore, the
observation of local bouton enlargement in response to stimulation may represent
involvement of PI(4,5)P
2
/WASP pathway, which has been shown to promote synaptic
bouton growth by promoting actin branching (Collins and DiAntonio, 2004; Khuong et
al., 2010). Precisely regulated activity-induced changes in synaptic structure and
function are crucial for the foundation of complex brain functions such as learning and
memory. As synapse has abilities to make precise adjustment to the dynamic changes
in synaptic efficacy, understanding how Shv functions in regulating activity-dependent
structural modification may shed a light on cellular mechanism implicated in psychiatric
disorders.

131
References
Albin SD, Davis GW (2004) Coordinating Structural and Functional Synapse
Development: Postsynaptic p21-Activated Kinase Independently Specifies
Glutamate Receptor Abundance and Postsynaptic Morphology. J Neurosci
24:6871–6879.
Allison DW, Gelfand VI, Spector I, Craig AM (1998) Role of actin in anchoring
postsynaptic receptors in cultured hippocampal neurons: differential attachment of
NMDA versus AMPA receptors. J Neurosci 18:2423–2436.
Amoyel M, Anderson J, Suisse A, Glasner J, Bach EA (2016) Socs36E Controls Niche
Competition by Repressing MAPK Signaling in the Drosophila Testis. PLoS Genet
12:e1005815.
Amoyel M, Bach EA (2012) Functions of the Drosophila JAK-STAT pathway. JAK-STAT
1:176–183.
Amoyel M, Sanny J, Burel M, Bach EA (2012) Hedgehog is required for CySC self-
renewal but does not contribute to the GSC niche in the Drosophila testis.
Development 140:56–65.
Amoyel M, Simons BD, Bach EA (2014) Neutral competition of stem cells is skewed by
proliferative changes downstream of Hh and Hpo. The EMBO Journal 33:2295–
2313.
Arai F, Hirao A, Ohmura M, Sato H, Matsuoka S, Takubo K, Ito K, Koh GY, Suda T
(2004) Tie2/Angiopoietin-1 Signaling Regulates Hematopoietic Stem Cell
Quiescence in the Bone Marrow Niche. Cell 118:149–161.
Arikkath J (2010) N-cadherin: stabilizing synapses. J Cell Biol 189:397–398.
Ataman B, Ashley J, Gorczyca D, Gorczyca D, Gorczyca M, Mathew D, Mathew D,
Wichmann C, Sigrist SJ, Budnik V (2006) Nuclear trafficking of Drosophila Frizzled-
2 during synapse development requires the PDZ protein dGRIP. Proc Natl Acad Sci
USA 103:7841–7846.
Ataman B, Ashley J, Gorczyca M, Ramachandran P, Fouquet W, Sigrist SJ, Budnik V
(2008) Rapid activity-dependent modifications in synaptic structure and function
require bidirectional Wnt signaling. Neuron 57:705–718.
Bahr BA, Stäubli U, Xiao P, Chun D, Ji ZX, Esteban ET, Lynch G (1997) Arg-Gly-Asp-
Ser-selective adhesion and the stabilization of long-term potentiation:
pharmacological studies and the characterization of a candidate matrix receptor. J
Neurosci 17:1320–1329.
Barja-Fidalgo C, Coelho ALJ, Saldanha-Gama R, Helal-Neto E, Mariano-Oliveira A,
Freitas MS de (2005) Disintegrins: integrin selective ligands which activate integrin-
132
coupled signaling and modulate leukocyte functions. Brazilian Journal of Medical
and Biological Research 38:1513–1520.
Benson DL, Schnapp LM, Shapiro L, Huntley GW (2000) Making memories stick: cell-
adhesion molecules in synaptic plasticity. Trends in Cell Biology 10:473–482.
Berninger B, Bi GQ (2002) Synaptic modification in neural circuits: a timely action.
Bioessays 24:212–222.
Beumer KJ, Rohrbough J, Prokop A, Broadie K (1999) A role for PS integrins in
morphological growth and synaptic function at the postembryonic neuromuscular
junction of Drosophila. Development 126:5833–5846.
Beumer KK, Matthies HJGH, Bradshaw AA, Broadie KK (2002) Integrins regulate
DLG/FAS2 via a CaM kinase II-dependent pathway to mediate synapse elaboration
and stabilization during postembryonic development. Development 129:3381–3391.
Bourne JN, Harris KM (2008) Balancing structure and function at hippocampal dendritic
spines. Annu Rev Neurosci 31:47–67.
Boyle M, Wong C, Rocha M, Jones DL (2007) Decline in Self-Renewal Factors
Contributes to Aging of the Stem Cell Niche in the Drosophila Testis. Cell Stem Cell
1:470–478.
Brakebusch C, Fässler R (2003) The integrin-actin connection, an eternal love affair.
The EMBO Journal 22:2324–2333.
Bramham CR (2008) Local protein synthesis, actin dynamics, and LTP consolidation.
Current Opinion in Neurobiology 18:524–531.
Brawley C (2004) Regeneration of Male Germline Stem Cells by Spermatogonial
Dedifferentiation in Vivo. Science 304:1331–1334.
Broadie K, Baumgartner S, Prokop A (2011) Extracellular matrix and its receptors in
Drosophila neural development. Prokop A, Reichardt LF, eds. Devel Neurobio
71:1102–1130.
Brown NH (1993) Integrins hold Drosophila together. Bioessays 15:383–390.
Brown NH, Gregory SL, Martin-Bermudo MD (2000) Integrins as mediators of
morphogenesis in Drosophila. Developmental Biology 223:1–16.
Bulgakova NA, Klapholz B, Brown NH (2012) Cell adhesion in Drosophila: versatility of
cadherin and integrin complexes during development. Current Opinion in Cell
Biology 24:702–712.
Bunch TA, Brower DL (1992) Drosophila PS2 integrin mediates RGD-dependent cell-
matrix interactions. Development 116:239–247.
133
Bussolati B (2014) Stem cells for organ repair. Organogenesis 7:95–95.
Calderwood DA (1999) The Talin Head Domain Binds to Integrin beta Subunit
Cytoplasmic Tails and Regulates Integrin Activation. J Biol Chem 274:28071–
28074.
Calderwood DAD, Ginsberg MHM (2003) Talin forges the links between integrins and
actin. Nat Cell Biol 5:694–697.
Campbell ID, Humphries MJ (2011) Integrin structure, activation, and interactions. Cold
Spring Harbor Perspectives in Biology 3:a004994.
Caroni P, Donato F, Muller D (2012) Structural plasticity upon learning: regulation and
functions. Nat Rev Neurosci 13:478–490.
Chan C-S, Weeber EJ, Zong L, Fuchs E, Sweatt JD, Davis RL (2006) Beta 1-integrins
are required for hippocampal AMPA receptor-dependent synaptic transmission,
synaptic plasticity, and working memory. Journal of Neuroscience 26:223–232.
Chavis P, Westbrook G (2001) Integrins mediate functional pre- and postsynaptic
maturation at a hippocampal synapse. Nature 411:317–321.
Chen D (2003) A discrete transcriptional silencer in the bam gene determines
asymmetric division of the Drosophila germline stem cell. Development 130:1159–
1170.
Chen J-H, Kellner Y, Zagrebelsky M, Grunwald M, Korte M, Walla PJ (2015) Two-
Photon Correlation Spectroscopy in Single Dendritic Spines Reveals Fast Actin
Filament Reorganization during Activity-Dependent Growth. PLoS ONE
10:e0128241.
Chen K, Featherstone DE (2005) Discs-large (DLG) is clustered by presynaptic
innervation and regulates postsynaptic glutamate receptor subunit composition in
Drosophila. BMC Biol 3:1.
Chen K, Merino C, Sigrist SJ, Featherstone DE (2005) The 4.1 protein coracle mediates
subunit-selective anchoring of Drosophila glutamate receptors to the postsynaptic
actin cytoskeleton. Journal of Neuroscience 25:6667–6675.
Chen S, Lewallen M, Xie T (2012) Adhesion in the stem cell niche: biological roles and
regulation. Development 140:255–265.
Chen X (2008) Stem cells - What can we learn from flies? Fly (Austin) 2:19–28.
Cheng J, Türkel N, Hemati N, Fuller MT, Hunt AJ, Yamashita YM (2008) Centrosome
misorientation reduces stem cell division during ageing. Nature 456:599–604.
Chicurel ME, Harris KM (1992) Three‐dimensional analysis of the structure and
134
composition of CA3 branched dendritic spines and their synaptic relationships with
mossy fiber boutons in the rat hippocampus. J Comp Neurol 325:169–182.
Chklovskii DB, Mel BW, Svoboda K (2004) Cortical rewiring and information storage.
Nature 431:782–788.
Chun D, Gall CM, Bi X, Lynch G (2001) Evidence that integrins contribute to multiple
stages in the consolidation of long term potentiation in rat hippocampus. NSC
105:815–829.
Cingolani LA, Thalhammer A, Yu LMY, Catalano M, Ramos T, Colicos MA, Goda Y
(2008) Activity-Dependent Regulation of Synaptic AMPA Receptor Composition and
Abundance by β3 Integrins. Neuron 58:749–762.
Cohen S, Greenberg ME (2008) Communication between the synapse and the nucleus
in neuronal development, plasticity, and disease. Annu Rev Cell Dev Biol 24:183–
209.
Collins CA, DiAntonio A (2004) Coordinating synaptic growth without being a nervous
wreck. Neuron 41:489–491.
Dani N, Zhu H, Broadie K (2014) Two protein N-acetylgalactosaminyl transferases
regulate synaptic plasticity by activity-dependent regulation of integrin signaling.
Journal of Neuroscience 34:13047–13065.
de Cuevas M, Matunis EL (2011) The stem cell niche: lessons from the Drosophila
testis. Development 138:2861–2869.
Desgrosellier JS, Cheresh DA (2010) Integrins in cancer: biological implications and
therapeutic opportunities. Nature Reviews Cancer 10:890–890.
DiNardo S, Okegbe T, Wingert L, Freilich S, Terry N (2011) lines and bowl affect the
specification of cyst stem cells and niche cells in the Drosophila testis. Development
138:1687–1696.
Dityatev A, Schachner M (2003) Extracellular matrix molecules and synaptic plasticity.
Nat Rev Neurosci 4:456–468.
Dityatev A, Schachner M, Sonderegger P (2010) The dual role of the extracellular
matrix in synaptic plasticity and homeostasis. Nature Publishing Group 11:735–746.
Doren MV, Williamson AL, Lehmann R (1998) Regulation of zygotic gene expression in
Drosophila primordial germ cells. Current Biology 8:243–246.
D’Alterio C, Loza-Coll M, Toledano H, Jones DL (2012) Dual fluorescence detection of
protein and RNA in Drosophila tissues. Nature Protocols 7:1808–1817.
Eaton BA, Davis GW (2005) LIM Kinase1 controls synaptic stability downstream of the
135
type II BMP receptor. Neuron 47:695–708.
Eaton BA, Fetter RD, Davis GW (2002) Dynactin is necessary for synapse stabilization.
Neuron 34:729–741.
Fiala JC, Spacek J, Harris KM (2002) Dendritic spine pathology: cause or consequence
of neurological disorders? Brain Res Brain Res Rev 39:29–54.
Fischer M, Kaech S, Knutti D, Matus A (1998) Rapid actin-based plasticity in dendritic
spines. Neuron 20:847–854.
Fleming HE, Janzen V, Celso Lo C, Guo J, Leahy KM, Kronenberg HM, Scadden DT
(2008) Wnt Signaling in the Niche Enforces Hematopoietic Stem Cell Quiescence
and Is Necessary to Preserve Self-Renewal In Vivo. Cell Stem Cell 2:274–283.
Fogerty FJ, Fessler LI, Bunch TA, Yaron Y, Parker CG, Nelson RE, Brower DL,
Gullberg D, Fessler JH (1994) Tiggrin, a novel Drosophila extracellular matrix
protein that functions as a ligand for Drosophila alpha PS2 beta PS integrins.
Development 120:1747–1758.
Fuentes-Medel Y, Logan MA, Ashley J, Ataman B, Budnik V, Freeman MR (2009) Glia
and muscle sculpt neuromuscular arbors by engulfing destabilized synaptic boutons
and shed presynaptic debris. Plos Biol 7:e1000184.
Fuller MT (1998) Genetic control of cell proliferation and differentiation in Drosophila
spermatogenesis. Semin Cell Dev Biol 9:433–444.
Fuller MT, Spradling AC (2007) Male and Female Drosophila Germline Stem Cells: Two
Versions of Immortality. Science 316:402–404.
Gall CM, Lynch G (2005) Consolidation: A View from the Synapse. In: Synaptic
Plasticity and Transsynaptic Signaling, pp 469–494. New York: Springer US.
Garey LJ, Ong WY, Patel TS, Kanani M, Davis A, Mortimer AM, Barnes TR, Hirsch SR
(1998) Reduced dendritic spine density on cerebral cortical pyramidal neurons in
schizophrenia. J Neurol Neurosurg Psychiatr 65:446–453.
Gattazzo F, Urciuolo A, Bonaldo P (2014) Extracellular matrix: A dynamic
microenvironment for stem cell niche. BBA - General Subjects 1840:2506–2519.
Genereux JC, Qu S, Zhou M, Ryno LM, Wang S, Shoulders MD, Kaufman RJ,
Lasmézas CI, Kelly JW, Luke Wiseman R (2014) Unfolded protein response-
induced ERdj3 secretion links ER stress to extracellular proteostasis. The EMBO
Journal.
Glantz LA, Lewis DA (2000) Decreased dendritic spine density on prefrontal cortical
pyramidal neurons in schizophrenia. Arch Gen Psychiatry 57:65–73.
136
Gotwals PJ, Paine-Saunders SE, Stark KA, Hynes RO (1994) Drosophila integrins and
their ligands. Current Opinion in Cell Biology 6:734–739.
Graner MW, Bunch TA, Baumgartner S, Kerschen A, Brower DL (1998) Splice Variants
of the Drosophila PS2 Integrins Differentially Interact with RGD-containing
Fragments of the Extracellular Proteins Tiggrin, Ten-m, and D-Laminin 2. J Biol
Chem 273:18235–18241.
Graupner M (2010) Mechanisms of induction and maintenance of spike-timing
dependent plasticity in biophysical synapse models. Front Comput Neurosci 4:1–19.
Grooms SY, Jones LS (1997) RGDS tetrapeptide and hippocampal in vitro kindling in
rats: evidence for integrin-mediated physiological stability. Neurosci Lett 231:139–
142.
Grotewiel MS, Beck CD, Wu KH, Zhu XR, Davis RL (1998) Integrin-mediated short-term
memory in Drosophila. Nature 391:455–460.
Guo F, Snapp EL (2013) ERdj3 regulates BiP occupancy in living cells. Journal of Cell
Science 126:1429–1439.
Hamilton BA, Palazzolo MJ, Chang JH, VijayRaghavan K, Mayeda CA, Whitney MA,
Meyerowitz EM (1991) Large scale screen for transposon insertions into cloned
genes. Proc Natl Acad Sci USA 88:2731–2735.
Harburger DS, Calderwood DA (2009) Integrin signalling at a glance. Journal of Cell
Science 122:159–163.
Hardy RW, Tokuyasu KT, Lindsley DL, Garavito M (1979) The germinal proliferation
center in the testis of Drosophila melanogaster. Journal of Ultrastructure Research
69:180–190.
Hetz C (2012) The unfolded protein response: controlling cell fate decisions under ER
stress and beyond. Nature Publishing Group 13:89–102.
Holtmaat A, Svoboda K (2009) Experience-dependent structural synaptic plasticity in
the mammalian brain. Nat Rev Neurosci 10:647–658.
Honkura N, Matsuzaki M, Noguchi J, Ellis-Davies GCR, Kasai H (2008) The Subspine
Organization of Actin Fibers Regulates the Structure and Plasticity of Dendritic
Spines. Neuron 57:719–729.
Hoozemans JJM, van Haastert ES, Nijholt DAT, Rozemuller AJM, Scheper W (2012)
Activation of the Unfolded Protein Response Is an Early Event in Alzheimer’s and
Parkinson’s Disease. Neurodegenerative Dis 10:212–215.
Hoshino M, Suzuki E, Miyake T, Sone M, Komatsu A, Nabeshima Y, Hama C (1999)
Neural expression of hikaru genki protein during embryonic and larval development
137
of Drosophila melanogaster. Dev Genes Evol 209:1–9.
Hoshino M, Suzuki E, Nabeshima Y, Hama C (1996) Hikaru genki protein is secreted
into synaptic clefts from an early stage of synapse formation in Drosophila.
Development 122:589–597.
Hrdlicka L, Gibson M, Kiger A, Micchelli C, Schober M, Schöck F, Perrimon N (2002)
Analysis of twenty‐four Gal4 lines in Drosophila melanogaster. genesis 34:51–57.
Hsu C-W, Yu J-S, Peng P-H, Liu S-C, Chang Y-S, Chang K-P, Wu C-C (2014)
Secretome Profiling of Primary Cells Reveals That THBS2 Is a Salivary Biomarker
of Oral Cavity Squamous Cell Carcinoma. J Proteome Res 13:4796–4807.
Hsu Y-C, Fuchs E (2012) A family business: stem cell progeny join the niche to regulate
homeostasis. Nature Publishing Group 13:103–114.
Huang Z, Shimazu K, Woo NH, Zang K, Müller U, Lu B, Reichardt LF (2006) Distinct
roles of the beta 1-class integrins at the developing and the mature hippocampal
excitatory synapse. Journal of Neuroscience 26:11208–11219.
Humphries JD (2006) Integrin ligands at a glance. Journal of Cell Science 119:3901–
3903.
Hynes RO (2002) Integrins: Bidirectional, allosteric signaling machines. Cell 110:673–
687.
Inaba M, Yuan H, Salzmann V, Fuller MT, Yamashita YM (2010) E-Cadherin Is
Required for Centrosome and Spindle Orientation in Drosophila Male Germline
Stem Cells Hansen IA, ed. PLoS ONE 5:e12473.
Inoue Y, Hayashi S (2007) Tissue-specific laminin expression facilitates integrin-
dependent association of the embryonic wing disc with the trachea in Drosophila.
Developmental Biology 304:90–101.
Issigonis M, Tulina N, de Cuevas M, Brawley C, Sandler L, Matunis E (2009) JAK-STAT
Signal Inhibition Regulates Competition in the Drosophila Testis Stem Cell Niche.
Science 326:153–156.
Je H-S, Yang F, Zhou J, Lu B (2006) Neurotrophin 3 induces structural and functional
modification of synapses through distinct molecular mechanisms. J Cell Biol
175:1029–1042.
Jenkins AB, McCaffery JM, Van Doren M (2003) Drosophila E-cadherin is essential for
proper germ cell-soma interaction during gonad morphogenesis. Development
130:4417–4426.
Jia J-M, Chen Q, Zhou Y, Miao S, Zheng J, Zhang C, Xiong Z-Q (2008) Brain-derived
neurotrophic factor-tropomyosin-related kinase B signaling contributes to activity-
138
dependent changes in synaptic proteins. J Biol Chem 283:21242–21250.
Jiang M, Lee CL, Smith KL, Swann JW (1998) Spine loss and other persistent
alterations of hippocampal pyramidal cell dendrites in a model of early-onset
epilepsy. J Neurosci 18:8356–8368.
Jin Z, Kirilly D, Weng C, Kawase E, Song X, Smith S, Schwartz J, Xie T (2008)
Differentiation-defective stem cells outcompete normal stem cells for niche
occupancy in the Drosophila ovary. Cell Stem Cell 2:39–49.
Johnson KG, Tenney AP, Ghose A, Duckworth AM, Higashi ME, Parfitt K, Marcu O,
Heslip TR, Marsh JL, Schwarz TL, Flanagan JG, Van Vactor D (2006) The HSPGs
Syndecan and Dallylike bind the receptor phosphatase LAR and exert distinct
effects on synaptic development. Neuron 49:517–531.
Jones DL, Wagers AJ (2008) No place like home: anatomy and function of the stem cell
niche. Nat Rev Mol Cell Biol 9:11–21.
Joti P, Ghosh-Roy A, Ray K (2011) Dynein light chain 1 functions in somatic cyst cells
regulate spermatogonial divisions in Drosophila. Sci Rep 1.
Jou TS, Stewart DB, Stappert J, Nelson WJ, Marrs JA (1995) Genetic and biochemical
dissection of protein linkages in the cadherin-catenin complex. Proc Natl Acad Sci
USA 92:5067–5071.
Kampinga HH, Craig EA (2010) The HSP70 chaperone machinery: J proteins as drivers
of functional specificity. :1–14.
Kanatsu-Shinohara M, Takehashi M, Takashima S, Lee J, Morimoto H, Chuma S,
Raducanu A, Nakatsuji N, Fässler R, Shinohara T (2008) Homing of Mouse
Spermatogonial Stem Cells to Germline Niche Depends on β1-Integrin. Stem Cell
3:533–542.
Kawase E (2004) Gbb/Bmp signaling is essential for maintaining germline stem cells
and for repressing bam transcription in the Drosophila testis. Development
131:1365–1375.
Kazama H, Morimoto-Tanifuji T, Nose A (2003) Postsynaptic activation of
calcium/calmodulin-dependent protein kinase ii promotes coordinated pre- and
postsynaptic maturation of drosophila neuromuscular junctions. Neuroscience
117:615–625.
Kelley JJEA (1997) The Effect of Chronic Haloperidol Treatment on Dendritic Spines in
the Rat Striatum. :1–8.
Khuong TM, Habets RLP, Slabbaert JR, Verstreken P (2010) WASP is activated by
phosphatidylinositol-4,5-bisphosphate to restrict synapse growth in a pathway
parallel to bone morphogenetic protein signaling. Proc Natl Acad Sci USA
139
107:17379–17384.
Kiger AA (2001) Stem Cell Self-Renewal Specified by JAK-STAT Activation in
Response to a Support Cell Cue. Science 294:2542–2545.
Kiger AA, White-Cooper H, Fuller MT (2000) Somatic support cells restrict germline
stem cell self-renewal and promote differentiation. Nature 407:750–754.
Kim C, Ye F, Ginsberg MH (2011) Regulation of Integrin Activation. Annu Rev Cell Dev
Biol 27:321–345.
Kim K, Lakhanpal G, Lu HE, Khan M, Suzuki A, Hayashi MK, Narayanan R, Luyben TT,
Matsuda T, Nagai T, Blanpied TA, Hayashi Y, Okamoto K (2015) A Temporary
Gating of Actin Remodeling during Synaptic Plasticity Consists of the Interplay
between the Kinase and Structural Functions of CaMKII. Neuron 87:813–826.
Koh YH, Popova E, Thomas U, Griffith LC, Budnik V (1999) Regulation of DLG
localization at synapses by CaMKII-dependent phosphorylation. Cell 98:353–363.
Kramar EA, Bernard JA, Gall CM, Lynch G (2002) Alpha3 integrin receptors contribute
to the consolidation of long-term potentiation. NSC 110:29–39.
Kramár EA, Lin B, Rex CS, Gall CM, Lynch G (2006) Integrin-driven actin
polymerization consolidates long-term potentiation. Proc Natl Acad Sci USA
103:5579–5584.
Laird DJ, Andrian von UH, Wagers AJ (2008) Stem Cell Trafficking in Tissue
Development, Growth, and Disease. Cell 132:612–630.
Lamprecht R, LeDoux J (2004) Structural plasticity and memory. Nat Rev Neurosci
5:45–54.
Lapidot T (2005) How do stem cells find their way home? Blood 106:1901–1910.
Le Bras S, Van Doren M (2006) Development of the male germline stem cell niche in
Drosophila. Developmental Biology 294:92–103.
Leatherman JL, DiNardo S (2010) Germline self-renewal requires cyst stem cells and
stat regulates niche adhesion in Drosophila testes. Nature Publishing Group
12:806–811.
Lee JY, Chen JY, Shaw JL, Chang KT (2016) Maintenance of Stem Cell Niche Integrity
by a Novel Activator of Integrin Signaling Leatherman JL, ed. PLoS Genet
12:e1006043.
Lee S, Zhou L, Kim J, Kalbfleisch S, Schöck F (2008) Lasp anchors the Drosophila
male stem cell niche and mediates spermatid individualization. Mechanisms of
Development 125:768–776.
140
Legate KR, Wickström SA, Fässler R (2009) Genetic and cell biological analysis of
integrin outside-in signaling. Genes & Development 23:397–418.
Lehmann R (2012) Germline Stem Cells: Origin and Destiny. Stem Cell 10:729–739.
Leptin M, Bogaert T, Lehmann R, Wilcox M (1989) The function of PS integrins during
Drosophila embryogenesis. Cell 56:401–408.
Levi BP, Ghabrial AS, Krasnow MA (2006) Drosophila talin and integrin genes are
required for maintenance of tracheal terminal branches and luminal organization.
Development 133:2383–2393.
Lewis DA (2009) Neuroplasticity of excitatory and inhibitory cortical circuits in
schizophrenia. Dialogues Clin Neurosci 11:269–280.
Li C-Y, Guo Z, Wang Z (2007) TGFβ receptor saxophone non-autonomously regulates
germline proliferation in a Smox/dSmad2-dependent manner in Drosophila testis.
Developmental Biology 309:70–77.
Liebl FLW, Featherstone DE (2005) Genes involved in Drosophila glutamate receptor
expression and localization. BMC Neurosci 6:44.
Lin C-Y, Lynch G, Gall CM (2005) AMPA receptor stimulation increases alpha5beta1
integrin surface expression, adhesive function and signaling. J Neurochem 94:531–
546.
Lin H (2002) The stem-cell niche theory: lessons from flies. Nat Rev Genet 3:931–940.
Lisman J, Schulman H, Cline H (2002) The molecular basis of CaMKII function in
synaptic and behavioural memory. Nat Rev Neurosci 3:175–190.
Losick VP, Morris LX, Fox DT, Spradling A (2011) Drosophila Stem Cell Niches: A
Decade of Discovery Suggests a Unified View of Stem Cell Regulation.
Developmental Cell 21:159–171.
Ludvigsen M, Østergaard M, Vorum H, Jacobsen C, Honoré B (2009) Identification and
characterization of endonuclein binding proteins: evidence of modulatory effects on
signal transduction and chaperone activity. BMC Biochem 10:34.
Majewska A, Brown E, Ross J, Yuste R (2000) Mechanisms of calcium decay kinetics in
hippocampal spines: role of spine calcium pumps and calcium diffusion through the
spine neck in biochemical compartmentalization. Journal of Neuroscience 20:1722–
1734.
Malinow R, Malenka RC (2002) AMPA R ECEPTORT RAFFICKING ANDS YNAPTICP
LASTICITY. Annu Rev Neurosci 25:103–126.
Martin SJ, Morris RGM (2002) New life in an old idea: the synaptic plasticity and
141
memory hypothesis revisited. Hippocampus 12:609–636.
Matsuzaki M, Ellis-Davies GC, Nemoto T, Miyashita Y, Iino M, Kasai H (2001) Dendritic
spine geometry is critical for AMPA receptor expression in hippocampal CA1
pyramidal neurons. Nat Neurosci 4:1086–1092.
Matsuzaki M, Honkura N, Ellis-Davies GCR, Kasai H (2004) Structural basis of long-
term potentiation in single dendritic spines. Nature 429:761–766.
McGeachie AB, Cingolani LA, Goda Y (2011) Stabilising influence: integrins in
regulation of synaptic plasticity. Neurosci Res 70:24–29.
Menon KP, Carrillo RA, Zinn K (2013) Development and plasticity of the Drosophila
larval neuromuscular junction. Wiley Interdiscip Rev Dev Biol 2:647–670.
Michel M, Raabe I, Kupinski AP, Pérez-Palencia R, Bökel C (2011) Local BMP receptor
activation at adherens junctions in the Drosophila germline stem cell niche. Nature
Communications 2:415–12.
Ming G-L, Song H (2005) Adult neurogenesis in the mammalian central nervous
system. Annu Rev Neurosci 28:223–250.
Mitra SK, Hanson DA, Schlaepfer DD (2005) Focal adhesion kinase: in command and
control of cell motility. Nat Rev Mol Cell Biol 6:56–68.
Montanez E, Ussar S, Schifferer M, Bosl M, Zent R, Moser M, Fässler R (2008) Kindlin-
2 controls bidirectional signaling of integrins. Genes & Development 22:1325–1330.
Montell C, Jones K, Hafen E, Rubin G (1985) Rescue of the Drosophila
phototransduction mutation trp by germline transformation. Science 230:1040–1043.
Morimoto T, Nobechi M, Komatsu A, Miyakawa H, Nose A (2010) Subunit-specific and
homeostatic regulation of glutamate receptor localization by CaMKII in Drosophila
neuromuscular junctions. NSC 165:1284–1292.
Morrison SJ, Spradling AC (2008) Stem Cells and Niches: Mechanisms That Promote
Stem Cell Maintenance throughout Life. Cell 132:598–611.
Mosca TJ (2015) On the Teneurin track: a new synaptic organization molecule
emerges. Front Cell Neurosci 9:392.
Multani P, Myers RH, Blume HW, Schomer DL, Sotrel A (1994) Neocortical dendritic
pathology in human partial epilepsy: a quantitative Golgi study. Epilepsia 35:728–
736.
Murase S, Mosser E, Schuman EM (2002) Depolarization drives beta-Catenin into
neuronal spines promoting changes in synaptic structure and function. Neuron
35:91–105.
142
Ohtsuka K, Hata M (2000) Mammalian HSP40/DNAJ homologs: cloning of novel cDNAs
and a proposal for their classification and nomenclature. Cell Stress & Chaperones
5:98–112.
Okamoto K-I, Nagai T, Miyawaki A, Hayashi Y (2004) Rapid and persistent modulation
of actin dynamics regulates postsynaptic reorganization underlying bidirectional
plasticity. Nat Neurosci 7:1104–1112.
Ostroff LE, Fiala JC, Allwardt B, Harris KM (2002) Polyribosomes redistribute from
dendritic shafts into spines with enlarged synapses during LTP in developing rat
hippocampal slices. Neuron 35:535–545.
Padrick SB, Doolittle LK, Brautigam CA, King DS, Rosen MK (2011) Arp2/3 complex is
bound and activated by two WASP proteins. Proc Natl Acad Sci USA 108:E472–
E479.
Papagiannouli F, Mechler BM (2009) discs large regulates somatic cyst cell survival and
expansion in Drosophila testis. Cell Research:1139–1149.
Papagiannouli F, Schardt L, Grajcarek J, Ha N, Lohmann I (2014) The Hox gene Abd-B
controls stem cell niche function in the Drosophila testis. Developmental Cell
28:189–202.
Pfeiffer BE, Huber KM (2006) Current advances in local protein synthesis and synaptic
plasticity. Journal of Neuroscience 26:7147–7150.
Postel R, Vakeel P, Topczewski J, Knöll R, Bakkers J (2008) Zebrafish integrin-linked
kinase is required in skeletal muscles for strengthening the integrin–ECM adhesion
complex. Developmental Biology 318:92–101.
Potocnik AJ, Brakebusch C, Fässler R (2000) Fetal and adult hematopoietic stem cells
require beta1 integrin function for colonizing fetal liver, spleen, and bone marrow.
Immunity 12:653–663.
Qin J, Vinogradova O, Plow EF (2004) Integrin Bidirectional Signaling: A Molecular
View. Plos Biol 2:e169.
Rohrbough J, Grotewiel MS, Davis RL, Broadie K (2000) Integrin-mediated regulation of
synaptic morphology, transmission, and plasticity. J Neurosci 20:6868–6878.
Rohrbough J, Rushton E, Woodruff E, Fergestad T, Vigneswaran K, Broadie K (2007)
Presynaptic establishment of the synaptic cleft extracellular matrix is required for
post-synaptic differentiation. Genes & Development 21:2607–2628.
Rushton E, Rohrbough J, Broadie K (2009) Presynaptic secretion of mind-the-gap
organizes the synaptic extracellular matrix-integrin interface and postsynaptic
environments. Dev Dyn 238:554–571.
143
Sahin AO, Buitenhuis M (2012) Molecular mechanisms underlying adhesion and
migration of hematopoietic stem cells. Cell Adh Migr 6:39–48.
Sanes JR, Lichtman JW (1999) Can molecules explain long-term potentiation? Nat
Neurosci 2:597–604.
Schofield R (1978) The relationship between the spleen colony-forming cell and the
haemopoietic stem cell. Blood Cells 4:7–25.
Schulz C, Wood CG, Jones DL, Tazuke SI, Fuller MT (2002) Signaling from germ cells
mediated by the rhomboid homolog stet organizes encapsulation by somatic support
cells. Development 129:4523–4534.
Serrels B, Serrels A, Brunton VG, Holt M, McLean GW, Gray CH, Jones GE, Frame MC
(2007) Focal adhesion kinase controls actin assembly via a FERM-mediated
interaction with the Arp2/3 complex. Nat Cell Biol 9:1046–1056.
Shattil SJ, Kim C, Ginsberg MH (2010) The final steps of integrin activation:the end
game. :1–13.
Shen Y, Hendershot LM (2005) ERdj3, a stress-inducible endoplasmic reticulum DnaJ
homologue, serves as a cofactor for BiP's interactions with unfolded substrates. Mol
Biol Cell 16:40–50.
Sindi IA, Tannenberg RK, Dodd PR (2014) Role for the neurexin-neuroligin complex in
Alzheimer's disease. Neurobiol Aging 35:746–756.
Singh SR, Chen X, Hou SX (2005) JAK/STAT signaling regulates tissue outgrowth and
male germline stem cell fate in Drosophila. Cell Research 15:1–5.
Song X, Xie T (2002) DE-cadherin-mediated cell adhesion is essential for maintaining
somatic stem cells in the Drosophila ovary. Proc Natl Acad Sci USA 99:14813–
14818.
Song X, Zhu C-H, Doan C, Xie T (2002) Germline stem cells anchored by adherens
junctions in the Drosophila ovary niches. Science 296:1855–1857.
Spradling A, Fuller MT, Braun RE, Yoshida S (2011) Germline Stem Cells. Cold Spring
Harbor Perspectives in Biology 3:a002642–a002642.
Srinivasan S, Mahowald AP, Fuller MT (2012) The receptor tyrosine phosphatase Lar
regulates adhesion between Drosophila male germline stem cells and the niche.
Development 139:1381–1390.
Stäubli U, Chun D, Lynch G (1998) Time-dependent reversal of long-term potentiation
by an integrin antagonist. J Neurosci 18:3460–3469.
Stefen H, Chaichim C, Power J, Fath T (2016) Regulation of the Postsynaptic
144
Compartment of Excitatory Synapses by the Actin Cytoskeleton in Health and Its
Disruption in Disease. Neural Plasticity 2016:1–19.
Sugiyama T, Kohara H, Noda M, Nagasawa T (2006) Maintenance of the Hematopoietic
Stem Cell Pool by CXCL12-CXCR4 Chemokine Signaling in Bone Marrow Stromal
Cell Niches. Immunity 25:977–988.
Swaminathan V, Fischer RS, Waterman CM (2016) The FAK-Arp2/3 interaction
promotes leading edge advance and haptosensing by coupling nascent adhesions
to lamellipodia actin. Mol Biol Cell 27:1085–1100.
Swann JW, Al-Noori S, Jiang M, Lee CL (2000) Spine loss and other dendritic
abnormalities in epilepsy. Hippocampus 10:617–625.
Takagi J (2007) Structural basis for ligand recognition by integrins. Current Opinion in
Cell Biology 19:557–564.
Takeuchi T, Duszkiewicz AJ, Morris RGM (2014) The synaptic plasticity and memory
hypothesis: encoding, storage and persistence. Philos Trans R Soc Lond B Biol Sci
369:20130288–20130288.
Tanentzapf G, Devenport D, Godt D, Brown NH (2007) Integrin-dependent anchoring of
a stem-cell niche. Nature Publishing Group 9:1413–1418.
Tarayrah L, Herz HM, Shilatifard A, Chen X (2013) Histone demethylase dUTX
antagonizes JAK-STAT signaling to maintain proper gene expression and
architecture of the Drosophila testis niche. Development 140:1014–1023.
Teixeira PF, Cerca F, Santos SD, Saraiva MJ (2006) Endoplasmic reticulum stress
associated with extracellular aggregates. Evidence from transthyretin deposition in
familial amyloid polyneuropathy. J Biol Chem 281:21998–22003.
Togashi H, Abe K, Mizoguchi A, Takaoka K, Chisaka O, Takeichi M (2002) Cadherin
regulates dendritic spine morphogenesis. Neuron 35:77–89.
Trachtenberg JT, Chen BE, Knott GW, Feng G, Sanes JR, Welker E, Svoboda K (2002)
Long-term in vivo imaging of experience-dependent synaptic plasticity in adult
cortex. Nature 420:788–794.
Tran J, Brenner TJ, DiNardo S (2000) Somatic control over the germline stem cell
lineage during Drosophila spermatogenesis. Nature 407:754–757.
Tsai P-I, Kao H-H, Grabbe C, Lee Y-T, Ghose A, Lai T-T, Peng K-P, Van Vactor D,
Palmer RH, Chen R-H, Yeh S-R, Chien C-T (2008) Fak56 functions downstream of
integrin alphaPS3betanu and suppresses MAPK activation in neuromuscular
junction growth. Neural Dev 3:26.
Tsai P-I, Wang M, Kao H-H, Cheng Y-J, Lin Y-J, Chen R-H, Chien C-T (2012a) Activity-
145
dependent retrograde laminin A signaling regulates synapse growth at Drosophila
neuromuscular junctions. Proc Natl Acad Sci USA 109:17699–17704.
Tsai P-I, Wang M, Kao H-H, Cheng Y-J, Walker JA, Chen R-H, Chien C-T (2012b)
Neurofibromin mediates FAK signaling in confining synapse growth at Drosophila
neuromuscular junctions. Journal of Neuroscience 32:16971–16981.
Tulina N, Matunis E (2001) Control of stem cell self-renewal in Drosophila
spermatogenesis by JAK-STAT signaling. Science 294:2546–2549.
Vallejo D, Codocedo JF, Inestrosa NC (2016) Posttranslational Modifications Regulate
the Postsynaptic Localization of PSD-95. Mol Neurobiol:1–18.
Van Harreveld A, Fifkova E (1975) Swelling of dendritic spines in the fascia dentata
after stimulation of the perforant fibers as a mechanism of post-tetanic potentiation.
Experimental Neurology 49:736–749.
van Roy F, Berx G (2008) The cell-cell adhesion molecule E-cadherin. Cell Mol Life Sci
65:3756–3788.
Vasin A, Zueva L, Torrez C, Volfson D, Littleton JT, Bykhovskaia M (2014) Synapsin
regulates activity-dependent outgrowth of synaptic boutons at the Drosophila
neuromuscular junction. Journal of Neuroscience 34:10554–10563.
Voog J, D’Alterio C, Jones DL (2008) Multipotent somatic stem cells contribute to the
stem cell niche in the Drosophila testis. Nature 454:1132–1136.
Wang H, Singh SR, Zheng Z, Oh S-W, Chen X, Edwards K, Hou SX (2006) Rap-GEF
Signaling Controls Stem Cell Anchoring to Their Niche through Regulating DE-
Cadherin-Mediated Cell Adhesion in the Drosophila Testis. Developmental Cell
10:117–126.
Wang LD, Wagers AJ (2011) Dynamic niches in the origination and differentiation of
haematopoietic stem cells. Nat Rev Mol Cell Biol 12:643–655.
Wang Y-C, Juan H-C, Wong Y-H, Kuo W-C, Lu Y-L, Lin S-F, Lu C-J, Fann M-J (2013)
Protogenin prevents premature apoptosis of rostral cephalic neural crest cells by
activating the α5β1-integrin. Cell Death Dis 4:e651.
Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya T, Yates JR,
Nusse R (2003) Wnt proteins are lipid-modified and can act as stem cell growth
factors. Nature 423:448–452.
Wong Y-H, Lu A-C, Wang Y-C, Cheng H-C, Chang C, Chen P-H, Yu J-Y, Fann M-J
(2010) Protogenin defines a transition stage during embryonic neurogenesis and
prevents precocious neuronal differentiation. Journal of Neuroscience 30:4428–
4439.
146
Xi R (2009) Anchoring stem cells in the niche by cell adhesion molecules. Cell Adh Migr
3:396–401.
Xie T, Spradling AC (1998) decapentaplegic Is Essential for the Maintenance and
Division of Germline Stem Cells in the Drosophila Ovary. Cell 94:251–260.
Xie Z, Srivastava DP, Photowala H, Kai L, Cahill ME, Woolfrey KM, Shum CY, Surmeier
DJ, Penzes P (2007) Kalirin-7 controls activity-dependent structural and functional
plasticity of dendritic spines. Neuron 56:640–656.
Xu T, Yu X, Perlik AJ, Tobin WF, Zweig JA, Tennant K, Jones T, Zuo Y (2009) Rapid
formation and selective stabilization of synapses for enduring motor memories.
Nature 462:915–919.
Yamagata M, Sanes JR, Weiner JA (2003) Synaptic adhesion molecules. Current
Opinion in Cell Biology 15:621–632.
Yamashita YM, Fuller MT, Jones DL (2005) Signaling in stem cell niches: lessons from
the Drosophila germline. Journal of Cell Science 118:665–672.
Yamashita YM, Jones DL, Fuller MT (2003) Orientation of asymmetric stem cell division
by the APC tumor suppressor and centrosome. Science 301:1547–1550.
Yu M, Haslam RH, Haslam DB (2000) HEDJ, an Hsp40 co-chaperone localized to the
endoplasmic reticulum of human cells. J Biol Chem 275:24984–24992.
Yuste R, Bonhoeffer T (2001) Morphological changes in dendritic spines associated
with long-term synaptic plasticity. Annu Rev Neurosci 24:1071–1089.
Zheng Q, Wang Y, Vargas E, DiNardo S (2011) magu is required for germline stem cell
self-renewal through BMP signaling in the Drosophila testis. Developmental Biology
357:202–210.
Zhou Q, Xiao M, Nicoll RA (2001) Contribution of cytoskeleton to the internalization of
AMPA receptors. Proc Natl Acad Sci USA 98:1261–1266.

Abstract (if available)

Abstract Cell adhesion molecules (CAM) mediate adhesion between cellular components and play pivotal roles during the development and throughout the adult life of an organism. Integrin, a heterodimer of α and β subunits, modulates adhesion between cells and the extracellular matrix (ECM) to support physical structure of the cells and mediate intracellular signaling cascade that govern cell survival and differentiation. Cell surface integrin receptors have been implicated in optimizing stem cell niche environment, and synaptic plasticity by stabilizing synaptic strength. The goal of this dissertation is to identify the roles of a novel DNAJ domain protein, Shriveled (Shv), in maintaining cellular integrity through integrin signaling pathway. Over the course of my research, I investigated the genetic and functional interactions between Shv and integrin by utilizing two different Drosophila model systems: 1) germline stem cell niche, and 2) neuromuscular junction (NMJ). My data reveal that Shv activates integrin signaling in vivo to enhance stem cell survival and modulate synaptic growth

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Translational regulation and endosomal trafficking in synaptic adaptation to stress

PDF

Dual genetic screens for mutants in synaptic homeostatic plasticity and a characterization of insomniac as a regulator for retrograde homeostatic signaling

PDF

Harnessing the power of stem cell self-renewal pathways in cancer: dissecting the role of BMI-1 in Ewing’s sarcoma initiation and maintenance

PDF

Role of the bone marrow niche components in B cell malignancies

PDF

The role of endoplasmic reticulum chaperones in regulating hematopoietic stem cells and hematological malignancies

PDF

Novel roles for Maf1 in embryonic stem cell differentiation and adipogenesis

PDF

The calcium-sensing receptor in the specification of normal and malignant hematopoietic cell localization in the bone marrow microenvironment

PDF

LINC00261 induces a G2/M cell cycle arrest and activation of the DNA damage response in lung adenocarcinoma

PDF

Role of purinergic P2X4 receptors in regulation of dopamine homeostasis in the basal ganglia and associated behaviors

PDF

Using chemical biology approaches to investigate the consequences of protein concentration and activity in cancer cells

PDF

Dissecting the role of a non-canonical interleukin-6 cytokine signaling in systemic chronic inflammation and multimorbidity induced by a high-fat diet

Asset Metadata

Creator Lee, Joo Yeun (author)

Core Title The role of a novel integrin activator in the maintenance of stem cell niche and activity-induced synaptic structural modifications in Drosophila

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 02/23/2017

Defense Date 12/08/2016

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag activity-dependent,cadherin,cell-adhesion molecule,DNAJB11,Drosophila,germline stem cell niche,integrin,OAI-PMH Harvest,shriveled,stem cell niche,structural modification,structural plasticity,synaptic plasticity

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Chang, Karen T. (committee chair)

Creator Email jooyeun@usc.edu,jooyeunlee.jl@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-342248

Unique identifier UC11258282

Identifier etd-LeeJooYeun-5094.pdf (filename),usctheses-c40-342248 (legacy record id)

Legacy Identifier etd-LeeJooYeun-5094.pdf

Dmrecord 342248

Document Type Dissertation

Rights Lee, Joo Yeun

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA