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

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

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Dual Genetic Screens for Mutants in Synaptic
Homeostatic Plasticity and a Characterization of
Insomniac as a Regulator for Retrograde
Homeostatic Signaling

Koto Kikuma

A dissertation
presented to the faculty
of the USC Graduate School
in candidacy for the degree
Doctor of Philosophy
(Neuroscience)

August 2018

© Copyright by Koto Kikuma, 2018.
All rights reserved.
i

This dissertation is dedicated to Okaasan, Otousan, Chuuchan, Hugo, and Taiga.

ii
Acknowledgements

I wish to give my deepest acknowledgement to my adviser, Dr. Dion Dickman, for his
mentoring throughout my graduate study. Thank you for accommodating my pregnancy
(twice!) and family needs. Also, I wish to extend my gratefulness to my committee
members, Dr. Chien-Ping Ko and Dr. Karen Chang for their guidance throughout my
graduate degree.

Special thanks to Xiling Li, who helped me with all the TEVC experiments. Many many
thanks to Dan Kim, who patiently helped me with my experiments. Tons of thanks to my
colleague Xun and Beril. I’m glad it was you two who started together, and end together
this crazy journey of graduate school. Many thanks to all the lab members.

Special Arigato to my husband Chuuchan, who knows me and understands me the best
in the world. He gave me emotional support when I needed. Deep Arigato to my mom,
who has been the best friend to Hugo and a caring baba/okasan to ALL my family, Matcha
and Mochi included. I wouldn’t have been able to remain sane without you. True Arigato
to my dad, who is most excited about my research, possibly more than my PI. Daisuki
Arigato to my 3-year old son, Hugo, who learned to respect mama’s “obenkyo time,” at
least sometimes. Kawaii Arigato to my 5-month old son, Taiga, who smiles at me
whenever he finds me.

iii
Table of Contents

Acknowledgements ......................................................................................................... ii
Table of Contents ............................................................................................................ iii
List of Figures and Tables ...............................................................................................iv

Abstract ........................................................................................................................... 1

Chapter 1: Introduction .................................................................................................... 2
1.1 Synaptic plasticity – Hebbian plasticity vs. homeostatic plasticity ............................. 3
1.2 Physiological relevance of homeostatic plasticity ...................................................... 3
1.3 Model systems to study homeostatic synaptic plasticity ............................................ 5
1.4 Major questions in the field ........................................................................................ 7
1.5 Summary ................................................................................................................. 11

Chapter 2: Cul3-Inc dependent ubiquitination drives retrograde homeostatic signaling 12
2.1 Abstract ................................................................................................................... 13
2.2 Introduction ............................................................................................................. 14
2.3 Results .................................................................................................................... 17
2.4 Discussion ............................................................................................................... 29
2.5 Materials and Methods ............................................................................................ 33

Chapter 3: Extended Synaptotagmin localizes to presynaptic ER and promotes
neurotransmission and synaptic growth in Drosophila .................................................. 65
3.1 Abstract ................................................................................................................... 66
3.2 Introduction ............................................................................................................. 67
3.3 Results .................................................................................................................... 69
3.4 Discussion ............................................................................................................... 79
3.5 Materials and Methods ............................................................................................ 83

Chapter 4: Conclusions ............................................................................................... 105
4.1 Insomniac .............................................................................................................. 106
4.2 Extended Synaptotagmin ...................................................................................... 107

References .................................................................................................................. 110

iv
List of Figures and Tables
Chapter 2
Figure 2-1. Dual forward genetic screens identify 6 genes necessary for PHP induction
and/or expression in distinct synaptic compartments. ............................................ 38
Figure 2-2. inc is required for the acute and chronic expression of PHP. ...................... 39
Figure 2-3: inc expression is required in the postsynaptic cell to drive retrograde PHP
signaling. ................................................................................................................ 41
Figure 2-4: cul3 genetically interacts with inc and is required postsynaptically for
retrograde PHP signaling. ...................................................................................... 42
Figure 2-5: Inc-smFP rapidly traffics to PSDs after PhTx application. ........................... 43
Figure 2-6: Postsynaptic glutamate receptors are unperturbed in inc mutants. ............. 44
Figure 2-7: Inc does not regulate CamKII phosphorylation or abundance at postsynaptic
densities. ................................................................................................................ 45
Figure 2-8: Inc functions downstream of CamKII and does not occlude Tor-OE mediated
retrograde signaling. .............................................................................................. 46

Figure 2-S1: Baseline synaptic transmission is largely unperturbed in inc
kk
mutants. ... 47
Figure 2-S2: Loss of inc does not influence synaptic structure. .................................... 48
Figure 2-S3: inc overexpression does not alter physiology. .......................................... 49
Figure 2-S4: Endogenously tagged smFP does not impact synaptic function or PHP. . 50
Figure 2-S5: Model for Inc function. .............................................................................. 51

Table 2-S1: List of synaptic homeostatic mutants ......................................................... 52
Table 2-S2: List of neurotransmission mutants ............................................................. 53
Table 2-S3: List of all mutants screened ....................................................................... 54
Table 2-S4: Absolute and additional values for normalized and presented data. .......... 61

Chapter 3
Figure 3-1: Genetic analysis and generation of null mutations in Drosophila Esyt. ....... 91
Figure 3-2: The hydrophobic stretch is necessary to localize Esyt to axonal ER. ......... 93
Figure 3-3: PI(4,5)P2 and PI(3)P phospholipid levels are unchanged at presynaptic
terminals in Esyt mutants and Esyt-OE. ................................................................. 95
Figure 3-4: Presynaptic overexpression of Esyt promotes synaptic growth. ................. 96
Figure 3-5: Esyt promotes presynaptic neurotransmitter release. ................................. 97
Figure 3-6: Depletion of the synaptic vesicle pool in Esyt mutants and Esyt-OE. ......... 99
Figure 3-7: Synaptic vesicle density and endocytic pools are unchanged in Esyt mutants
and Esyt-OE. ........................................................................................................ 100
Figure 3-8: Esyt is dispensable for presynaptic homeostatic plasticity. ....................... 101

Table 3-S1: Absolute and additional values for normalized and presented data. ........ 102
1
Abstract

At synapses, homeostatic plasticity maintains the synaptic activities in the physiologically
appropriate range. Impaired synaptic homeostatic plasticity is associated with complex
neurological diseases such as Schizophrenia. At the Drosophila neuromuscular junction (NMJ),
postsynaptic glutamate receptor inhibition induces retrograde signaling, leading to a
compensatory increase in presynaptic neurotransmitter release, thereby precisely restoring
baseline levels of synaptic strength. Despite the recent advances in knowledge for presynaptic
mechanisms involved in this process, mechanisms that drive the retrograde homeostatic
signaling have remained elusive. Here, we conducted dual electrophysiology-based forward-
genetic screens, and identified insomniac (inc) as an essential regulator for retrograde signaling.
Upon the induction of homeostatic synaptic plasticity, insomniac is recruited to the NMJ,
functions together with ubiquitin ligase Cul3 downstream of pCamKII activity. We conclude that
ubiquitination is a key regulator for the homeostatic retrograde signaling.
In the second project, I investigated how presynaptic endoplasmic reticulum influences
synaptic physiology. We characterized mutations in Extended Synaptotagmin (Esyt),
evolutionarily conserved ER proteins with Ca
2+
-sensing domains. We found that Esyt localizes
to presynaptic ER structures. While synaptic structure, membrane lipid balance, and
homeostatic plasticity are surprisingly unperturbed, neurotransmission is reduced in Esyt
mutants. Surprisingly, neuronal overexpression of Esyt enhances synaptic growth and the
sustainment of the vesicle pool during intense activity. Thus, we identify Esyt as a presynaptic
ER protein that can promote neurotransmission and synaptic growth, revealing the first in vivo
neuronal functions of this conserved gene family.
2
Chapter 1: Introduction

3
1.1 Synaptic plasticity – Hebbian plasticity vs. homeostatic plasticity
Neurons communicate with other cells in the form of electrical or chemical signal
across connections called synapses. During development, neurons form billions of
synapses in a precise manner and build extremely intricate network that account for
neural function and animal behavior (McAllister, 2007). Strikingly, these synapses are
not static; after formation, they continuously undergo dynamic adaptations throughout
life in response to external stimuli (Turrigiano and Nelson, 2000, 2004; Davis, 2013).
The best studied form of such adaptations is Hebbian plasticity. In this form of
adaptation, synaptic strength can be enhanced or reduced in response to a change in
activity levels (Song et al., 2000). While Hebbian plasticity is the basis for various
essential functions in the nervous system including learning memory, this is not the only
player governing the synaptic adaptation. Two decades ago, Gina Turrigiano introduced
the concept and evidence of homeostatic plasticity, the ability of synapse to stabilize
and maintain the neural activity in physiologically appropriate range in the face of
perturbations (Turrigiano et al., 1998). Since the introduction of homeostatic synaptic
plasticity, there has been a growing number of studies elucidating molecules and
mechanisms involved. Now we know that Hebbian plasticity and homeostatic plasticity
coexist in the nervous system to ensure proper, stable, yet flexible brain functions
(Turrigiano and Nelson, 2004; Davis, 2006).

1.2 Physiological relevance of homeostatic plasticity
What happens if homeostatic synaptic plasticity is impaired? Genetic mutants
that disrupt homeostatic plasticity are viable, and often display normal synaptic
4
transmission under baseline condition (Dickman and Davis, 2009). However,
homeostatic synaptic plasticity plays an essential role when the nervous system is
confronted with destabilizing forces such as experiential, genetic, developmental, and
environmental variations. For example, when muscles develop, motor neurons must
operate synaptic homeostasis by increasing their input accordingly to ensure that the
muscle excitation levels pass the threshold for contraction. Without synaptic
homeostasis, developing muscle would have too little input from motor neuron and
result in hypoexcitation and inability to contract (Turrigiano, 2012).
Synaptic homeostasis is also essential for the expression of Hebbian plasticity.
Hebbian plasticity is operated through a positive feedback mechanism, which adjusts
the synaptic strength in the same direction of the activity. Thus, enhanced activity
strengthens the synaptic strength, and reduced activity weakens the synaptic
connections. In contrast, homeostatic plasticity is a negative feedback mechanism, in
which the synaptic strength is adjusted in the opposite direction of the persistent
perturbation. Therefore, if Hebbian plasticity is turned on in the absence of homeostatic
plasticity, it could lead to “run-away” plasticity, resulting in extreme levels of synaptic
activity because there is no “break” to keep the activity levels in the physiological range
(Turrigiano, 2012; Davis, 2013). Thus, homeostatic plasticity prevents neurons from
reaching to extreme levels of synaptic activity that can cause synaptic instability and
ultimately result in neurological diseases.
Accordingly, it is now known that homeostatic synaptic plasticity is essential for
proper synaptic function, and that disruption in this plasticity can lead to neurological
diseases. Indeed, a schizophrenia susceptibility gene, dysbindin, was identified as
5
required for synaptic homeostasis at the Drosophila neuromuscular junction (NMJ)
(Dickman and Davis, 2009). Furthermore, fmr1, lack of which causes Fragile X
Syndrome, was found to be essential for homeostatic adjustment of postsynaptic
receptor internalization in mammalian system (Soden and Chen, 2010). Synaptic
homeostasis is also implicated in epilepsy (Chang et al., 2010) and Alzheimer’s disease
(Kamenetz et al., 2003). Although the mechanisms delineating how disruption in
homeostatic synaptic plasticity contributes to these complex neurological disorders has
not been elucidated, these studies clearly established a link between synaptic
homeostasis and neurological disease.

1.3 Model systems to study homeostatic synaptic plasticity
Homeostatic synaptic plasticity is evolutionally conserved from invertebrates to
primates both in the peripherally and central nervous systems (Davis, 2006; Turrigiano,
2012). Here, I discuss two most established model systems to study homeostatic
plasticity at synapses; dissociated rodent neuronal cell culture and Drosophila
neuromuscular junctions (NMJ).
In rodent dissociated neuronal cell culture, pharmacologically blocking action
potential by application of tetrodotoxin (TTX), sodium channel antagonist, leads to an
compensatory increase in postsynaptic receptor level to restore normal
neurotransmission. By contrast, increasing synaptic activity by application of GABA
antagonist, bicuculline, results in homeostatic reduction in postsynaptic receptor,
restoring the baseline levels of neurotransmission (Turrigiano et al., 1998). This bi-
directional postsynaptic receptor levels is termed synaptic scaling. While rodent cell
6
culture might be most ideal system to dissect cellular and molecular mechanisms
underlying synaptic homeostatic plasticity at individual synapses (Pozo and Goda,
2010), the biggest limitation is that it lacks the endogenous environment and network,
which we now know is important to understand the synaptic homeostatic plasticity
(Timmerman and Sanyal, 2012).
The Drosophila NMJ is a glutamatergic synapse, which provides a powerful
model system to dissect the molecules and mechanisms underlying homeostatic
plasticity at synapses. At the Drosophila NMJ, pharmacological or genetic perturbation
in glutamate receptors in the postsynaptic cell causes a compensatory increase in
presynaptic neurotransmitter release, precisely offsetting the perturbation and restore
baseline neurotransmission, which is termed presynaptic homeostatic potentiation
(PHP) (Petersen et al., 1997; Frank et al., 2006) because of its presynaptic effectors.
The Drosophila NMJ has its advantages in its tractable genetics, strong genetic tools,
simpler neuronal networks, and accessibility for in vivo electrophysiological recordings
(Harris and Littleton, 2015). The Drosophila NMJ is particularly an ideal system to
screen a large number of genes for its ease of electrophysiology recordings and
tractable genetics. Accordingly, previous forward-genetic screens utilizing the
Drosophila NMJ system have identified a number of molecules involved in synaptic
homeostasis (Dickman and Davis, 2009; Muller et al., 2011; Hauswirth et al., 2018).

The
limitation of this system is that molecules found in Drosophila might not be conserved
with mammals, yet this model system is still powerful to provide new concepts and
signaling strategies, such as retrograde signaling and post-translational modifications,
which is conserved through evolution (Davis, 2013). In Chapter 2, I therefore employ
7
this established screening strategy to identify molecules and mechanisms required for
presynaptic homeostatic potentiation.

1.4 Major questions in the field
Over the past decades, number of molecules and mechanisms necessary for the
expression of homeostatic synaptic plasticity at the Drosophila NMJ has been revealed;
however, there are still major questions yet to be answered. Here, I discuss what we
learned to date, and what are the top remaining questions in the field of presynaptic
homeostatic plasticity.

Expression of PHP in the presynaptic cells
At the Drosophila NMJ, inhibition of postsynaptic glutamate receptors lead to
increase in presynaptic neurotransmitter release, restoring the baseline transmission.
How does this happen? Two mechanisms were identified as essential to potentiate
presynaptic release during PHP; increase in Ca
2+
influx (Muller and Davis, 2012) and
increase in readily releasable pool (RRP) (Muller et al., 2011; Weyhersmuller et al.,
2011). First, Ca
2+
imaging studies demonstrated that Ca
2+
influx increases upon the
glutamate receptor inhibition. Further, the lack of alpha1 subunit of CaV2-type Ca
2+

channels, cacophony (cac) blocks the expression of PHP. Therefore, these results
suggest that an increase in Ca
2+
influx through cac is essential for the presynaptic
potentiation following glutamate receptor perturbation.
Second, pool of RRP also increases upon the PHP induction. Electrophysiology
analyses on stimulus train identified that the size of RRP is increased by 150-200%
8
upon the induction of PHP (Muller et al., 2011; Weyhersmuller et al., 2011). While it
could be a secondary results due to the increase in Ca
2+
, there are some evidences
against it. In the RIM (Rab3 Interacting Molecule) mutants, which block PHP, it was
found that an increase in Ca
2+
influx was normal while the increase in RRP was
blocked. Thus, these two modulation are genetically distinct, yet ultimately converge to
enhance presynaptic release for PHP. How these two mechanisms as well as other
molecules identified to be required in the presynaptic cell for the expression of PHP
coordinate together to ultimately increase presynaptic release needs further
examination.
Presynaptic modification for the expression of synaptic homeostasis was also
observed in cultured mammalian hippocampal cells. As similar to the Drosophila NMJ,
chronic inhibition of synaptic activity was reported to lead to increased Ca
2+
influx and
presynaptic neurotransmitter release (Zhao et al., 2011). Also, application of TTX
chronically increases active zone area and the number of readily releasable pool
(Murthy et al., 2001; Moulder et al., 2006). Therefore, presynaptic remodeling seems to
be a conserved strategy for homeostatic adaptation at synapses across evolution.

Retrograde signaling
Once postsynaptic receptor perturbation is detected in the muscle, this
information must be transmitted by retrograde signaling to the presynaptic cells, which
subsequently enhances synaptic release. Recent studies identified two putative
retrograde signals: multiplexin and semaphorine 2B (Sema2B). Multiplexin is the
Drosophila homolog of mammalian Collagens XV and XVIII, extracellular matrix protein.
This study found that C-terminal of this protein, Endostatin, can be cleaved from the rest
9
of the protein, and is essential to upregulate presynaptic Ca
2+
channels, enhancing
presynaptic Ca
2+
influx upon PHP induction (Wang et al., 2014). Lack of endostatin
blocks the expression of PHP and this block is rescued either by pre- or postsynaptic
expression of endostatin, consistent with its putative role as a retrograde signal.
Subsequently, Semaphorin 2B (Sema2B) and its receptor Plexin B (PlexB), previously
identified as synaptic signaling molecule associated with axon guidance, were identified
as a retrograde signal and its receptor for the expression of PHP (Orr et al., 2017).
sema2B mutants block PHP, and this block was rescued by application of Sema2B
protein. However, it is not clear whether these two signals interact, if they transmit
different types of information or if there are any other molecules that can function as
retrograde signaling.

Induction of PHP in the postsynaptic cells
What is the sensor and what is it sensing?
In order to initiate PHP, cells must detect the perturbation in synaptic activities.
To date, what the sensor is, and what it is sensing for is not known. Based on the fact
that postsynaptic receptor perturbation initiates PHP, and Ca
2+
-sensing molecule
CamKII was shown to be involved in the regulation of retrograde signaling for chronic
PHP (Haghighi et al., 2003; Newman et al., 2017), it was speculated that the
postsynaptic cell senses Ca
2+
(Davis, 2013). However, recent study showed that
pharmacologically induced acute homeostasis is extracellular Ca
2+
independent (Goel
et al., 2017). While other detection mechanism such as receptor conformational change
and signaling through postsynaptic scaffolds were suggested (Goel et al., 2017),
10
Identity of the sensor and the target of the sensor still remain as major questions in the
field.
How is the sensed information translated into retrograde signaling?
How is the detection of perturbation translated into retrograde signaling? Recent
studies identified PI3K-cII to be required in the postsynaptic cell for the expression of
PHP (Hauswirth et al., 2018). PI3K-cII is localized on Golgi-derived clathrine coated
vesicles, and putatively regulates endocytic pathways. This is so far the only molecule
known to be involved in the generation of retrograde PHP signaling, but a direct role
PI3K-cII plays in the generation of retrograde signaling is not clear.

Postsynaptic remodeling
Postsynaptic density remodeling and AMPA receptor trafficking is extensively
studied for homeostatic plasticity in mammals. While it is less studied at the Drosophila
NMJ, there have been some evidences that postsynaptic form of homeostatic plasticity
is expressed at the Drosophila NMJ (Davis and Goodman, 1998).

Glia
Glia cells have not been extensively studied in the context of homeostatic synaptic
plasticity. At the Drosophila NMJ, glia cells express glutamate transporter, EAAT, which
is responsible for reuptake of excess glutamate in the synaptic cleft (Soustelle et al.,
2002). This suggests that Glia cells could potentially play an active role in glutamate
signaling at the synapses. In fact, the lack of EAAT causes aberrant behaviors in adult
flies (Rival et al., 2006). Future studies will identify the role of glia cells in PHP.

11
1.5 Summary
To fill the gap in knowledge in the PHP induction mechanism, I conducted dual
screens to identify genes postsynaptically required for acute and sustained expression
of synaptic homeostatic plasticity utilizing the proven screening method. I identified
insomniac (inc) a gene involved in sleep homeostasis to be required for retrograde PHP
signal generation. I have characterized this gene further. In addition, I investigated the
function of synaptic ER in the synaptic transmission and structure by characterizing
extended synaptotagmin using the Drosophila NMJ.
Identification of these genes will shed light on the mechanisms underlying
homeostatic modulation of presynaptic release, which can contribute to understanding
of the etiology and progression of complex neurological diseases linked to synaptic
homeostasis.

12
Chapter 2:
Cul3-Inc dependent ubiquitination drives
retrograde homeostatic signaling

13
2.1 Abstract
The stability of nervous system function is constantly confronted with challenges due to
experiential, genetic, developmental, and environmental variations. To accommodate
these variations, organisms are equipped with a remarkable ability to adaptively adjust
synaptic strength to stabilize neural function, a process referred to as homeostatic
synaptic plasticity. It is now known that homeostatic synaptic plasticity is essential for
proper and reliable synaptic function, and that disruption of this process can lead to
neurological diseases such as epilepsy, autism, and schizophrenia. However,
molecules and mechanisms specifically required in the postsynaptic cell for the
induction of retrograde plasticity are largely unknown. We therefore conducted
complementary electrophysiology-based forward genetic screens to find genes needed
for induction of PHP. This screen revealed the sleep behavior gene insomniac to be
required in the postsynaptic muscle for the induction of retrograde PHP signaling.
Further, we find insomniac is accumulated at the NMJ upon synaptic homeostatic
challenge and is required for functions downstream of CamKII signaling together with
Cul-3 ubiquitin ligase complex. Thus, these findings suggest a novel link connecting
homeostatic modulation of synapse and sleep homeostasis.

14
2.2 Introduction
Maintaining the stable synaptic activity is essential to ensure healthy neural function
(Wondolowski and Dickman, 2013; Davis and Muller, 2015). However, the nervous
system is constantly confronted by experiential, developmental, and environmental
forms of stress that destabilize its function throughout life. To maintain synaptic
activities in physiologically acceptable range in the face of these challenges, the
nervous system is endowed with robust plasticity that homeostatically adjusts synaptic
strengths by altering various synaptic properties including presynaptic neurotransmitter
release efficacy (Davis, 2006; Jakawich et al., 2010) and postsynaptic receptor
sensitivity (Turrigiano and Nelson, 2004; Turrigiano, 2008; Pozo and Goda, 2010). This
homeostatic plasticity in the nervous system is conserved from invertebrates to
primates, suggesting it serves an important function selected and maintained
throughout evolution (Davis, 2013; Frank, 2014). Indeed, recent studies have revealed
intriguing links between homeostatic synaptic plasticity and complex neurological
diseases such as schizophrenia, autism spectrum disorder, and intellectual disabilities
(Dickman and Davis, 2009; Wondolowski and Dickman, 2013), further underscoring the
importance of synaptic homeostatic plasticity.
The Drosophila neuromuscular junction (NMJ) is an established model system to
study homeostatic modulation of presynaptic neurotransmitter release (Davis, 2013;
Frank, 2014). At the Drosophila NMJ, perturbation of postsynaptic glutamate receptors
induces retrograde signaling, leading to a compensatory increase in presynaptic
neurotransmitter release, thereby precisely restoring baseline levels of synaptic strength
(Petersen et al., 1997; Frank et al., 2006). This process is referred to as presynaptic
15
homeostatic potentiation (PHP). While recent studies have identified several genes and
mechanisms necessary for presynaptic potentiation (Frank, 2014) and putative
retrograde signaling molecules (Wang et al., 2014; Orr et al., 2017), far less is known
for the postsynaptic induction mechanism underlying the generation of retrograde
signaling (Chen and Dickman, 2017; Goel et al., 2017).
In the postsynaptic cells, PHP is initiated by two distinct perturbations to
glutamate receptors (GluRs). First, pharmacologically blocking GluRs with application of
philanthotoxin-433 (PhTx) acutely induces the PHP expression within 10 minutes (Frank
et al., 2006). Alternatively, genetically ablating a subunit of GluR that is persistent
throughout development (several days) also results in enhanced presynaptic
neurotransmitter release, which is interpreted as the expression of chronic PHP
(Petersen et al., 1997). We now know that these two distinct GluR perturbations lead to
reduced levels of pCamKII specifically in the compartmentalized zone in the
postsynaptic cell opposite of active zone, called subsynaptic reticulum (SSR)
(Weyhersmuller et al., 2011; Li et al., 2018). Following the pCamKII reduction, PhTx-
induced acute PHP and GluRIIA-induced chronic PHP appear to operate through
distinct signaling pathways, as PhTx-induced PHP is protein synthesis independent,
while GluRIIA-induced chronic PHP is protein synthesis dependent (Goold and Davis,
2007; Kauwe et al., 2016). However, postsynpatic mechanisms underlying the
generation of retrograde signaling following pCamKII reduction remains poorly
understood. Therefore, we have developed a forward genetic screen designed to
identify genes necessary for PHP induction.
16
Fragile X Syndrome (FXS) is the most common heritable cause of intellectual
disability (ID) and autism spectrum disorder (ASD). The condition stems from the loss or
altered expression of a single gene, fmr1 (Pieretti et al., 1991), highly conserved gene
from Drosophila (dfmr1) to human (Friedman et al., 2013). fmr1 encodes Fragile X
mental retardation protein (FMRP), an RNA-binding protein which regulates translation
of over 748 genes in mammals (Darnell and Klann, 2013). We hypothesized that
synaptic targets of FMRP would be a rich source of genes necessary for PHP for
several reasons. First, previous studies established a strong link between disruption in
PHP and complex neurological diseases including autism (Dickman and Davis, 2009;
Wondolowski and Dickman, 2013). Second, local protein synthesis at synaptic terminal
is required for proper homeostatic plasticity at the Drosophila NMJ as loss of the target
of rapamycin (TOR), a translational regulator, inhibits PHP (Penney et al., 2012; Goel et
al., 2017). This is also consistent with studies in mammals, where de novo protein
synthesis is required for proper synaptic homeostatic plasticity (Sutton et al., 2004;
Sutton et al., 2006). Lastly, FMRP is essential for homeostatic plasticity at both
excitatory and inhibitory synapses in mouse hippocampus (Soden and Chen, 2010;
Henry, 2011; Lee et al., 2018). We therefore screened FMRP synaptic targets for
mutants that disrupt the expression of acute and chronic PHP.
By screening, we identified insomniac (inc), to be postsynaptically required for
the expression of PHP. inc encodes a putative adaptor for the Cullin-3 (Cul3) E3
ubiquitin ligase complex, which recruits substrates for ubiquitination (Bayon et al.,
2008). Further, we showed that Inc is accumulated in the compartmentalized zone in
the postsynaptic cell upon induction of PHP, and functions together with Cul3
17
downstream of pCamKII activity and upstream of retrograde signaling. Intriguingly, inc
was initially identified and named in separate independent screens for genes required
for sleep regulation (Stavropoulos and Young, 2011). Thus, this study provides a novel
link between homeostatic control of synaptic activities and sleep regulation.

2.3 Results
Dual electrophysiology-based forward genetic screens identify inc
To systematically screen targets of FMRP for roles in synaptic function, we first
established a list of Drosophila homologs of putative FMRP targets in mammals (See
methods for details). This established a collection of 197 genetic mutants to screen for
defects in acute PHP and 341 RNAis to screen for defects in chronic PHP at the third-
instar larval NMJ. First, we screened 197 genetic mutants for disruption in the acute
PHP expression. At the Drosophila NMJ, application of Philanthotoxin (PhTx), a
glutamate receptor antagonist, initially causes a ~50% decrease in miniature excitatory
postsynaptic potential (mEPSP) amplitude (quantal size) and a parallel decrease in
excitatory postsynaptic potential (EPSP) amplitude. After incubation in PhTx for 10
minutes, EPSP amplitude is restored to the baseline values due to an enhancement of
presynaptic neurotransmitter release (Frank et al., 2006). For each mutant, we recoded
EPSP amplitude following 10 minute PhTx incubation. If the gene is essential for acute
PHP, it would fail to increase QC, and therefore EPSP amplitude remains reduced
(Figure 1A). This screen isolated ten mutants that demonstrated average EPSP
amplitude smaller than two standard deviations away from the mean EPSP amplitude of
wild type animals (Figure 1B and C). Of these ten mutants, I have excluded four
18
mutants with the baseline EPSP defects, as their defects in synaptic release machinery
may interfere with their ability to express PHP (Figure 1B). To rule out the possibility
that off target mutations cause the block in synaptic homeostasis, the remaining six
genes were re-screened for acute PHP in the animal with a mutant allele put in trans to
a deficiency that uncovers the entire inc locus. This screening approach has established
six novel genes essential for acute PHP.
Separately, we conducted a complementary screen to identify genes responsible
for chronic PHP expression. Recently, an RNAi-based genetic tool to investigate chronic
PHP was developed, allowing large-scale genetic screen for mutants that disrupt
chronic PHP (Brusich et al., 2015). With this tool, we knocked down the expression of
each target gene both pre- and postsynaptically throughout development while a
glutamate receptor subunit GluRIII is simultaneously knocked down. Similarly to PhTx,
GluRIII knockdown results in reduced mEPSP, which triggers PHP; however, this
homeostatic challenge persists throughout development. For each mutant, we
measured EPSP under this condition and isolated 12 genes that showed a mean EPSP
smaller than two standard deviations away from the mean EPSP of GluRIII knocked
down animals (Figure 1E and F). Among them, two mutants showed a decline in EPSP
amplitude in GluRIII knock down background compared to baseline (without GluRIII
knock down), suggesting that these genes are involved in sustained expression of PHP
(Figure 1E). Thus, these two screens discovered six acute PHP mutants and two
chronic PHP mutants. The two chronic PHP mutants identified were also identified in
PHP screen, suggesting the robustness of the screen.
19
Finally, we used a combination of strategies to determine which synaptic
compartment each genes was required for the expression of PHP. The combination of
RNA-seq profile analyses (Chen and Dickman, 2017), known expression, and tissues
specific RNAi knockdown and/or genetic rescue experiments identified six genes
required in the presynaptic cell for the expression of acute PHP, including the two genes
that are also required for chronic PHP expression. However, only a single gene,
insomniac (inc), was found to be required in the postsynaptic cell (Figure 1D and G).
Therefore, we chose inc for further investigation.

inc is required for the acute and chronic expression of PHP
inc encodes a protein that belongs to a BTB/POZ family, many of which serve as
an adaptor for Cullin3 (Cul3) ubiquitin ligase complexes involved in ubiquitination and
protein degradation (Geyer et al., 2003; Xu et al., 2003). There are two previously
published null alleles for inc, inc
1
and inc
2
(Stavropoulos and Young, 2011). However,
inc
1
affects an adjacent gene CG14795 (~60% reduction). inc
2
introduces a PBac
transposon containing an UAS/TATA sequence in the 5’UTR of the inc gene. This
results in inc expression when a Gal4 is expressed. To obtain a clean null mutant
without a disruption in another gene or unintended gene expression, we generated
novel null alleles, inc
kk3
and inc
kk4
, using CRISPR/Cas9 genome editing technology
(Gratz et al., 2013b). inc
kk3
and inc
kk4
introduced an early stop codon at the 50
th
amino
acid and 57
th
amino acid respectively in all three insomniac isoforms, truncating the
insomniac protein after 1/3 of the BTB/POZ domain on the N-terminal and completely
deletes the C-terminal domain which is speculated to identity substrates (Bayon et al.,
20
2008) (Figure 2A). The BTB/POZ domain was found to be essential for associating with
Cul3 in mammalian homolog of Inc, KCTD5 (Pinkas et al., 2017) (Bayon et al., 2008).
Thus, these mutants are unlikely to be able to bind Cul3. By Western blot analyses
using an anti-Inc antibody (Stavropoulos and Young, 2011), we confirmed that inc
kk3

and inc
kk4
are protein nulls (Figure 2B). Further, behavioral analyses showed that inc
kk3

animals demonstrated a similar sleep behavior deficits as previously reported with inc
1

and inc
2
(Stavropoulos and Young, 2011)(unpublished data). Thus, we concluded that
inc
kk
alleles are protein nulls, and used these alleles for further experiments.
To validate our screen finding, we examined the expression of acute and chronic
PHP in inc
kk
null mutants using two-electrode voltage clamp. First, we tested the
expression of acute PHP. Consistent with the screen, inc
kk
mutants failed to express
homeostatic increase in presynaptic release following PhTx incubation, resulting in
reduced EPSC (Figure 2C and D). This result was replicated with animals with inc
kk3

heterozygotes in trance to a deficiency allele and inc
kk4
animals, further validating the
results. Next, we tested inc mutants for the expression of chronic PHP by genetically
mutating glutamate receptor subunit GluRIIA in inc null background. Genetic mutation of
GluRIIA reduces postsynaptic receptor sensitivity, which induces PHP throughout larval
life (Petersen et al., 1997), and was employed to examine the chronic PHP expression
(Davis and Muller, 2015). While inc was not initially identified in the chronic PHP screen,
inc and GluRIIA double mutant resulted in failure to express chronic PHP (Figure 2E
and F). We speculate that the inc knock down by RNAi did not reduce the inc
expression sufficiently, and as a result, the animal retained the capability to express
21
PHP. Thus, Inc is a part of core machinery necessary for the two PHP mechanisms of
distinct temporal requirements.

Baseline synaptic transmission and structure are largely unperturbed by loss of
inc
We considered the possibility that the absence of inc might alter synaptic
development, which may in turn interfere with the ability to express PHP. We therefore
characterized basal synaptic transmission and structure in inc mutants. While we
observed reduced synaptic transmission in inc
1/2
mutants as previously reported (Li et
al., 2017), inc
2
, inc
2
in trans to a deficiency (inc
2/Df
), and inc
kk3
mutants showed largely
normal synaptic transmissions except for minor, but significant, reduction in mEPSC
frequency (Figure S1). Therefore, we conclude that previously reported alteration in
synaptic transmission in inc
1/2
mutants is likely caused by genetic background, and
basal synaptic transmission in inc mutants are largely normal.
We next examined synaptic structure in inc mutants by quantifying the level and
density of active zone marker Bruchpilot (BRP) and postsynaptic scaffold marker DLG
in inc
kk
animals. While inc mutants were previously reported to display increased bouton
numbers (Li et al., 2017), we found no significant difference in these key pre- and
postsynaptic markers in inc mutants compared to wild type (Figure S2). Thus, loss of inc
does not significantly influence basal synaptic transmission or structures, and we have
no evidence suggesting that the block in PHP in inc mutants is due to the secondary
effect caused by altered synaptic development.

22
inc is required in the postsynaptic muscle to drive retrograde PHP signaling
We next sought to confirm that Inc function is required in postsynaptic cell for
PHP signaling using inc
kk
null alleles. Requirement in postsynaptic cell suggests its
involvement in the induction of PHP and generation of retrograde signaling, while
requirement in presynaptic cells suggests its role in the remodeling of the presynaptic
neurotransmitter release machinery for PHP expression. First, we sought to define the
expression profile of inc at the NMJ using an inc promoter fused with Gal4 (inc-Gal4)
(Stavropoulos and Young, 2011). Driving the expression of GFP under UAS control with
inc-Gal4 revealed GFP signals both at NMJ and muscle (Figure 3A) as previously
shown (Li et al., 2017). Thus, inc is expressed in both presynaptic neurons and
postsynaptic muscles. We then examined which compartment Inc function is required
for PHP by a tissue-specific rescue experiment. First, we confirmed that neuronal or
muscle overexpression does not cause any overexpression artifacts (Figure S3). We
then found that presynaptic expression of smFP-tagged inc under UAS control (smFP-
inc) in inc
kk3
null background using Ok371-Gal4 driver did not restore the block in the
PHP expression. However, postsynaptic expression of smFP-inc using MHC-Gal4 driver
in the same null background fully restored the PHP expression, consistent with the
screen finding earlier (Figure 3B and C). These results were replicated with inc
kk4

background. Thus, inc is required in the postsynaptic cell for the expression of PHP,
suggesting that it is involved in the PHP induction mechanism and the regulation of
retrograde signaling.

23
cul3 genetically interacts with inc and is required postsynaptically for retrograde
PHP signaling
inc encodes a protein that contains a BTB/POZ domain, a recognition motif for
Cul3 ubiquitin ligase complex (Xu et al., 2003). Therefore, inc was speculated to be an
adaptor for cul3 ubiquitin ligase complex, which identifies and recruits substrates for
ubiquitination by Cul3 (Figure 4A). Indeed, Inc physically interacts with Cul3 in
Drosophila S2 cells demonstrated by copreciptation studies (Stavropoulos and Young,
2011; Pfeiffenberger and Allada, 2012), and this interaction is highly conserved as
observed in C. elegans (TAG-303) (Xu et al., 2003) and mammalian (KCTD5) (Bayon et
al., 2008) homolog of Inc. Furthermore, reduction of Cul3 results in sleep defect
reminiscent of inc mutants (Stavropoulos and Young, 2011). Given the putative function
of Inc as a Cul3 substrate specifying adaptor, we speculated that inc mutants may affect
PHP in a cul3-dependent manner, and went on to determine whether cul3 is also
required for the PHP expression. cul3 null mutants are embryonic lethal (Mistry et al.,
2004), therefore, we knocked down inc expression in neurons or muscles using a cul3
RNAi (Stavropoulos and Young, 2011) together with Dcr-2 RNAi (Dietzl et al., 2007).
Reduced expression of cul3 in neurons using Ok371-Gal4 driver did not affect PHP. By
contrast, cul3 knock down in muscles using MHC-Gal4 driver phenocopied the loss of
inc; the animal failed to enhance presynaptic release following PhTx application, unable
to restore EPSC to the baseline levels (Figure 4B and C). Next, we assayed genetic
interaction between inc and cul3. Here, individual heterozygous mutations (inc/+ and
cul3/+) did not significantly affect the expression of PHP. However, inc and cul3 trans-
heterozygous animals (inc/+; cul3/+) completely blocked PHP, indicating that Inc and
Cul3 act in the same pathway for the expression of PHP (Figure 4D and E). Therefore,
24
these results, together with the evidence that Cul3 and Inc physically interacts, identify
Cul3-Inc ubiquitin ligase as an important regulator for the induction of PHP.
Cul3-Inc complex could regulate proteasome-dependent protein degradation by
poly-ubiquitination, or it could change the function, stability or localization of the
substrate by mono-ubiquitination. (Hicke and Dunn, 2003; Mukhopadhyay and
Riezman, 2007) . A recent paper demonstrated that proteasome-regulated protein
degradation is presynaptically required for the expression of PHP, yet postsynaptically
dispensable (Wentzel et al., 2018). Thus, proteasome-dependent protein degradation is
unlikely a key function of Cul3-Inc for the induction of PHP in the postsynaptic cell.
Instead, Cul3-Inc drives retrograde signaling for PHP, most likely by mono-
ubiquitination.

Inc-smFP rapidly traffics to PSDs after PhTx application
Thus far, we have identified the requirement of Cul3-Inc ubiquitin complex in the
postsnypatic cell for the induction of PHP. Recently, it was demonstrated that the
ubiquitination level significantly increases at the NMJ when PHP is chronically induced
(Wentzel et al., 2018). We, therefore, sought to determine whether subcellular
localization of Inc is confined at the NMJ and whether localization or abundance of Inc
might be altered upon the PHP expression. Muscle overexpression of smFP-tagged inc
filled the whole muscle and did not identify any specific subcellular localization (data not
shown). We, therefore, generated an inc transgene that is endogenously tagged with
smFP (GFP with 10x Flag tag) on the C-terminal (inc-smFP), and confirmed that this tag
does not affect the basal synaptic transmission or the expression of PHP (Figure S5).
25
Consistent with the reported ubiquitination levels at the NMJ (Wentzel et al., 2018),
observation of inc-smFP revealed accumulated signal at the NMJ with diffused signal
throughout muscle (Figure 5A and B). Strikingly, we found that the intensity of the inc-
smFP signal at the NMJ was significantly enhanced after PHP induction by PhTx
application. Thus, this finding indicates that Cul3-Inc-dependent ubiquitination required
for the induction of PHP takes place at the NMJ in the postsynaptic cell in response to
GluR inhibition. This is consistent with a finding in mammals, where Cul3 is found to
translocate to dendritic spines in activity-dependent manner (Shen et al., 2007).
Together, this finding suggests that Inc couples synaptic activity with local
ubiquitination-dependent signal transduction, and adjusts synaptic proteins in an
activity-dependent manner.

Postsynaptic glutamate receptors and scaffolding are unperturbed in inc mutants
PhTx-induced enhancement of the Inc levels at the NMJ suggests that the Cul3-
Inc-dependent ubiquitination takes place at the postsynaptic SSR to generate
retrograde signaling. Although little is known about the postsynaptic signal transduction
necessary to drive the induction of PHP, it is clear that PHP is initiated by loss or
inhibition of postsynaptic glutamate receptors at the postsynaptic density. In mammals,
postsynaptic glutamate receptor internalization is a key mechanism in homeostatic
synaptic plasticity in mammals(Turrigiano, 2008). In addition, mono-ubiquitination was
reported to regulate glutamate receptor internalization and intracellular trafficking in an
activity-dependent manner (Schwarz et al., 2010; Widagdo et al., 2017). We, therefore,
hypothesized that Inc may ubiquitinate glutamate receptors following PhTx application,
26
which might alter glutamate receptor abundance, function or composition at the
postsynaptic density, renders incapable to transduce PHP signaling.
To test this hypothesis, we first examined the localization and abundance of
postsynaptic glutamate receptor subunit A, B, and D at the postsynaptic terminal in wild
type and inc mutants before and after PhTx application. At the Drosophila NMJ, there
are two major types of receptors; GluRIIA subunit-containing receptors (GluRIIA) and
GluRIIB subunit-containing receptors (GluRIIB). Both types share common subunits
GluRIIC, GluRIID, and GluRIIE (Thomas and Sigrist, 2012). Importantly, loss of GluRIIA
leads to the expression of PHP, whereas loss of GluRIIB does not impact PHP (Frank et
al., 2006). However, we found no significant difference in GluR subunit intensity levels
and A vs. B composition between wild type and inc mutants before and after PhTx
application (Figure 6A-D). Thus, inc is not required for proper GluR development and
maintenance during PHP.
While morphologically normal, GluRs in inc mutants may change its functions.
We therefore, examined rise time, decay time, and total charge transfer of mEPSC in
wild type and inc mutants. GluRIIB desensitizes much faster than GluRIIA (DiAntonio et
al., 1999), resulting in faster decay time and charge transfer. If the GluRIIA/GluRIIB ratio
is altered, we expect to see a change in the decay time and charge transfer. However,
we found no significant difference between wild type and inc mutants in mEPSC
properties we quantified, consistent with intensity level analyses (Figure 6E-H). Thus,
there is no evidence that glutamate receptors is impacted by the loss of inc and Inc
appears to have functions downstream of GluR perturbations.

27
Inc does not regulate CamKII phosphorylation or abundance at postsynaptic
densities
Glutamate receptor perturbation results in reduced levels of phosphorylated
Ca2+/calmodulin-dependent protein kinase II (pCamKII), an active form of CamKII,
specifically in the local postsynaptic compartments when homeostasis was genetically
or pharmacologically induced (Goel et al., 2017; Newman et al., 2017). While it is yet
unclear the role of CamKII/pCamKII in the induction of PHP, we hypothesized that Inc
might ubiquitinate pCamKII, leading to a change in localization and/or abundance upon
the induction of PHP. In order to test this hypothesis, we observed pCamKII localization
and intensity at the postsynaptic density in wild type and inc mutants before and after
PhTx perturbation by immunostaining. First, we confirmed that in wild type, pCamKII
level was significantly reduced following 10 minutes of PhTx application as previously
reported (Figure 7A and B). Next, we observed that inc mutants demonstrate a similar
reduction of pCamKII intensity following PhTx application, indicating that a reduction in
pCamKII level following PhTx application is independent of Inc functions (Figure 7A and
B). Thus, Inc appears to function downstream of CamKII in the generation of retrograde
signaling for PHP.

inc is necessary for PHP signaling upstream of unitary retrograde PHP signaling
and does not occlude Tor-OE mediated retrograde signaling
Following the pCamKII reduction, PHP appears to operate through two parallel
pathways; PhTx-mediated rapid, translation independent pathway (Frank et al., 2006)
and GluRIIA-mediated chronic, translation dependent pathway (Goold and Davis, 2007).
Little is known about these two distinct pathways, but they ultimately converge to
generate retrograde signaling to remodel presynaptic terminal for the expression of PHP
28
(Goel et al., 2017). The biomarker for this remodeling is enhancement of active zone
scaffold BRP (Weyhersmuller et al., 2011; Goel et al., 2017). If Inc functions in the
postsynaptic cells to generate retrograde signal, this BRP enhancement should be
blocked in inc mutants. To test this hypothesis, we examined individual BRP puncta in
wild type and inc mutants and compared the level before and after PhTx application. At
the baseline level, there was no significant difference between wild type and inc mutants
(Figure 8A-D). However, following PhTx application, wild type animals displayed a
significant increase in both mean and sum puncta intensity as previously seen, while no
significant change was observed in inc mutants. (Figures 8A–D) This finding further
validates Inc function in the postsynaptic cell for the induction of PHP.
Recently, overexpression of the translational regulator Target of Rapamycin (Tor-OE) in
the postsynaptic muscle has been demonstrated to induce retrograde signal leading to
enhanced presynaptic release (Penney et al., 2012). Tor-OE leads to a chronic and
global increase in protein synthesis (Chen and Dickman, 2017), which appears to
artificially activate an instructive retrograde signal to increase presynaptic
neurotransmitter release without glutamate receptor perturbation. Importantly, GluR
perturbation in Tor-OE animals do not lead to further potentiation of presynaptic release
(Goel et al., 2017). This was interpreted to mean that PHP and Tor-OE share the same
retrograde signal, and therefore, one cannot be further expressed when the other is
already expressed. If inc is required for the generation of common retrograde signal
following glutamate receptor perturbation and Tor-OE, presynaptic potentiation caused
by Tor-OE would be blocked in inc mutants. We therefore tested whether inc mutants
retain the capacity to increase presynaptic release when retrograde signal is artificially
29
driven by Tor-OE. As previously seen, Tor-OE in wild type resulted in increase in EPSC
amplitude and quantal content, with no change in mEPSC amplitude (Figure 8E and F).
Surprisingly, postsynaptic Tor-OE in inc mutants (inc+Tor-OE) resulted in an increase in
presynaptic neurotransmitter release comparable to that of wild type (Figure 8E and F).
Consistent with this results, we also observed an enhanced BRP signal in inc
kk3
+ Tor-
OE animals compared to inc
kk3
alone (Figure 8C and D). Thus, inc mutants occludes
presynaptic remodeling following glutamate receptor perturbation, yet retains the
capacity to increase presynaptic release triggered by Tor-OE. This demonstrates that
Inc function is specifically responsible for the generation of retrograde signal caused by
glutamate receptor perturbation and is independent of Tor-OE-mediated increase in
presynaptic release. These evidences suggest that the Tor-dependent retrograde
signaling is independent of PHP retrograde signaling induced by glutamate receptor
inhibition.

2.4 Discussion
By screening more than 300 synaptic targets of FMRP, we have identified Cul3-
Inc as an essential regulator to drive retrograde signal for the PHP expression. Upon
the PHP induction, Inc is recruited to the PSD, and regulates ubiquitination of
postsynaptic proteins in a Cul3-dpendent manner. In the postsynaptic cell, this Cul3-Inc
ubiquitination appears to be required downstream of GluR perturbation and pCamKII
signaling, and is a part of the core machinery shared by acute and chronic PHP (see
Figure S5 for a model). Together, we have identified a novel post-translational signaling
30
system, ubiquitination, as necessary for the induction of PHP, filling the gap in
knowledge for the generation of retrograde PHP signaling.

Ubiquitination as a local, rapid, and finely-tuned signal transduction mechanism
to regulate synaptic plasticity
The expression of acute PHP precisely offsets the amount of glutamate receptor
perturbation within 10 minutes independent of new protein synthesis. This suggests that
the mechanism underlying this process must involve protein synthesis independent
modification that is rapid and finely-tuned. Accordingly in this study, we have identified a
novel regulating system ubiquitination to be required for the induction of PHP.
Ubiquitination is a post-translational modification by covalent attachment of 76-amino
acid protein, ubiquitin, to substrate, which can be tightly and rapidly regulated.
Strikingly, ubiquitinated status of synaptic proteins has been demonstrated to be
modified within less than 15 seconds upon depolarization-induced Ca
2+
influx in rodent
neuronal synaptosome (Chen et al., 2003). Furthermore, ubiquitination is a reversible
process by deubiquitination, which allows for flexible and fine-regulation of substrates
by modulating delicate balance of these two processes. For example, at the Drosophila
NMJ, synaptic growth has been shown to be regulated by the balance between E3
ubiquitnase highwire and deubiquitinase fat facets which antagonize each other
(DiAntonio et al., 2001). These qualities of ubiquitination makes it an ideal signaling
mechanism at synapse to acutely adjust in response to an external stimuli. In mammals
and Drosophila, indeed, ubiquitination has been found to be a common intracellular
communication strategy to modulate synaptic development, synaptic transmission,
synaptic plasticity (DiAntonio, Goodman, Nature, 2001; Wang, Chien, 2017; Sturgeon,
31
Liebl, 2016; Tian, Wu 2013). This suggest that the rapid and reversible control of
ubiquitination of synaptic proteins might be a conserved strategy to dynamically regulate
synaptic plasticity in an activity-dependent manner across evolutions.

Postsynaptic role of Inc in the generation of retrograde PHP signaling
The substrate of Cul3-Inc-dependent ubiquitination is yet known. We have
eliminated the possibility that Cul3-Inc-dependent ubiquitination alters localization,
abundance, or function of GluRs and pCamKII. Accordingly, Inc is expected to function
downstream of pCamKII signaling and upstream of the retrograde signal generation. We
have considered two potential mechanisms, by which Cul3-Inc ubiquitination could
contribute to the generation of retrograde signaling. One possible role Inc might play is
regulation of the membrane trafficking of retrograde signal. Homolog of human collagen
XV/XVIII, multiplexin, and Semaphorine 2B were recently identified as a part of
retrograde signaling to express PHP (Wang et al., 2014; Orr et al., 2017). These signals
need to be secreted from muscle cells. Interestingly, recent studies demonstrated that
Cul3-dependent mono-ubiquitination regulates collagen transport to plasma membrane
by enlarging the size of cargo budding from the endoplasmic reticulum in a Ca
2+
-
dependent manner (Jin et al., 2012; McGourty et al., 2016). One interesting hypothesis
is that Inc may modulate the secretion of the retrograde signal by directly regulating the
membrane trafficking.
Alternatively, Cul3-Inc dependent ubiquitination may direct postsynaptic terminal
remodeling by regulating postsynaptic membrane and scaffold protein availability and
function. While postsynaptic remodeling at the Drosophila NMJ for the expression of
32
PHP is not well studied, it is a key mechanism underlying homeostatic synaptic plasticity
in mammals. Furthermore, mono-ubiquitination has been well established to regulate
membrane protein availability and activity by signaling them for internalization into
endocytic pathway (Hicke and Dunn, 2003; Foot et al., 2017). For instance, glutamate
receptors in mammalian neurons have been reported to be ubiquitinated and
subsequently endocytosed (Widagdo et al., 2017). Consistent with this hypothesis, a
recent study implicated an involvement of recycling endosome in the generation of
retrograde PHP signaling (Hauswirth et al., 2018). Accordingly, the current model is that
Cul3-Inc-dependent ubiquitination might serve as a sorting signal for yet identified
membrane regulatory protein at the postsynaptic density. This directs them for
internalization and/or intracellular trafficking to endosome and lysosomes, which in turn
leads to the generation of retrograde PHP signaling.

Inc controls homeostatic regulation of sleep and synaptic activity
inc mutants have defects in sleep regulation. Intriguingly, it was reported to
disrupt sleep homeostasis, defined as the amount of sleep recovered above baseline
following deprivation (Pfeiffenberger and Allada, 2012). This raises the possibility that
defects in synaptic homeostatic plasticity in inc mutants might contribute to the sleep
disruption. An attractive model is that Inc regulates homeostatic plasticity at synapses,
which in turn regulates sleep homeostasis. Future studies on inc mutants will elucidate
how these two homeostatic regulations intersect, which will shed lights on
understanding sleep defects commonly found in neurological disorder patients
(Wondolowski and Dickman, 2013).
33
2.5 Materials and Methods
Screen: Over 700 mammalian genes have been identified through a variety of
approaches to be putative transcripts associated with FMRP (Darnell papers). We
started with a list of 327 top FMRP synaptic target genes, which constituted 27% of the
presynaptic proteome and 73% of the estimate postsynaptic proteome (ref; Table S1).
This list was further supplemented with an additional 176 genes associated with
schizophrenia and autism spectrum disorder (Gilman, Neuron, 2011; Sando, Cell, 2012;
Cross-Disorder Group of the Psychiatric Genomics Consortium, Lancet, 2017; Jurado,
Neuron, 2013, and Table S1). From this initial list of 503 total genes, we identified 352
homologs in Drosophila (Table S1). From this list, we obtained a combination of known
genetic mutations and/or putative transposon mutations (197) or RNA-interference
transgenes (341) targeting these genes (Bloomington, refs, Table S1). Finally, we
assessed the lethal phase of homozygous mutants and RNAi lines crossed to motor
neuron and muscle Gal4 drivers and removed any mutants that failed to survive to at
least the third-instar larval stage.

Fly Stocks: All Drosophila stocks were raised at 25°C on standard molasses food. The
following fly stocks were used in this study: inc
1
(Stavropoulos and Young, 2011); inc
2

(Stavropoulos and Young, 2011); inc deficiency (Df(2L)Exel7010 CHECK (Stavropoulos
and Young, 2011); Bloomington Drosophila Stock Center (BDGP)); OK371-Gal4 (Mahr
and Aberle, 2006); MHC-Gal4 (Schuster et al., 1996); UAS-Cul3 RNAi (NIG-Fly stock
11861R-2) (Stavropoulos and Young, 2011); UAS-Dcr2 (Dietzl et al., 2007); Cul3EY;
GluRIIA
SP16
(Petersen et al., 1997); CamKII Ntide(Haghighi et al., 2003); G14-Gal4
34
(Aberle et al., 2002); UAS-Tor (Hennig and Neufeld, 2002). The w
1118
strain was used
as the wild-type control unless otherwise noted because this is the genetic background
in which all genotype are bred.

Molecular Biology: inc
kk
mutants were generated using a CRISPR/Cas9 genome editing
strategy as described (Gratz et al., 2013b; Kikuma et al., 2017). Briefly, a target Cas-9
cleavage site in inc was chosen in earliest target of the first exon without obvious off
target sequences in the Drosophila genome (sgRNA target sequence: 5’
GTTCCTCTCCCGTCTGATTC AGG 3’, PAM underscored). DNA sequences covering
this target sequence were synthesized and subcloned into the pU6-BbsI-chiRNA
plasmid (Addgene 45946). To generate the sgRNA, pU6-BbsI-chiRNA was PCR
amplified and cloned into the pattB vector (Bischof et al., 2007). This construct was
injected and inserted into the VK18 target sequence and balanced. Screening of 10
lines with active CRISPR mutagenesis led to 9 independent deletions or insertions with
predicted frameshift mutations in the inc open reading frame. The line which introduced
an earliest stop codon (R50Stop) was chosen for further analyses and named the inc
kk

allele.
To generate UAS-smFP-inc, we subcloned the full-length inc cDNA from the
expressed sequence tag LD43051 (Drosophila Genomics Resources Center;
Bloomington, IN) into the pACU2 vector (Han et al., 2011a) using standard methods.
smFP-10xFlag tag was PCR amplified and placed in-frame at the C-terminal of inc
cDNA. Constructs were sequence verified and injected into the w
1118
strain by
BestGene Inc. (Chino Hill, CA).
35
Endogenously tagged inc-smFP was generated by WellGenetics Inc. (Taipei,
Taiwan) using CRISPR/Cas9 technology (Gratz et al., 2013b; Gratz et al., 2013a).
Briefly, smFP with DsRed flanked by Piggybac was inserted by single target CRISPR at
the C-terminal of inc. The construct was injected to w1118 strain and the insertion was
confirmed by DsRed eyes, which was subsequently excised out, leaving only smFP at
the inc c-terminal. The insertion was confirmed by sequencing.

Electrophysiology: Electrophysiology was performed as described (Kiragasi et al.,
2017). Briefly, wandering third-instar larvae were dissected in modified HL-3 saline
(70mM NaCl, 5mM KCl, 10mM MgCl2, 10mM NaHCO3, 115mM Sucrose, 5mM
Trehelose, 5mM HEPES, pH 7.2) and 0.4mM CaCl2 unless otherwise specified (Stewart
et al., 1994; Dickman et al., 2005). Muscle input resistance (Rin) was monitored for each
cell. Recordings were acquired by an Axonclamp 900A amplifier, Digidata 1550A
acquisition system, and pClamp 10.7 software (Molecular Devices). Electrophysiological
sweeps were digitized at 10 kHz with a 1 kHz filter. For all two electrode voltage clamp
(TEVC) recordings, muscles were clamped at -70 mV, with a leak current below 5 nA.
mEPSCs were recorded for 1 min from each muscle cell in the absence of stimulation.
20 EPSCs were acquired for each cell under stimulation with 0.5 msec duration by
using an ISO-Flex Stimulus Isolator (A.M.P.I.). To acutely block postsynaptic receptors,
larvae were incubated with or without philanthotocin-433 (20 µM; Sigma) in HL-3 for 10
minutes, as described (Frank et al., 2006). Data were analyzed using Clampfit 10.7
(Molecular Divices), MiniAnalysis (Synaptosoft), and Excel (Microsoft, Redmond, WA).

36
Immunocytochemistry: Third-instar larvae were dissected in ice cold 0 Ca2+ HL-3 and
immune-stained as described (Kikuma et. al., 2017). Briefly, wandering third-instar
larvae were dissected in ice cold 0 Ca2+ HL-3 and fixed in either Bouin’s fixative or 4%
paraformaldehyde. Larvae were then washed and incubated in primary antibodies at
4°C overnight. Larva were then washed and incubated in secondary antibodies and
mounted in VectaShield medium (Vector Laboratories) on a glass slide. The following
antibodies were used: mouse anti-Bruchpilot (BRP; nc82; 1:100; DSHB); mouse anti-
GluRIIA (1:100; 8B4D2; DSHB); rabbit anti-GluRIIB (1:1000; (Goel & Dickman, 2018));
guinea pig anti-GluRIID (1:1,000;(Perry et. al., 2017)); rabbit anti-DLG (1:5000; (Pielage
et al., 2005)); mouse anti-DLG (1:100; 4F3; DSHB); mouse anti-pCamKII (1:100; MA1-
047; Invitrogen); guinea pig anti-vGlut (1:2000; (Chen et al., 2017)). Donkey anti-mouse,
anti-guinea pig, and anti-rabbit Alexa Fluor 488-, DyLight 405-, and Cyanine 3 (Cy3)-
conjugated secondary antibodies (Jackson Immunoresearch) were used at 1:400. Alexa
Fluor 647 conjugated goat anti-HRP (Jackson ImmunoResearch) was used at 1:200.

Confocal imaging and analysis: Imaging was performed as described (Perry et al.,
2017). Briefly, samples were imaged using a Nikon A1R Resonant Scanning Confocal
microscope equipped with NIS Elements software using a 100x APO 1.4NA oil
immersion objective. For fluorescence quantifications of BRP and pCamKII intensities,
all genotypes were immunostained in the same tube with identical reagents, then
mounted and imaged in the same session. z-stacks were obtained using identical
settings for all genotypes with z-axis spacing between 0.15 µm to 0.2 µm within an
experiment and optimized for detection without saturation of the signal. Maximum
37
intensity projections were used for quantitative image analysis with the NIS Elements
software General Analysis toolkit. Type Ib and Is boutons were distinguished based on
size and intensity of the HRP and DLG signal on muscle 6/7 and muscle 4 of segments
A2 and A3. Synapse surface area was calculated by creating a mask around the HRP
channel that labels the neuronal membrane. Type Ib boutons were selected at individual
NMJs based on their typical morphological hallmark of higher DLG intensity and bigger
bouton area compared to Type Is boutons, and BRP and pCamKII quantifications were
performed only for Type Ib boutons. For analysis of pCamKII levels, only the pCamKII
signal that co-localized within DLG and HRP areas were summated for Ib and Is
boutons to obtain mean pCamKII intensity levels. BRP puncta number, area, sum
intensity and DLG intensity were quantified by applying intensity thresholds and filters to
binary layers on the BRP channel. Measurements based on confocal images were
taken from at least twelve synapses acquired from at least six different animals.

Statistical Analysis: All data are presented as mean +/-SEM. Data were compared using
either a one-way ANOVA and tested for significance using a 2-tailed Bonferroni post-
hoc test, or using a Student’s t-test (where specified), analyzed using Graphpad Prism
or Microsoft Excel software, and with varying levels of significance assessed as p<0.05
(*), p<0.01 (**), p<0.001 (***), ns=not significant.

38

Figure 2-1. Dual forward genetic screens identify 6 genes necessary for PHP induction
and/or expression in distinct synaptic compartments.
(A) Schematic of presynaptic homeostatic potentiation at the Drosophila NMJ. (B and E)
Electrophysiology-based forward genetic screen strategy and outcome for PhTx-based
acute PHP mutants (B) and for GluRIII RNAi-based chronic PHP mutants (E). (C and F)
Average EPSP amplitudes per genotype with postsynaptic receptor perturbation by
PhTx (C) and by GluRIII RNAi (F). Highlighted in red are mutants that showed EPEP
more than two standard deviations below control EPSP. (D and G) Pre vs. postsynaptic
requirement testing strategy and outcome for PhTx-based acute PHP mutants (D) and
for GluRIII RNAi-based chronic PHP mutants (G).

B
+ PhTx
D
139 mutants
screened
6 homeostatic
E
+ GluRIII
RNAi
10
15
# of mutants
0 20 40
323 RNAi lines
screened
2 homeostatic
control
EPSP amplitude + GluRIII
RNAi
(mV)
0
5
- RNA-seq expression
- Known expression
- RNAi pre vs. post
- Rescue
PHP
expression
acute PHP
induction
10
20
# of mutants
EPSP amplitude + PhTx (mV)
0 20 40
wild type
inc
0
6 PHP
genes
assay
acute PHP
assay chronic
PHP
G
- RNAi pre vs. post
PHP
expression
chronic PHP
induction
2 PHP
genes
2 presynaptic
0 postsynaptic
5 presynaptic
1 postsynaptic
C
F
10 candidate mutants
- PhTx
assay
baseline
12 candidate mutants
- GluRIII
RNAi assay
baseline
Philanthotoxin
(PhTx)
1 min
Set point muscle excitation mEPSP and EPSP amplitudes
reduced
PhTx
10 min
EPSP amplitude restored
EPSP
mEPSP
A
39

Figure 2-2. inc is required for the acute and chronic expression of PHP.
(A) (Top) The Drosophila inc gene locus. The CRISPR-induced cut site in inc
kk
(*) is
shown. (Bottom) The protein structure of wild type Inc and Inc mutant alleles. The Inc
protein contains a BTB and a CTD. inc
kk
mutations are predicted to truncate the BTB
domain. (B) Immunoblot analysis of inc expression in whole adult lysate confirms both
inc
kk3
and inc
kk4
are protein null alleles. (C) Acute expression of PHP requires inc.
Representative EPSC and mEPSC traces for wild type (w
1118
) and inc
kk3
mutants (inc
kk3
)
before and after PhTx application. inc
kk3
mutants fail to homeostatically increase
presynaptic release after PhTx application and EPSC amplitude remains reduced. (D)
Average mEPSC amplitude and presynaptic release (quantal content, QC) following
PhTx application relative to baseline (-PhTx) for indicated genotypes. (E) Chronic
expression of PHP requires inc. Representative EPSC and mEPSC traces for indicated
% Baseline (- PhTx)
D
wild type inc
kk3
0
100
200
E
C
mEPSP quantal
content
GluRIIA inc
kk3
,
GluRIIA
wild type
baseline
inc
kk3
baseline
wild type
+ PhTx
inc
kk3
+ PhTx
inc
kk4
inc
kk3/Df
ns
ns
**
1 kb
inc
kk
*
inc locus
BTB Inc
A
B
Inc
kk3
CTD
whole adult α-Inc
wild
type
inc
kk3
Insomniac
R50STOP
50 AA
150
50
inc
kk3/Df
,
GluRIIA
250
GluRIIA
% Baseline (- GluRIIA)
mEPSP quantal
content
F
0
50
100
150
200
inc
kk3
,
GluRIIA
inc
kk3/Df
,
GluRIIA
***
250
ns
ns
ns
Inc
kk4
C57STOP
inc
kk4
Actin
10nA
10 ms
500pA
100 ms
40
genotypes. inc
kk3
mutants and inc
kk3
mutants in trans to a deficiency (inc
kk3/Df
) fail to
restore baseline EPSC levels in the absence of GluRIIA. (F) Average mEPSC and QC
in (E) normalized to baseline (-GluRIIA). Error bars indicate ± SEM.

41

Figure 2-3: inc expression is required in the postsynaptic cell to drive retrograde PHP
signaling.
(A) Representative images of GFP expression (green) driven by endogenous inc
promoter fused to Gal4 (inc-Gal4/+;GFP/+) at neuromuscular junction (NMJ)
coimmunostained with a neuronal marker HRP (magenta) (Top) and at muscle
coimmunostained with a muscle marker phalloidin (magenta) (Bottom). Inc is present in
presynaptic neurons and postsynaptic muscles. Scale bar=20 μm. (B) Schematic and
representative EPSC and mEPSC traces of presynaptic rescue (inc
kk3
; Ok371-
Gal4/UAS-inc-smFP) and postsynaptic rescue (inc
kk3
; UAS-inc-smFP/+; MHC-Gal4/+)
by tissue-specific expression of inc-smFP in inc
kk3
null mutant background following
PhTx application. Postsynaptic expression of inc fully restores the expression of PHP.
(C) Average mEPSP and quantal content in (B) relative to baseline. Error bars indicate
± SEM.

inc-Gal4 > GFP
A
HRP
20 μm
+ inc
% baseline (no PhTx)
C
0
50
100
150
mEPSP quantal
content
ns
**
200
+ inc
10nA
10 ms
250pA
100 ms
presynaptic
rescue
postsynaptic
rescue
B
inc-Gal4 > GFP phalloidin
42

Figure 2-4: cul3 genetically interacts with inc and is required postsynaptically for
retrograde PHP signaling.
(A) Cul3 E3 ubiquitin ligase complex. Inc acts as a substrate-specifying adaptor for Cul3
ubiquitin ligase complex to identify target proteins for ubiquitination. (B) Representative
EPSC and mEPSC traces for animals with cul3 knock down in neurons (cul3 neuronal
KD: Cul3 RNAi/Ok371) and in muscles (cul3 muscle KD: Cul3 RNAi/+; MHC/+) before
and after PhTx application. Reduced cul3 expression in the muscle blocks the
expression of PHP. (C) Average mEPSC and QC after PhTx perturbation in (B)
normalized to baseline. (D) Representative EPSC and mEPSC traces for heterozygous
controls (inc
kk3
/+ and cul3/+) and double heterozygous mutants (inc
kk3
/+; cul3/+) before
and after PhTx application. The PHP expression is blocked in inc
kk3
and cul3 double
heterozygotes, but not in individual heterozygote controls, suggesting inc
kk3
and cul3
genetically interact. (E) Average mEPSC and QC after PhTx perturbation in (D) relative
to baseline. Error bars indicate ± SEM.
E
inc
kk3
/+;
cul3/+
cul3/+ inc
kk3
/+
mEPSP quantal
content
0
100
200
% baseline (- PhTx)
D
**
*
Inc
Substrate
Cul-3
Rbx1
E2
Ub
A
Poly-ubiquitination
Mono-ubiquitination
Degradation
Signaling
300
mEPSP
quantal
content
0
50
100
150
B
C
% baseline (-PhTx)
ns
**
**
cul3
neuronal KD
500pA
100 ms
10 ms
10nA
inckk3/+
; cul3/+
+ PhTx
cul3
muscle KD
+ PhTx
+ PhTx + PhTx + PhTx inckk3/+ cul3/+
500pA
100 ms
10 ms
10nA
43

Figure 2-5: Inc-smFP rapidly traffics to PSDs after PhTx application.
(A) Representative NMJ images of Inc endogenously tagged with smFP (inc-smFP)
before and after PhTx application immunostained for Flag (green) and postsynaptic
scaffold marker Dlg (magenta). Areas outlined by dashed-line are shown at higher
magnification (Bottom). Inc accumulates at the NMJ following PhTx application. Scale
bar=5 μm. (B) Quantification of GFP puncta intensity following PhTx application relative
to baseline (- PhTx). Error bars indicate ± SEM.

inc-smFP + PhTx
Flag
Dlg Flag
inc-smFP
0
50
100
B
% baseline (- PhTx)
***
150
Puncta intensity
A
Dlg
5 μm
Flag
5 μm
44

Figure 2-6: Postsynaptic glutamate receptors are unperturbed in inc mutants.
(A) Representative NMJ images of wild type, inc
kk3
, and inc
kk4
before and after PhTx
application coimmunostained for GluRIIA (green), GluRIIB (magenta), and GluRIID
(white). No alteration in glutamate receptor levels or composition is observed in inc
mutants. (B-D) Average intensity levels of GluRIIA (B), GluRIIB (C), and GluRIID (D) in
(A) relative to wild type baseline. (E) Representative mEPSC traces for wild type, inc
kk3
,
and inc
kk4
before and after PhTx application. (F-H) Average mEPSC rise time (F), decay
time (G), and charge transfer (H) in (E) normalized to wild type baseline. No functional
change in glutamate receptors is observed in inc mutants. Error bars indicate ± SEM.
A
GluRIIA GluRIIB GluRIID
wild type inc
kk3
inc
kk4
inc
kk3
+ PhTx
E
mEPSP Ƭ
rise
(% wild type)
mEPSP Ƭ
decay
(% wild type)
wild type + PhTx inc
kk4
+ PhTx
charge transfer
(% wild type)
0
50
100
B
GluRIIA intensity
(% wild type)
0
50
100
C
GluRIIB intensity
(% wild type)
0
50
100
GluRIID intensity
(% wild type)
D
5 μm
0
50
100
F
0
50
100
G
0
50
100
H
***
*
wild type
- PhTx + PhTx
inckk
3
inckk
4
500pA
100 ms
***
***
45

Figure 2-7: Inc does not regulate CamKII phosphorylation or abundance at postsynaptic
densities.
(A) Representative NMJ images of wild type, inc
kk3
, and inc
kk4
before and after PhTx
application immunostained for pCamKII (green) and Dlg (magenta). pCamKII level is
reduced upon PhTx application in wild type and inc mutants. (B) Average pCamKII
intensity levels in (A) relative to wild type baseline. (C) Representative EPSC and
mEPSC traces for wild type, muscle overexpression of CamKII
Ntide
(G14/+; UAS-
CamKII
Ntide
/+), inc
kk3
, and muscle overexpression of CamKII
Ntide
in inc
kk3
background
(inc
kk3
; G14/+; UAS-CamKII
Ntide
/+). Presynaptic potentiation caused by postsynaptic
overexpression of CamKII
Ntide
is blocked in inc mutants. (D) Average mEPSC and QC in
(C) relative to baseline. Error bars indicate ± SEM.

pCamKII DLG merge
wild type + PhTx
A
wild type inc
kk3
inc
kk3
+ PhTx inc
kk4
inc
kk4
+ PhTx
5 μm
0
50
100
B
pCamKII intensity
% wild type
46

Figure 2-8: Inc functions downstream of CamKII and does not occlude Tor-OE mediated
retrograde signaling.
(A) Representative NMJ images of wild type, wild type following PhTx application, and
muscle overexpression or Tor (Tor-OE: G14-Gal4/+; UAS-Tor/+) immunostained for
BRP (green) and HRP (white). BRP levels increase upon PhTx application and Tor-OE
in wild type. (B) Average BRP intensity and area in (A) relative to wild type. (C)
Representative NMJ images of inc
kk3
, inc
kk3
following PhTx application, and Tor-OE in
inc
kk3
background. inc
kk3
mutants block the increase in BRP levels induced by PhTx
application, but not by Tor-OE. (D) Average BRP intensity and area in (C) relative to
wild type. (E) Representative EPSC and mEPSC traces in wild type, Tor-OE, inc
kk3
, and
Tor-OE in inc
kk3
background. inc
kk3
mutants retain the capacity to increase BRP levels
induced by Tor-OE. (F) Average mEPSP and QC in (E) relative to baseline.

Tor-OE wild type + PhTx
A
wild type
BRP HRP
B
0
50
100
BRP Sum
Intensity
BRP
Area
inc
kk3
inc
kk3
+ PhTx
BRP HRP
150
200
% wild type
C
**
**
**
D
0
50
100
BRP Sum
Intensity
BRP
Area
150
200
% wild type
ns
**
5 μm
ns
**
**
inc
kk3
, Tor-OE
500pA
100 ms
10 ms
10nA
wild type
F
0
50
100
150
200
mEPSP quantal
content
% baseline (no Tor-OE)
**
**
Tor-OE inc
kk3
inc
kk3
, Tor-OE E
47

Figure 2-S1: Baseline synaptic transmission is largely unperturbed in inc
kk
mutants.
(A) The Drosophila inc gene locus. The deletion site of inc
1
, the PBac transposon
insertion site of inc
2
(red triangle), and the CRISPR-induced early stop codon in inc
kk3

and inc
kk4
(*) are shown. (B) Representative electrophysiological traces showing EPSC
and mEPSC for wild type (w
1118
), inc
kk3
, inc
2
, inc
2/Df
, and inc
2/1
mutants. While inc
2/1

mutants show reduced synaptic transmission, base line synaptic transmission is largely
normal in other inc mutants. Average EPSC amplitude (C), mEPSC amplitude (D),
mEPSC frequency (E), and quantal content (F) in (B).

inc
1
mEPSC freq (Hz)
mEPSC amplitude (nA)
EPSC amplitude (nA)
**
E
C
D
0.2
0
0
1.0
2.0
0
20
40
***
*
ns
B
**
3.0
*
**
0.4
quantal content
F
0
50
100
150 ns
60
ns
*
wild type inc
2/1
inc
kk3
inc
2
inc
2/Df
10 ms
500pA
100 ms
10nA
1 kb
inc
2
inc
kk
*
inc locus
A
CG14795 locus
48

Figure 2-S2: Loss of inc does not influence synaptic structure.
(A) Representative NMJ images of wild type, inc
kk3
, and inc
kk4
mutants
coimmunostained for active zone marker BRP (green), postsynaptic density marker Dlg
(red), and pan-neuronal marker HRP (white). Scale bar=5 μm. Quantification of BRP
puncta density (B), BRP area (C), BRP intensity (D), Dlg density (E), and neuronal
membrane intensity (F) of (A).

wild type A
BRP density (/µm
2
)
(% wild type)
BRP area (µm
2
)
(% wild type)
50
100
0
50
100
0
B C
150
E F
inc
kk4
BRP intensity
(% wild type)
0
50
100
D
150
DLG intensity
(% wild type)
0
50
100
150
HRP intensity
(% wild type)
0
50
100
150 ns
ns
ns ns ns
5 μm
BRP
DLG
HRP
inc
kk3
49

Figure 2-S3: inc overexpression does not alter physiology.
(A) Representative electrophysiological traces showing EPSC and mEPSC for wild type
(w
1118
) neuronal overexpression (Ok371-Gal4/UAS-inc-smFP), and muscle
overexpression (UAS-inc-smFP/+; MHC-Gal4 /+) of UAS-inc-smFP. Quantification of
mEPSC amplitude (B), EPSC amplitude (C), and quantal content (D) for (A).

A
C B
0.2
0.4
0
0
20
40
60
mEPSC amplitude (nA)
EPSC amplitude (nA)
D
0
100
quantal content
ns
ns
50
150
ns
wild type
muscle >
inc-smFP
neuronal >
inc-smFP
500pA
100 ms
10 ms
10nA
50

Figure 2-S4: Endogenously tagged smFP does not impact synaptic function or PHP.
(A) Representative EPSC and mEPSC traces for wild type (w
1118
) and inc-smFP
animals (inc-smFP) before and after PhTx application. Synaptic transmission and the
PHP expression are normal in inc-smFP animals. (B) Average mEPSC amplitude and
presynaptic release (quantal content, QC) following PhTx application relative to baseline
(-PhTx) for (A).
mEPSP
quantal
content
0
50
100
150
A
% baseline (-PhTx)
***
**
B
wild type inc-smFP + PhTx + PhTx
500pA
100 ms
10 ms
10nA
200
51

Figure 2-S5: Model for Inc function.
Inc functions downstream of CamKII and upstream of retrograde signal in the
compartmentalized area (SSR) in the postsynaptic cell for the induction of PHP.
GluR Perturbation
pCamKII
Tor
Retrograde signal
PHP Expression
Generation of
retrograde
signal
Acute PHP
induction
Chronic PHP
induction
Inc
SSR
52
Table 2-S1: List of synaptic homeostatic mutants

PhTx-Based Acute Synaptic Homeostasis Screen
CG # Gene Name Putative function Source PhTx mEPSP EPSP QC
32810 Inc Regulation of sleep, ubiquitination BL18307 + 0.4 17 42.50
32810 Inc Regulation of sleep, ubiquitination BL18307 - 0.98 38 38.78
43955 Fife Synaptic scaffold protein Bruckner, et al., 2012 + 0.47 20.9 44.47
43955 Fife Synaptic scaffold protein Bruckner, et al., 2012- 0.89 39 43.82
16725 Smn NMJ development, skeletal muscle thin filament assembly + 0.3 10 33.33
16725 Smn NMJ development, skeletal muscle thin filament assembly - 0.7 25 35.71
6703 CASK NMJ synaptic transmission, synaptic growth, terminal button organizationBL22662, BL8105 + 0.6 20 33.33
6703 CASK NMJ synaptic transmission, synaptic growth, terminal button organizationBL22662, BL8105 - 0.98 33 33.67
42250 Lqfr Cell proliferation, positive regualtion of Wnt signaling pathway BL29925 + 0.3 18 60.00
42250 Lqfr Cell proliferation, positive regualtion of Wnt signaling pathway BL29925 - 0.7 41 58.57
11430 olf186-F Store-operated calcium entry BL20119, BL25430 + 0.5 20.83 41.66
11430 olf186-F Store-operated calcium entry BL20119, BL25430 - 0.9 33.1 36.78
GluRIIIRNAi-Based Chronic Synaptic Homeostasis Screen
CG # Gene Name Putative function Source PhTx mEPSP EPSP QC
6703 CASK NMJ synaptic transmission, synaptic growth, terminal button organization X307/X313 + 0.44 22.633 51.44
6703 CASK NMJ synaptic transmission, synaptic growth, terminal button organization X307/X313 - 1.28 38.571 30.13
42250 Lqfr Cell proliferation, positive regualtion of Wnt signaling pathway BL28987 + 0.65 35.333 54.36
42250 Lqfr Cell proliferation, positive regualtion of Wnt signaling pathway BL28987 - 1.09 35.8 32.84
11430 olf186-F Store-operated calcium entry v12221 + 0.51 11 21.58
11430 olf186-F Store-operated calcium entry v12221 - 0.90 25.5 28.24
53
Table 2-S2: List of neurotransmission mutants

PhTx-Based Acute Synaptic Homeostasis Screen
CG # Gene Name Putative function Source PhTx mEPSP EPSP QC
10523 Park Protein ubiquitination, negative regulation of JNK pathway, mitochondrion organization BL34747 + 0.3 22 73.33
10523 Park Protein ubiquitination, negative regulation of JNK pathway, mitochondrion organization BL34747 - 0.8 24 30.00
10295 Pak kinase for the small GTPases Rac and Cdc42 BL8804 + 0.4 24 60.00
10295 Pak kinase for the small GTPases Rac and Cdc42 BL8804 - 1 20 20.00
43657 Myo10A Dorsal closure, intracellular protein transport BL29919 + 0.4 24 60.00
43657 Myo10A Dorsal closure, intracellular protein transport BL29919 - 0.8 33 41.25
NA MAPT Expresses human MAPT (tau) under the control of UAS, VTR. BL33819 + 0.5 26 52.00
NA MAPT Expresses human MAPT (tau) under the control of UAS, VTR. BL33819 - 0.9 25.8 28.67
33516 Dpr3 Store-operated calcium entry BL25315 + 0.3 23 76.67
33516 Dpr3 Store-operated calcium entry BL25315 - 0.6 32 53.33
1839 Fbxl4 Deactivation of rhodopsin-mediated signaling, ubiquitin-protein transferase activity BL18041 + 0.3 5 16.67
1839 Fbxl4 Deactivation of rhodopsin-mediated signaling, ubiquitin-protein transferase activity BL18041 - 1.1 10 9.09
6210 Evi Peptide secretion, positive regulation of Wnt signaling pathway BL15363, BL8068 + 0.5 20 40.00
6210 Evi Peptide secretion, positive regulation of Wnt signaling pathway BL15363, BL8068 - 1 25 25.00
42799 Dikar Long-term memory, olfactory learning BL19174 + 0.4 15 37.50
42799 Dikar Long-term memory, olfactory learning BL19174 - 0.7 20 28.57
GluRIIIRNAi-Based Chronic Synaptic Homeostasis Screen
CG # Gene Name Putative function Source PhTx mEPSP EPSP QC
7223 Htl CNS developmeent, muscle fiber development, germ cell development BL35024 + 0.5 18 36.00
7223 Htl CNS developmeent, muscle fiber development, germ cell development BL35024 - 1.1 21 19.09
7765 Khc Microtubule motor activity, ATP binding BL25898 + 0.4 20 50.00
7765 Khc Microtubule motor activity, ATP binding BL25898 - 0.9 20 22.22
7986 Atg18 Autophagy BL34714 + 0.5 21 42.00
7986 Atg18 Autophagy BL34714 - 1.1 25 22.73
2658 CG2658 Proteolysis, ATPase activity BL31223 + 0.4 22 55.00
2658 CG2658 Proteolysis, ATPase activity BL31223 - 0.8 25 31.25
11154 ATPsyn-beta ATP hyrdolysis coupled proton transport BL27712 + 0.4 23 57.50
11154 ATPsyn-beta ATP hyrdolysis coupled proton transport BL27712 - 0.9 28 31.11
4894 Ca-α1D Voltage-gated calcium channel activity BL33413 + 0.16 24 150.00
4894 Ca-α1D Voltage-gated calcium channel activity BL33413 - 0.5 26 52.00
42317 Csk Cell migration, negative regulation of growth BL41712 + 0.4 24 60.00
42317 Csk Cell migration, negative regulation of growth BL41712 - 0.9 23 25.56
9695 Dab Motor neuron axon guidance, cellularization (Song et al., 2010), synaptic vesicle endocytosis, clathrin-mediated endocytosis (Kawasaki et al, 2011) BL42646 + 0.5 24 48.00
9695 Dab Motor neuron axon guidance, cellularization (Song et al., 2010), synaptic vesicle endocytosis, clathrin-mediated endocytosis (Kawasaki et al, 2011) BL42646 - 1.2 29 24.17
31646 DIP-θ (Hit; in old screen, called neurotrimin not DPR) Unknown BL28654 + 0.46 25.4 55.22
31646 DIP-θ (Hit; in old screen, called neurotrimin not DPR) Unknown BL28654 - 1.19 28 23.53
33198 Pen-2 Gamma-secretase complex, recycling endosome BL27298 + 0.4 24 60.00
33198 Pen-2 Gamma-secretase complex, recycling endosome BL27298 - 0.9 27 30.00
11217 CanB2 Sarcomere organization, sleep BL38971 + 0.4 25 62.50
11217 CanB2 Sarcomere organization, sleep BL38971 - 1.1 16 14.55
54
Table 2-S3: List of all mutants screened

PhTx-Based Acute Synaptic Homeostasis Screen
CG # Gene Name Putative function Source PhTx mEPSP EPSP QC
6875 Asp Calmodulin binding, mictrotubule organization BL29202 + 0.44 45 102.3
12505 Arc1 Muscle system process, Behavioral response to starvation BL37530 + 0.35 34 97.1
13941 Arc2 Unknown BL22466 + 0.4 30 75.0
9753 AdoR G-protein coupled adenosine receptor activity, calcium ion import regulation BL35940 + 0.4 34.25 85.6
4006 Akt1 Protein kinase activity, circadian rhythm BL19894 + 0.51 27.6 54.1
NA Hsap\SNCA human mutant A53T form of alpha-synuclein under UAS control BL8148 + 0.3 30 100.0
NA Hsap\SNCA Expresses human mutant A30P form of alpha-synuclein under UAS control, N.B BL8147 + 0.46 35 76.1
13533 Asrij Cytoplasmic vesicle component, recycling endosome component BL14935 + 0.65 29 44.6
4547 Atx-1 Photoreceptor cell maintenance BL18418 + 0.5 30 60.0
5166 Atx2 Circadian regulation of translation, phagocytosis, regulation of synapse structural plasticity BL11688 + 0.5 33.25 66.5
3624 Babos Reglation of dendrite morphogenesis BL13626 + 0.5 24.5 49.0
5680 Bsk JUN kinase activity BL14876 + 0.39 32 82.1
34417 Bsn BL17378 + 0.4 38.3 95.8
15899 Ca-α1T Voltage-gated calcium channel activity BL19179 + 0.5 30.5 61.0
5809 CaBP1 Positive regulation of apoptotic cell clearance, sleep BL20346 + 0.65 30.75 47.3
33653 Cadps Synaptic tansmission, synaptic vesicle exocytosis BL24279 + 0.4 28.25 70.6
CG8639 Cirl G-protein coupled receptor signaling pathway, veicle-mediated transport, neurotransmitter secretion BL21398, BL24989 + 0.5 27 54.0
33344 CcapRAPCcapR BL37788 + 0.3 40 133.3
30106 CCha1r Neuropeptide signaling pathway BL16476 + 0.4 39.6 99.0
16787 CG16787 Unknown BL15918 + 0.51 35 68.6
2118 CG2118 Leucine metabolism BL19368 + 0.4 17 42.5
32137 CG32137 Neuron projection morphogenesis BL19563 + 0.4 31 77.5
32732 CG32732 Later inhibition BL15879 + 0.53 38.6 72.8
42235/ 6111 CGBCG42236111 unknown BL37788 + 0.8 41.25 51.6
42673 CG42673 Unknown BL30630 + 0.5 36 72.0
42788 CG42788 Unknown BL22905 + 0.5 31.8 63.6
4839/ 44153 CG4839/CG44153 Protein phosphorylation BL29130 + 0.5 27.33 54.7
5621 CG565621 Kainate selective glutamate receptor activity BL24332 + 0.4 21.4 53.5
9267 CG9267 Enzyme binding BL16914 + 0.55 33 60.0
3924 Chi Axon guidance, dendrite guidance, phagocytosis, positive regulation of transcription BL16378 + 0.3 42.67 142.2
7391 Clk Circadian regulation and rhythms, sleep/wake BL24513 + 0.3 33.5 111.7
6493 Dcr-2 Cellular response to virus, RNA interfference BL33098 + 0.5 39 78.0
1725 Dlg1 Positive regulation of NMJ synaptic growth, synaptic transmission, locomotor rhythm BL36280 + 0.5 31 62.0
6646, 1349 DJ-1α, Dj-1β Response to oxidative stress, regulation of protien kinase B signaling BL33602 + 0.5 40.83 81.7
5036 Dhit Regulation of G-protein coupled receptor protein signaling pathway BL28396 + 0.3 35.4 118.0
33196 Dpy Extracellular matrix structural constituent, lateral inhibition BL22804 + 0.4 26 65.0
32498 Dnc Synaptic transmission, associative learing, axon extension BL6020 + 0.5 37 74.0
3525 Eas Brain development, mushroom body development, mechanosensory behavior BL10135 + 0.5 44.25 88.5
4035 eIF-4E Translation initiation factor activity, RNA cap binding BL8648 + 0.5 32.67 65.3
34392 Epac Regulation of Rap protein signal transduction BL19033 + 0.925 29.5 31.9
9935 Ekar Glutamatergic synaptic transmission, regulation of photoreceptor membrane potential BL22661 + 0.5 40 80.0
8256 Gpo-1 Neurogenesis, glycerophosphate shuttle, flight behavior BL10577 + 0.43 44.3 103.0
3694 Gγ30A GPCR signaling pathway, regulation of glucose metabolism, phototransduction BL3694 + 0.4 35 87.5
7223 Htl CNS developmeent, muscle fiber development, germ cell development BL23332 + 0.5 26 52.0
44015 Hph Cellular rresponse to hypoxia, positive regulation of cell growth BL20142 + 0.5 26.2 52.4
1770 HDAC4 Histone acetylation, long-term memory BL16708 + 0.6 26.3 43.8
11324 Homer Positive regulation of circadian sleep/wake, regulation of locomotion BL9564 + 0.5 44.6 89.2
18285 Igl Calmodulin binding, myosin light chain binding BL34310 + 0.35 34.6 98.9
9623 If Axon guidance, CNS morphogenesis, sarcomere organization BL29896 + 0.5 32.67 65.3
44159 Irk1 Locomotor rhythm, potassium ion transport, regulation of membrane potential BL26372 + 0.5 32 64.0
7210 Kel Acton cytoskeleton organization, oogenesis BL13269 + 0.5 31 62.0
7765 Khc Microtubule motor activity, ATP binding BL31994 + 0.5 42 84.0
3861 Kdn Carbohydrate metabolism, tricarboxylic acid cycle BL14436 + 0.5 49 98.0
7147 Kuz Axon guidance, muscle tissue development, phagocytosis BL24048 + 0.4 36 90.0
42683 Kcnip4 Calcium ion binding BL25360 + 0.5 36 72.0
5483 Lrrk Protein kinase activity, synapse organization, regulation of synaptic vesicle endocytosis and dendrite morphogenesis BL34750 + 0.5 35 70.0
8909 Lrp4 BMP signaling pathway, cacium ion binding BL23835 + 0.3 28 93.3
30388 Magi Muscle cell postsynaptic density, terminal bouton component BL22093 + 0.55 34 61.8
30361 Mtt Adult feeding behavior, GPCR activity BL34158 + 0.425 35.6 83.8
11144 mGluR GPCR signaling pathway, NMJ development, regulation of synaptic transmission, terminal button organization BL32830 + 0.5 35 70.0
15669 MESK2 Positive regulation of Ras protein signal transduction BL10992 + 0.5 28.67 57.3
5747 Mfr Spermatogenesis, fertilization BL1102 + 0.5 34 68.0
18657 NetA Axon guidance, dendrite guidance BL22841 + 0.5 31.3 62.6
6827 Nrx-IV Synaptic target recognition, septate junction assembly BL4380 + 0.5 33 66.0
1634 Nrg Axonogenesis, nerve maturation, synapse organization BL5595 + 0.3 38 126.7
13772 Nlg2 Synapse maturation, synapse organization, NMJ synaptic growth, NMJ synaptic transmission BL25573 + 0.5 41 82.0
5722 Npc1a Molting cycle, regulation of cholesterol transport BL29886 + 0.5 32.5 65.0
6713 Nos Calmodulin binding, nitric-oxide synthase activity BL24283 + 0.4 26 65.0
55
Table 2-S3: List of all mutants screened (Cont.)

PhTx-Based Acute Synaptic Homeostasis Screen
CG # Gene Name Putative function Source PhTx mEPSP EPSP QC
33513 Nmdar2 Ionotropic glutamate receptor signaling pathway, NMDA glutamate receptor activity BL36432 + 0.6 48.2 80.3
3936 N Neurogenesis, motor neuron axon guidance, neuron development BL3092 + 0.4 40.25 100.6
5119 pAbp Synaptic transmission, neurogenesis, meiosis BL17261 + 0.6 39.75 66.3
12108 Ppt Endocytosis, regulation of axon guidance BL30878 + 0.5 38.5 77.0
8201 Par-1 NMJ synaptic growth, microtubule cytoskeleton organization BL10574 + 0.6 42 70.0
12002 Pxn ECM organization, phagocytosis BL34200 + 0.4 35 87.5
3682 PIP5K59B Regulation of NMJ synaptic growth BL23565 + 0.5 27 54.0
12358 Paip2 Negative regulation of translation, regulation of cell growth BL31788 + 0.5 27 54.0
9842 Pp2B-14D Regulation of emrbyonic development, sleep, wing disc development BL22025 + 0.43 25 58.1
18803 Psn + 0.4 32 80.0
33198 Pen-2 Gamma-secretase complex, late endosome component BL36019 + 0.5 38 76.0
30483 Prosap Postsynaptic density assembly BL13184 + 0.68 31.5 46.3
6622 Pkc53E Protein kinase C activity, zinc ion binding, positive regulation of clathrin-mediated endocytosis BL24443 + 0.5 24 48.0
10376 Ppm1e Protein serine/threonine phosphatase activity BL27461 + 0.5 39 78.0
4523 Pink1 Mitochondrion organization, NMJ synaptic transmission BL26611 + 0.4 35 87.5
12156 Rab39 Rab protein signal transduction, vesicle-mediated transport BL9823 + 0.6 44.25 73.8
1956 Rap1 GTPase activity, regulation of cell shape BL22899 + 0.3 31.67 105.6
42678 Reep1 Response to endoplasmic reticulum stress BL13889 + 0.5 33 66.0
33113 Rtnl1 Endoplasmic reticulum organization, olfactory behavior BL14065 + 0.5 29 58.0
5481 Robo2 Synaptic target recognition, axon guidance, neuron migration BL3102 + 0.5 38.33 76.7
6775 Rg NMJ development, olfactory learning BL23371 + 0.4 32 80.0
43398 Scrib Regulation of synapse structure or activity, regulation of endocytosis BL34112 + 0.5 40 80.0
1865, 11084 Spn43Ab, pk Multicellular organism reproduction, axon guidance BL36156 + 0.4 29.75 74.4
34358 ShakB Gap junction assembly, phototransduction + 0.5 30 60.0
3722 Shg Nervous system development, gastrulation, axonogenesis BL41436 + 0.5 39 78.0
43758 Sli Dendrite morphogenesis, axon guidance BL37047 + 0.3 30 100.0
10706 SK Calmodulin binding, potassium ion transport, regulation of membrane potential in photoreceptor cell BL12729 + 0.3 28 93.3
6410 Snx16 Positive regulation of NMJ synaptic growth, receptor recycling BL38992 + 0.64 28.5 44.5
4931 Sra-1 Axon guidance, phagocytosis, regulation of synapse organization BL16738 + 0.72 31.8 44.2
12292 Spict Negative regulation of NMJ synaptic growth BL17033 + 0.5 32 64.0
11895 Stan GPCR activity, axon guidance, dendrite morphogenesis, regulation of synapse assembly BL6969 + 0.25 26.6 106.4
33141 Sns Actin cytoskeleton organization, myoblast fusion BL35916 + 0.4 39 97.5
11793 Sod Regulation of terminal button organization BL24490 + 0.5 28 56.0
8884 Sap47 Synaptic transmission + 0.5 35 70.0
3985 Syn NMJ synaptic growth, terminal button organization BL18767 + 0.5 28.14 56.3
3985, 6281 Syn, Timp NMJ synaptic growth, terminal button organization; Wing disc morphogenesis, photoaxis BL23762 + 0.5 28 56.0
42684 Syngap1 GTPase activator activity, Ras protein signal transduction BL14006 + 0.3 32.75 109.2
3168 Sv2a Transmembrane transporter activity BL12516 + 0.43 30.3 70.5
10808 Syngr Synaptic vesicle transport, neurogenesis 56314 + 0.5 30.9 61.8
2381 Syt7 Neurotransmitter secretion, synaptic vesicle exocytosis, vesicle-mediated transport BL23394 + 0.5 36.5 73.0
2715 Syx4 SNARE binding, inter-male aggression BL12543 + 0.5 34 68.0
33950 Trol Motor neuron axon guidance, defasciculation of motor neurona xons BL34144 + 0.5 42.2 84.4
12143 Tspan42 Endocytosis, lysosome BL27335 + 0.5 39 78.0
5492 Tsp74F Integral membrane component BL15729 + 0.5 29 58.0
12443 Ths Myoblast migration, glial cell differentiation BL12536 + 0.3 30 100.0
17762 Tomosyn Regulation of NMJ synaptic transmission BL11357 + 0.43 30.3 70.5
11761 Trsn DNA binding, RNAi, protein stabilization BL16774 + 0.6 40 66.7
1693 Tty Chloride channel activity, chloride transport BL13639 + 0.3 37 123.3
4502 Ube2ql1 Unknown BL14644 + 0.5 26.5 53.0
5014 Vap33 Synapse maturation, terminal button organization, NMJ development 39684 + 0.5 26.6 53.2
9326 Vari Septate junction assembly, tracheal system development BL5319 + 0.56 34 60.7
7670 WRNexo 3'-5' exonuclease, DNA catabolism BL18267 + 0.4 39 97.5
5675 X11L Neurotransmitter secretion, synaptic vesicle docking and targetting BL22647 + 0.53 33.6 63.4
14168 Zasp67 Myofibril assembly BL23053 + 0.5 29 58.0
7727 Appl Synapse organization BL43632 + 0.4 39.6 99.0
33670, 11566 stg1, CG11566 Channel regulator activity BL16528 + 0.3 32.7 109.0
7337 Wdr62 Mitotic spindle organization BL17581 + 0.5 38.33 76.7
56
Table 2-S3: List of all mutants screened (Cont.)

GluRIIIRNAi-Based Chronic Synaptic Homeostasis Screen
CG # Gene Name Putative function Source PhTx mEPSP EPSP QC
6493 Dcr-2 Cellular response to virus, RNA interfference BL33656 + 0.4 25 62.5
6438 Amon Peptidase activity, carbohydrate homeostasis, proteolysis BL28583 + 0.5 29 58.0
3664 Rab5 Axonogenesis, dendrite morphogenesis, synaptic vesicle to endosome fusion, regulation of synaptic structure or activity BL34832 + 0.5 30 60.0
1915 Sls Sarcomere organization, skeletal muscle tissue development, myoblast fusion BL31538 + 0.5 30 60.0
1044 Dos Regulation of Ras protein signal transduction BL31766 + 0.5 36 72.0
18803 Psn BL33813 + 0.5 39 78.0
18408 CAPaR Actin filament organization, cell adhesion BL36663 + 0.4 38 95.0
7540 M6 Compound eye morphogenesis, positive phototaxis BL37503 + 0.4 36 90.0
43368 Cac Voltage-gated calcium channel activity, exocytosis, neuron cellular homeostasis, synaptic transmission BL27244 + 0.4 24 60.0
31132 BRWD3 Negative regulation of chromatin binding, phagocytosis BL33421 + 0.4 18 45.0
7727 Appl Synapse organization BL29862 + 0.5 18.4 36.8
12292 Spict Negative regulation of NMJ synaptic growth BL37505 + 0.5 19 38.0
18803 Psn Copper ion import, cytoskeleton organization, Notch signaling pathway BL33814 + 0.5 19.8 39.6
10295 Pak Regulation of synapse structure or activity, positive regulation of synaptic growth at NMJ, axon guidance BL8804 + 0.5 22.6 45.2
1725 Dlg1 Positive regulation of NMJ synaptic growth, synaptic transmission, locomotor rhythm BL36771 + 0.3 23 76.7
40452 Snap25 Synaptic vesicle transport, vesicle fusion, synaptic vesical priming, regulation of short-term neuronal plasticity, NMJ synaptic transmission BL34377 + 0.3 25 83.3
2330 Neurochondrin Muscle system process BL40903 + 0.5 26.4 52.8
3924 Chi Axon guidance, dendrite guidance, phagocytosis, positive regulation of transcription BL31049 + 0.4 26.4 66.0
1099 Dap 160 Positive regulation of protein kinase and neuroblast activity, synaptic growth at NMJ, synaptic vesicle endocytosis BL25879 + 0.5 26.4 52.8
3615 Atg9 Autophagy BL34901 + 0.5 26.5 53.0
14791 Rab27 GTPase activity, exosomal secretion, sleep, synaptic vesicle component BL35774 + 0.5 26.6 53.2
5092 Tor NMJ synaptic growth, regulation of terminal button organization, somatic muscle development, dendrite morphogenesis, axon guidance BL7012 + 0.5 26.75 53.5
2727 Emp Autophagic cell death, scavenger cell death BL40947 + 0.5 26.75 53.5
2715 Syx4 SNARE binding, inter-male aggression BL44054 + 0.5 26.75 53.5
14994 Gad1 NMJ development, neurotransmitter reeptor metabolism, synapse assembly BL28079 + 0.4 26.75 66.9
17248 nSyb Synaptic vesicle fusion, SNARE complex BL31983 + 0.4 26.75 66.9
17762 Tomosyn Regulation of NMJ synaptic transmission BL31980 + 0.5 26.75 53.5
5102 Da Neurogenesis BL38382 + 0.5 27 54.0
16725 Smn NMJ development, skeletal muscle thin filament assembly BL26288 + 0.2 27 135.0
11144 mGluR GPCR signaling pathway, NMJ development, regulation of synaptic transmission, terminal button organization BL34872 + 0.63 27.25 43.3
1241 Atg2 BL34719 + 1 27.3 27.3
42314 PMCA Cellular calcium ion homeostasis, calcium-transporting ATPase activity, metal ion binding BL31572 + 0.35 27.3 78.0
43976 RhoGEF3 Melanotic encapsulation of foreign target BL42526 + 0.5 27.4 54.8
15010 Ago Uiquitin transferase activity, Negative regulation of growth BL34802 + 0.5 27.5 55.0
10478 αKap4 Proteasome assembly, protein import to nucleus BL31640 + 0.5 27.5 55.0
9994 Rab9 Vesicle-mediated transport, retrograde transport, endosome to Golgi, GTPase activity BL42942 + 0.56 27.75 49.6
31137 Twin mRNA catabolism, oogenesis BL32490 + 0.4 27.75 69.4
7050 Nrx-1 NMJ synaptic transmission, synapse assembly and organization and growth, regulation of terminal button organization BL32408 + 0.55 27.8 50.5
11579 Arm Nervous system development BL35004 + 0.4 28 70.0
4428 Atg4 Regulation of autophagy BL35740 + 0.5 28 56.0
6224 Dbo Regulation of synapse organization, cell growth BL38940 + 0.5 28 56.0
1429 Mef2 Myoblast fusion, locomotor rhythm BL38247 + 0.375 28 74.7
17369 Vha55 ATP hydrolysis coupled proton transport BL40884 + 0.5 28 56.0
5621 CG5621 Kainate selective glutamate receptor activity BL25822 + 0.4 28 70.0
10295 Pak Regulation of synapse structure or activity, positive regulation of synaptic growth at NMJ, axon guidance BL28945 + 0.5 28 56.0
7152 Syn1 Locomotion, regulation of NMJ synaptic growth BL27504 + 0.4 28 70.0
33513 Nmdar2 Ionotropic glutamate receptor signaling pathway, NMDA glutamate receptor activity BL40846 + 0.5 28.25 56.5
34403 Pan Regulation of Wnt signaling pathway, wing morphogenesis, embryonic pattern specification BL40848 + 0.5 28.25 56.5
14548 E(spl)mβ-HLH Negative regulation of transcripition from RNA polymerase II promotor BL26206 + 0.4 28.25 70.6
3827 Sc DNA binding, nervous system development BL26206 + 0.4 28.25 70.6
11173 Snap29 SNARE binding, regulation of endocytosis BL25862 + 0.375 28.25 75.3
5771 Rab11 Endocytosis, vesicle-mediated transport, regulation of synapse organization, negative regulation of NMJ synaptic growth BL27730 + 0.4 28.3 70.8
12598 Adar ds-RNA adenosine deaminase activity BL28311 + 0.5 28.33 56.7
33208 Mical Axon guidance, sarcomere organization, synapse assembly involved in innervation BL31148 + 0.4 28.33 70.8
31118 RabX4 Rab protein signal transduction, vesicle-mediated transport BL28704 + 0.5 28.33 56.7
6601 Rab6 Axon guidance, regulation of postsynaptic membrane potential, exocytosis BL35744 + 0.4 28.4 71.0
32134 Btl Activation of MAPK activity, negative regulation of axon extension BL40871 + 0.4 28.6 71.5
9907 Para Sodium ion transport, voltage-gated sodium channel activity, response to hypoxia BL33923 + 0.4 28.67 71.7
3725 SERCA Celular calcium ion homeostasis, NMJ synaptic tranmission BL25928 + 0.5 28.67 57.3
8385 Arf79F Positive regulation of endocytosis BL29538 + 0.5 29 58.0
14575 CapaR Neuropeptide signaling pathway, positive regulation of calcium ion transport, GPCR signaling pathway BL27275 + 0.5 29 58.0
12143 Tsp42EJ Endocytosis, lysosome BL29392 + 0.4 29 72.5
31999 CG31999 Cell adhesion BL31587 + 0.5 29 58.0
32031 Argk Arginine kinase activity, Phosphorylation BL41697 + 0.4 29.25 73.1
45057 CG484893 Neuron projection morphogenesis BL38287 + 0.5 29.25 58.5
1725 Dlg1 Positive regulation of NMJ synaptic growth, synaptic transmission, locomotor rhythm BL35772 + 0.575 29.25 50.9
9778 Syt14 Neuron projection morphogenesis BL28365 + 0.4 29.3 73.3
10808 Syngr Synaptic vesicle transport, neurogenesis BL38274 + 0.42 29.4 70.0
57
Table 2-S3: List of all mutants screened (Cont.)

GluRIIIRNAi-Based Chronic Synaptic Homeostasis Screen
CG # Gene Name Putative function Source PhTx mEPSP EPSP QC
42768 Msp300 Actin filament binding, cytoskeletal protein binding BL9766 + 0.5 29.429 58.9
17077 Pnt Phagocytosis, regulation of neurogenesis BL35038 + 0.4 29.5 73.8
17654 Eno Magnesium ion binding, regulation of glucose metabolism BL26300 + 0.5 29.5 59.0
33955 Eys ECM structural constituent, cell morphogenesis, rhabdomere development BL33764 + 0.43 29.6 68.8
7103 Pvf1 Heparin binding, cell projection assembly BL39038 + 0.5 29.6 59.2
1389 Tor Terminal region determination, gastrulation BL33627 + 0.4 29.6 74.0
43067 FoxP Locmotion, male courtship, operant conditioning BL26774 + 0.33 29.6 89.7
9579 AnxB10 Calcium-dependent phospholipid binding BL38272 + 0.5 29.67 59.3
9267 CG9267 Enzyme binding BL34800 + 0.5 30 60.0
8261 Gγ1 Neurogenesis, actin filament organization, GPCR signaling pathway BL34372 + 0.4 30 75.0
8937 HC71 Lipid particle, microtubule associated complex BL34527 + 0.4 30 75.0
44159 Irk1 Locomotor rhythm, potassium ion transport, regulation of membrane potential BL42644 + 0.5 30 60.0
7138 R2d2 Defense response to virus, production of siRNA involved in RNAi BL34784 + 0.5 30 60.0
1395 Stg Gastrulation, regulation of mitotic cell cycle BL34831 + 0.5 30 60.0
31136 Syx1A SNARE binding, vesicle fusion, synaptic vesicle fusion to presynaptic active zone membrane, NMJ synaptic transmission, neurotransmitter secretion BL25811 + 0.4 30 75.0
42349 Pkcδ Intracellular signal transduction BL28355 + 0.5 30.25 60.5
6827 Nrx-IV Synaptic target recognition, septate junction assembly BL32424 + 0.3 30.3 101.0
7670 WRNexo 3'-5' exonuclease, DNA catabolism BL38297 + 0.47 30.3 64.5
31359 Hsp70 Response to heat BL32997 + 0.5 30.5 61.0
4889 Wg Extracellular matrix binding, morphogen activity BL32994 + 0.575 30.5 53.0
17870 14-3-3zeta Unknown BL28327 + 0.53 30.6 57.7
42333 Sytβ Neurotransmitter secretion, synaptic vesicle exocytosis, vesicle-mediated transport BL27293 + 0.5 30.66 61.3
2945 Cin and CG42376 Embryonic development and Cytochrome-c oxidase activity BL32425 + 0.5 30.666 61.3
44128 Src42A Negative regulation of NMJ synaptic growth, TM receptor protein tyrosine kinase signaling pathway BL44039 + 0.5 30.67 61.3
3204 Rap21 Neurogenesis, GTPase activity BL29568 + 0.5 30.67 61.3
11513 Armi Long-term memory, Negative regulation of DNA damage checkpoint, oogenesis BL34789 + 0.33 31 93.9
10861 Atg12 Autophagy BL34675 + 0.5 31 62.0
6703 CASK NMJ synaptic transmission regulation, neurotransmitter secretion regulation, NMJ synaptic growth regulation, regulation of terminal button organization BL32857 + 0.4 31 77.5
1084 Cont Nerve maturation, septate junction assembly, axon ensheathment (Banerjee et al., 2006) BL34867 + 0.5 31 62.0
5837 Hem Axogenesis, CNS development, myoblast fusion, NMJ development, phagocytosis BL41688 + 0.4 31 77.5
5119 pAbp Synaptic transmission, neurogenesis, meiosis BL36127 + 0.5 31 62.0
10523 Park Protein ubiquitination, negative regulation of JNK pathway, mitochondrion organization BL38333 + 0.5 31 62.0
6622 Pkc53E Protein kinase C activity, zinc ion binding, positive regulation of clathrin-mediated endocytosis BL34716 + 0.5 31 62.0
5518 Sda Mechanosensory behavior, regulation of membrane potential BL37494 + 0.5 31 62.0
2835 Gαs GPCR signaling pathway, NMJ development, synaptic transmission BL29576 + 0.45 31 68.9
10693 Slo Circadian behavior, NMJ synaptic growth regulation, negative regulation of synaptic transmission at NMJ BL26247 + 0.4 31 77.5
12295 Stj Synaptic vesicle endocytosis, synaptic vesicle fusion, NMJ synaptic transmission, negative regulation of NMJ synaptic growth BL25807 + 0.4 31 77.5
8390 Vlc Imaginal disc-derived wing morphogenesis BL40925 + 0.4 31.25 78.1
42403 Ca-β Voltage-gate calcium channel activity, regulation of glucose metabolism BL43292 + 0.4 31.33 78.3
9575 Rab35 Exosomal secretion, phagocytosis BL28342 + 0.4 31.33 78.3
2902 Nmdar1 Ionotropic glutamate receptor signaling pathway, NMDA glutamate receptor activity BL41666 + 0.5 31.333 62.7
10538 CdGAPr GTPase activity, retinal ganglion cell axon guidance BL38279 + 0.5 31.3333 62.7
9575 Rab35 Vesicle-mediated transport, phagocytosis, GTPase activity BL9818 + 0.5 31.5 63.0
43398 Scrib Regulation of synapse structure or activity, regulation of endocytosis BL35748 + 0.5 31.6 63.2
4905 Syn2 Regulation of NMJ synaptic growth BL28363 + 0.56 31.6 56.4
10023 Fak Negative regulation of synaptic growth at NMJ BL44075 + 0.5 31.67 63.3
32149 RhoGAP71E Rho protein signal transduction, GTPase activator activity BL32417 + 0.4 31.67 79.2
4969 Wnt6 Receptor binding, regulation of imaginal disc-derived wing size BL30493 + 0.5 31.67 63.3
18069 CaMKII Calmodulin-dependent protein kinase activity, synaptic transmission BL29401 + 0.52 31.75 61.1
10295 Pak Regulation of synapse structure or activity, positive regulation of synaptic growth at NMJ, axon guidance BL35141 + 0.5 31.83 63.7
7392 Cka Phagocytosis, positive regulation of JNK cascade BL34522 + 0.4 32 80.0
4792 Dcr-1 Dendrite morphogenesis, pre-miRNA binding BL34826 + 0.4 32 80.0
9834 EndoB Regulation of endocytosis, regulation of glucose metabolism, membrane organization BL34935 + 0.5 32 64.0
30388 Magi Muscle cell postsynaptic density, terminal bouton component BL33411 + 0.3 32 106.7
1954 Pkc98E Positive regulation of peptide hormone secretion, neuron projection morphogenesis BL44074 + 0.4 32 80.0
11173 Snap29 Autophagosome maturation, endocytosis regulation BL34088 + 0.3 32 106.7
1515 Ykt6 SNAP receptor activity, vesicle-mediated transport, retrograde Golgi to ER vesicle-mediated transport BL38314 + 0.5 32 64.0
6625 αSnap SNARE complex disassembly, NMJ synaptic transmission, synaptic vesicle fusion to presynaptic active zone membrane BL34088 + 0.3 32 106.7
7662 Veli Regulation of NMJ synaptic growth BL29590 + 0.4 32 80.0
2108 mRpS29 Apoptosis BL9805 + 0.5 32.17 64.3
9995 Htt Synaptic vesicle transport, axo-dendritic transport BL33808 + 0.3 32.3 107.7
33196 Dpy Extracellular matrix structural constituent, lateral inhibition BL36673 + 0.4 32.3 80.8
6702 Cbp53E Cellular io homeostasis, calcium ion binding BL28302 + 0.3 32.3 107.7
16944 SesB Synaptic transmission, synaptic vesicle transport, NMJ synaptic growth, neuron cellular homeostasis, mitochondrial calcium ion homeostasis BL36661 + 0.4 32.33 80.8
33547 Rim Neurotransmitter secretion, synaptic vesicle exocytosis, vesicle-mediated transport BL27300 + 0.5 32.4 64.8
10844 RyR Muscle contraction, microtubule associated complex BL31540 + 0.5 32.4 64.8
42734 Ank2 NMJ development, Regulation of synaptic transmission BL33414 + 0.4 32.5 81.3
42665 Exn Regulation of neurotransmitter secretion BL33373 + 0.4 32.5 81.3
58
Table 2-S3: List of all mutants screened (Cont.)

GluRIIIRNAi-Based Chronic Synaptic Homeostasis Screen
CG # Gene Name Putative function Source PhTx mEPSP EPSP QC
43398 Scrib Regulation of synapse structure or activity, regulation of endocytosis BL39073 + 0.36 32.6 90.6
8318 Nf1 Negative regulation of NMJ synaptic growth, cAMP-mediated signaling BL25845 + 0.5 32.6 65.2
6114 Sff NMJ development BL36656 + 0.4 32.67 81.7
7925 Tko Response to hyoxia, response to mechanical stimulus BL38251 + 0.5 32.67 65.3
5685 Calx Calcium ion binding, calcium:sodium antiporter activity BL28306 + 0.53 32.67 61.6
10844 Rya-r44F Muscle contraction, calcium ion transmembrane transport BL29445 + 0.4 32.67 81.7
8183 Khc73 ATP binding, microtubule motor activity BL22058 + 0.5 32.75 65.5
3664 Rab5 Endocytosis, synaptic vesicle to endosome fusion BL9771 + 0.5 32.75 65.5
1663 CG1663 Unknown BL27078 + 0.5 32.75 65.5
45110 Tau Negative regulation of neuron death, microtubule cytoskeleton organization BL28891 + 0.5 32.75 65.5
1500 Fw Imaginal disc-derived wing morphogenesis BL38975 + 0.6 33 55.0
1770 HDAC4 Histone acetylation, long-term memory BL34774 + 0.5 33 66.0
16973 Msn JNK cascade, axon guidance, negative regulation of Ras protein signal transduction BL42518 + 0.4 33 82.5
42279 Nedd4 NMJ development, positive regulation of endocytosis, positive regulation of synapse assembly BL34741 + 0.4 33 82.5
5492 Tsp74F Integral membrane component BL39046 + 0.4 33 82.5
42403 Ca-β Voltage-gated calcium channel activity BL29575 + 0.4 33 82.5
8203 Cdk5 Cyclin-dependent protein serine/threonine activity, axonogenesis, adult locomotion, motor neuron axon guidance, regulation of NMJ synaptic growth BL27517 + 0.425 33 77.6
7034 Sec15 Endocytic recycling, vesicle-mediated transport, axon guidance BL27499 + 0.5 33 66.0
11084 Swm Positive reglation of mRNA polydeylation and gene expression BL28548 + 0.3 33 110.0
4898 Tm1 Dendrite morphogenesis, actin filament binding BL38232 + 0.5 33.25 66.5
5387 Cdk5α Adult locomotion, axogenesis, motor neuron axon guidance BL27048 + 0.6 33.25 55.4
6396 Csp Brain morphogenesis, exocytosis, locomotion, neuron celular homeostasis, regulation of NMJ synaptic transmission BL33645 + 0.5 33.3 66.6
8183 Khc73 Microtubule motor activity, ATP binding BL36733 + 0.4 33.3 83.3
6167 PICK1 Regulated exocytosis, positive regulation of developmental growth BL31258 + 0.4 33.3 83.3
1107 Aux Notch signaling pathway BL39017 + 0.5 33.33 66.7
44533 NnaD Larval development, mitochondrion organization, neural retina development BL33549 + 0.4 33.33 83.3
4977 Kek2 Integral component of plasma membrane BL31874 + 0.4 33.33 83.3
5675 X11L Neurotransmitter secretion, synaptic vesicle docking and targetting BL29309 + 0.43 33.375 77.6
NA pmBYF5CA Expresses a constitutively active, YFP-tagged Rab5 protein under UAS control, H.B. BL9774 + 0.5 33.5 67.0
1511 Eph Peripheral nervous system developmenet, transmembrane receptor protein tyrosine kinase activity, mushroom body development BL39066 + 0.5 33.5 67.0
8430 Got1 Glutamate biosynthesis, imaginal disc-derived wing morphogenesis BL36768 + 0.5 33.5 67.0
18076 Shot Dendrite morphogenesis, axonogenesis, regulation of axon extension BL28336 + 0.5 33.5 67.0
18285 Igl Calmodulin binding, myosin light chain binding BL 29598 + 0.5 33.518 67.0
17134 Base1 BL42868 + 0.4 33.6 84.0
5680 Bsk JUN kinase activity BL32977 + 0.5 33.6 67.2
5227 Sdk Pigment cell differentiation BL33412 + 0.5 33.6 67.2
4931 Sra-1 Axon guidance, phagocytosis, regulation of synapse organization BL38294 + 0.5 33.66 67.3
1817 Ptp10D Motor neuron axon guidance, CNS development, long-term memory BL39001 + 0.5 33.67 67.3
9326 Vari Septate junction assembly, tracheal system development BL28599 + 0.4 33.67 84.2
6556 Cnk Signal transduction, Ras protein signal transduction BL33366 + 0.4 33.75 84.4
6064 Crtc Response to oxidative stress and starvation, CREB binding BL42561 + 0.5 33.75 67.5
10376 Ppm1e Protein dephosphorylation BL41907 + 0.3 33.75 112.5
4971 Wnt10 Receptor binding, Wnt signaling pathway BL31989 + 0.5 33.75 67.5
8884 Nap1 Nucleosome assembly, chromatin remodeling BL35445 + 0.5 34 68.0
1242 Hsp83 Actin filament organization, centrosome cycle, response to heat BL32996 + 0.5 34 68.0
18405 Sema-1a Synapse assembly, synaptic target recognition, motor neuron axon guidance BL34320 + 0.4 34 85.0
7793 Sos Actin filament organization, regulation of cell shape BL34833 + 0.5 34 68.0
6323 Tsp97E Integral membrane component BL39047 + 0.4 34 85.0
34392 Epac Cyclic nucleotide-dependent guanyl-nucleotide exchange factor activity BL29317 + 0.5 34 68.0
34358 ShakB Gap junction assembly, phototransduction BL27292 + 0.5 34 68.0
7050 Nrx-1 NMJ synaptic transmission, synapse assembly and organization and growth, regulation of terminal button organization BL27502 + 0.5 34 68.0
5092 Tor NMJ synaptic growth, regulation of terminal button organization, dendrite morphogenesis, axon guidance BL34639 + 0.3 34.2 114.0
12559 Rl Modulation of synaptic transmission, transmembrane receptor protein tyrosine kinase signalign pathway BL34855 + 0.5 34.25 68.5
5723 Ten-m NMJ synaptic growth, synaptic target attraction, motor neuron axon guidance BL29390 + 0.5 34.33 68.7
9195 Scamp NMJ synaptic transmission, locomotion BL38277 + 0.5 34.5 69.0
14447 Grip Muscle attachment, synapse organization BL28334 + 0.36 34.6 96.1
15669 MESK2 Positive regulation of Ras protein signal transduction BL29380 + 0.4 34.6 86.5
Disc1 + 0.5 34.75 69.5
5166 Atx2 Circadian regulation of translation, phagocytosis, regulation of synapse structural plasticity BL36114 + 0.5 34.75 69.5
44015 Hph Cellular rresponse to hypoxia, positive regulation of cell growth BL34717 + 0.3 34.75 115.8
10047 Syt4 Regulation of synaptic plasticity and neurotransmitter secretion and regulation of vesicle fusion BL39016 + 0.4 34.75 86.9
8442 GluRIA Extracellular-glutamate-gated ion channel activity BL27521 + 0.5 34.75 69.5
10295 Pak Regulation of synapse structure or activity, axogenesis, positive regulation of NMJ synaptic growth BL8804 + 0.5 35 70.0
40300 AGO3 RNA binding BL34815 + 0.5 35 70.0
Disc1 + 0.5 35 70.0
8639 Cirl G-protein coupled receptor signaling pathway, veicle-mediated transport, neurotransmitter secretion BL34821 + 0.5 35 70.0
12737 Crag Calmodulin and RabGTPase binding, basement membrane assembly BL33594 + 0.5 35 70.0
3694 Gγ30A GPCR signaling pathway, regulation of glucose metabolism, phototransduction BL34484 + 0.4 35 87.5
59
Table 2-S3: List of all mutants screened (Cont.)

GluRIIIRNAi-Based Chronic Synaptic Homeostasis Screen
CG # Gene Name Putative function Source PhTx mEPSP EPSP QC
5637 Nos Dendrite morphogenesis, germ cell development BL32985 + 0.4 35 87.5
1976 RhoGAP100F Synaptic vesicle localization, axon extension involved in axon guidance BL32946 + 0.375 35 93.3
10687 AsnRS Asparagine-tRNA aminoacylation BL28317 + 0.5 35 70.0
32540 CCKLR-17D3 G-protein coupled receptor activity, integral membrane component, neuropeptide receptor activity BL28333 + 0.46 35 76.1
1621 Coop Negative regulation of Wnt signaling pathway BL23260 + 0.5 35.167 70.3
7727 Appl BL29862 + 0.5 35.25 70.5
12001 Spartin NMJ synaptic transmission, negative regulation of NMJ synaptic growth BL37499 + 0.5 35.25 70.5
9214 Tob Negative regulation of cell proliferation BL38299 + 0.5 35.25 70.5
14741 and 42321 CG42321 Phospholipid translocation, integral membrane component BL28558 + 0.5 35.25 70.5
8909 Lrp4 Unknown BL31155 + 0.4 35.33 88.3
4698 Wnt4 Wnt signaling pathway, motor neuron axon guidance BL29442 + 0.45 35.33 78.5
3917 Grip84 Microtubule binding, centrosome organization, cell cycle, meiosis BL33548 + 0.3 35.6 118.7
16935 CG16935 Fatty acid metabolic process, microtubule associated complex BL36671 + 0.5 35.66 71.3
3861 Kdn Carbohydrate metabolism, tricarboxylic acid cycle BL36740 + 0.5 35.67 71.3
4589 Letm1 Mitochondrial calcium ion transport, neurotransmitter secretion BL37502 + 0.5 35.75 71.5
8363 Papss ATP binding, imaginal disc-derived wing morphogenesis BL9764 + 0.5 36 72.0
3267 Atg8a Autophagy BL34340 + 0.53 36 67.9
8472 Cam Calcium ion binding, mitotic sindple organization BL34609 + 0.35 36 102.9
42332 Camta Calmodulin-binding, positive regulation of transcription BL40849 + 0.5 36 72.0
12199 Kek5 Regulation of BMP signaling pathway BL40830 + 0.5 36 72.0
10443 Lar Axon guidance, NMJ synaptic growth BL34965 + 0.5 36 72.0
18028 Lt Endocytosis, Notch receptor processing BL34871 + 0.5 36 72.0
34127 Nlg3 Neurogenesis, NMJ junction development, synaptic vesicle endocytosis, phagocytosis BL38264 + 0.5 36 72.0
30483 Prosap Postsynaptic density assembly BL40929 + 0.33 36 109.1
6464 Salm Somatic muscle development BL33714 + 0.375 36 96.0
2621 Sgg Negative regulation of NMJ synaptic growth, habituation, JUN kinase activity regulation BL38293 + 0.5 36 72.0
10823 SIFaR GPCR activity, neuropeptide receptor activity BL34947 + 0.43 36 83.7
43738 Cpo Olfactory behavior, dormancy process, synaptic transmission BL28360 + 0.4 36 90.0
11136 Lrt Negative regulation of muscle cell chemotaxis BL28893 + 0.33 36 109.1
9108 RSG7 Regulation of GPCR protein sinaling pathway, intracellular signal transduction BL28574 + 0.46 36 78.3
10844 Rya-r44F Muscle contraction, calcium ion transmembrane transport BL28919 + 0.43 36 83.7
15899 Ca-α1T Voltage-gated calcium channel activity BL26251 + 0.5 36.25 72.5
30483 Prosap Postsynaptic density assembly BL27284 + 0.5 36.33 72.7
33653 Cadps Synaptic transmission, synaptic vesicle exocytosis BL31984 + 0.4 36.33 90.8
1063 Itp-r83A Cellular calcium ion homeostasis BL25937 + 0.45 36.33 80.7
3985 Syn NMJ synaptic growth, terminal button organization BL27304 + 0.53 36.33 68.5
6713 Nos Calmodulin binding, nitric-oxide synthase activity BL50675 + 0.5 36.4 72.8
4147 Hsc70-3 Centrosome duplication, regulation of glucose metabolism, response to heat BL32402 + 0.4 36.67 91.7
6805 CG686805 Dephosphorylation BL32380 + 0.5 37 74.0
6783 Fabp Fatty acid binding, long-term memory BL33976 + 0.5 37 74.0
9958 Snap Synaptic vesicle priming, synaptic vesicle exocytosis, neurotransmitter secretion BL27541 + 0.4 37 92.5
32809 Srcin1 Unknown BL28822 + 0.4 37 92.5
3168 Sv2a Transmembrane transporter activity BL29301 + 0.33 37 112.1
31118 RabX4 Rab protein signal transduction, vesicle-mediated transport BL23277 + 0.5 37.2 74.4
6827 Nrx-IV Synaptic target recognition, septate junction assembly BL28715 + 0.5 37.25 74.5
33950 Trol Motor neuron axon guidance, defasciculation of motor neurona xons BL38298 + 0.4 37.3 93.3
1725 Dlg1 Positive regulation of NMJ synaptic growth, synaptic transmission, locomotor rhythm BL34854 + 0.4 37.33 93.3
33196 Dpy Extracellular matrix structural constituent, lateral inhibition BL36122 + 0.5 37.33 74.7
43140 Pyd Wing disc development, Notch signaling pathway, chaeta development BL33386 + 0.4 37.33 93.3
3796 Ac Nervous system development BL42953 + 0.5 37.43 74.9
2381 Syt7 Neurotransmitter secretion, synaptic vesicle exocytosis, vesicle-mediated transport BL27279 + 0.5 37.8 75.6
NA Hsap\SNCA synuclein, alpha (non A4 component of amyloid precursor) FBst0008146, Locomotion, neuroanatomy BL8146 + 0.5 38 76.0
32062 Rbfox1 Transcription factor binding BL32476 + 0.45 38 84.4
3624 Babos FBgn0034724 BL36728 + 0.5 38 76.0
6407 Wnt5 Wnt signaling pathway, positive regulation of axon guidance, dendrite guidance BL34644 + 0.4 38 95.0
8732 Acsl Synaptic transmission, negative regulation of synaptic growth at NMJ (Liu et al, 2014) BL27729 + 0.5 38 76.0
7236 Cdkl5 Cyclin-dependent protein serine/threonine kinase activity BL27505 + 0.6 38 63.3
14296 EndoA Regulation of synapse structure or activity, synaptic vesicle endocytosis BL27679 + 0.5 38 76.0
9887 Vglut Neurotransmitter transporter activity, glutamatergic synaptic transmission BL27538 + 0.4 38 95.0
14447 Grip75 Centrosome organization, mitotic nuclear division, spindle organization BL31215 + 0.4 38 95.0
15520 CapaR Neuropeptide receptor binding, Nitric oxide-cGMP-mediated signaling pathway BL28345 + 0.6 38.25 63.8
1495 CaMKI Calmodulin binding, calmodulin-dependent protein kinase activity BL26726 + 0.4 38.3 95.8
7223 Htl CNS developmeent, muscle fiber development, germ cell development BL33808 + 0.4 38.4 96.0
11155 CG11155 Kainate selective glutamate receptor activity, neuron projection morphogenesis BL31991 + 0.3 38.4 128.0
15811 Rop Synaptic transmission, neurotransmitter secretion BL28929 + 0.56 38.6 68.9
5559 Sytα Neurotransmitter secretion, regulation of calcium ion-dependent exocytosis, vesicle-mediated transportBL29308 + 0.4 38.7 96.8
7439 AGO2 Gene silencing, autophagy BL34799 + 0.5 39 78.0
7727 Appl Synapse organization BL39013 + 0.5 39 78.0
60
Table 2-S3: List of all mutants screened (Cont.)

GluRIIIRNAi-Based Chronic Synaptic Homeostasis Screen
CG # Gene Name Putative function Source PhTx mEPSP EPSP QC
13772 Nlg2 Synapse maturation, synapse organization, NMJ synaptic growth, NMJ synaptic transmission BL28331 + 0.4 39 97.5
11324 Homer Positive regulation of circadian sleep/wake, regulation of locomotion BL41908 + 0.4 39.3 98.3
34358 ShakB Gap junction assembly, phototransduction BL27291 + 0.5 39.33 78.7
33094 Synd Positive regulation of NMJ synaptic growth, membrane organization, mitotic cytokinesis BL27297 + 0.5 39.33 78.7
43758 Sli MI03289 BL31468 + 0.46 39.6 86.1
NA alBAsyn Expresses human mutant A53T form of alpha-synuclein under UAS control, N.B BL8148 + 0.5 40 80.0
4843 Tm2 Actin binding, heart development BL41695 + 0.4 40 100.0
11324 Homer Positive regulation of circadian sleep/wake, regulation of locomotion BL27271 + 0.425 40 94.1
7210 Kel Acton cytoskeleton organization, oogenesis BL31251 + 0.46 40 87.0
4059 Ftz-f1 Dendrite morphogenesis, neuron remodeling BL33625 + 0.325 40.25 123.8
2108 Rab23 GTPase activity, mophogenesis of polarized epithelium, BL28025 + 0.5 40.3 80.6
4974 Dally Dendrite morphogenesis, regulation of BMP signaling pathway, motor neuron axon guidance BL33952 + 0.6 40.33 67.2
4839 CG4839 cGMP-dependent protein kinase activity BL27046 + 0.3 40.4 134.7
40494 RhoGAP1A Golgi organization, negative regulation of cell size BL33390 + 0.4 40.66 101.7
4173 42615 GTPase activity, neurogenesis, regulation of glucose metabolic process BL28004 + 0.5 41 82.0
4587 Itih3 Voltage-gated calcium channel activity BL25893 + 0.4 41.3 103.3
44422 CG44422 Calcium ion binding BL26020 + 0.33 41.33 125.2
42683 Kcnip4 Calcium ion binding BL26020 + 0.33 41.33 125.2
1956 Rap1 GTPase activity, regulation of cell shape BL35047 + 0.4 41.67 104.2
11086 Gadd45 JNK cascade, regulation of oviposition BL35023 + 0.5 42 84.0
8434 Lbk Chaeta morphogenesis, oogenesis BL28903 + 0.66 42 63.6
4523 Pink1 Mitochondrion organization, NMJ synaptic transmission BL38262 + 0.4 42 105.0
1634 Nrg Axonogenesis, nerve maturation, synapse organization BL28724 + 0.4 42 105.0
6890 Tollo Regulation of glucose metabolism, axis elongation BL28519 + 0.5 42.5 85.0
2999 Unc-13 Neurotransmitter secretion, synaptic vesicle exocytosis and priming, synaptic transmission BL29548 + 0.63 42.6 67.6
1725 Dlg1 Positive regulation of NMJ synaptic growth, synaptic transmission, locomotor rhythm BL33620 + 0.53 43 81.1
6134 Spz BL34699 + 0.52 43 82.7
3839 L(1)sc Neurogenesis, Positive regulation of transcription BL27058 + 0.45 43.25 96.1
NA SNBS30P Expresses human mutant A30P form of alpha-synuclein under UAS control, N.B BL8147 + 0.5 43.5 87.0
NA aES4rs Expresses a C-terminally Myc-tagged long form of human ACSL4 with a R570S mutation under the control of UAS, Z.W BL32329 + 0.4 43.67 109.2
34402 GluRIA Unknown BL8232 + 0.5 44 88.0
2520 Lap Neuron-neuron synaptic transmission, synaptic vesicle transport, synaptic vesicle endocytosis, receptor-mediated endocytosis, positive regulation of clathrin-mediated endocytosis BL39021 + 0.4 44 110.0
42595 Uex Cellular ion homeostasis BL36116 + 0.4 44 110.0
32434 Siz Myoblast fusion, CNS development, actin cytoskeleton organization BL39060 + 0.43 44.3 103.0
9748 Bel ATP-dependent RNA helicase activity BL28049 + 0.4 45 112.5
11556 Rph Synaptic vesicle endocytosis and exocytosis, vesicle-mediated transport, neurotransmitter secretion BL25950 + 0.5 47.1 94.2
30361 Mtt GPCR activity, dendrite component BL32376 + 0.56 47.66 85.1
42260 BL26723 + 0.4 27.1 67.8
61
Table 2-S4: Absolute and additional values for normalized and presented data.
The figure and panel, genotype, conditions (whether PhTx was applied or not), number of data samples (n), and p-values
from statistical test used are noted. For electrophysiological experiments, average values with standard error values noted
in parentheses are shown for mEPSC, EPSC, QC, and all passive membrane properties (input resistance, leak current).
For morphology data, average intensity values with standard error values noted in parentheses are shown.

Figure Label Genotype PhTx
mEPSC
amplitude
(nA)
EPSC
amplitude
(nA)
QC
mEPSC
frequency
(Hz)
Input
resistance
(MΩ)
Leak
current
(nA)
n
P Value
(significance:
mEPSC amp,
EPSC, QC,
mEPSC freq)
2c,d wild type w
1118
-
-0.416
(0.010)
-47.539
(6.205)
116.894
(15.715)
2.538
(0.297)
19.000
(2.154)
-4.403
(0.483)
17 -
2c,d wild type w
1118
+
-0.190
(0.014)
-40.084
(6.864)
209.751
(30.411)
1.818
(0.100)
11.364
(0.877)
-4.982
(0.669)
11
<0.0001 (****),
0.4359 (ns),
0.0062 (**),
0.0699 (ns)
2c,d inc
kk3
inc
kk3
-
-0.377
(0.018)
-40.591
(3.403)
109.991
(10.179)
1.765
(0.216)
14.188
(2.801)
-3.930
(0.532)
16 -
2c,d inc
kk3
inc
kk3
+
-0.203
(0.014)
-26.661
(4.994)
128.007
(20.924)
0.876
(0.159)
23.700
(3.113)
-1.506
(0.290)
10
<0.0001 (****),
0.0251 (*),
0.3963 (ns),
0.0072 (**)
2d inc
kk3/Df
inc
kk3
/inc
Df
-
-0.327
(0.009)
-42.173
(6.412)
129.429
(20.323)
1.750
(0.159)
6.900
(1.14)
-1.236
(0.319)
10 -
2d inc
kk3/Df
inc
kk3
/inc
Df
+
-0.143
(0.007)
-13.508
(2.437)
98.420
(21.895)
0.478
(0.064)
5.375
(0.263)
-3.603
(0.415)
8
<0.0001 (****),
0.0016 (**),
0.3167 (ns),
<0.0001 (****)
2d inc
kk4
inc
kk4
-
-0.475
(0.013)
-47.175
(4.913)
99.888
(10.597)
1.033
(0.119)
6.929
(0.529)
-2.165
(0.263)
14 -
2d inc
kk4
inc
kk4
+
-0.194
(0.009)
-20.077
(2.272)
108.39
(14.284)
1.689
(0.223)
5.857
(0.275)
-3.148
(0.264)
14
<0.0001 (****),
<0.0001 (****),
0.6366 (ns),
<0.0153 (*)

Figure Label Genotype PhTx
mEPSC
amplitude
(nA)
EPSC
amplitude
(nA)
QC
mEPSC
frequency
(Hz)
Input
resistance
(MΩ)
Leak
current
(nA)
n
P Value
(significance:
mEPSC amp,
EPSC, QC,
mEPSC freq)
2f,g
presynaptic
rescue
inc
kk3
; OK371-
Gal4/ UAS-
smFP-Inc
-
-0.342
(0.021)
-41.817
(2.837)
130.907
(13.521)
2.063
(0.279)
7.125
(0.730)
-2.321
(0.486)
16 -
2f,g
presynaptic
rescue
inc
kk3
; OK371-
Gal4/UAS-
smFP-Inc
+
-0.163
(0.011)
-22.532
(2.420)
148.699
(18.871)
0.884
(0.129)
5.714
(0.450)
-3.051
(0.211)
14
<0.0001 (****),
<0.0001 (****),
0.4418 (ns),
<0.0010 (**)
2f,g
postsynaptic
rescue
inc
kk3
; UAS-
smFP-Inc/+;
MHC-Gal4/+
-
-0.321
(0.023)
-46.367
(6.799)
149.643
(34.958)
3.410
(0.112)
5.750
(0.305)
-2.188
(0.238)
12 -
2f,g
postsynaptic
rescue
inc
kk3
; UAS-
smFP-Inc/+;
MHC-Gal4/+
+
-0.178
(0.010)
-42.524
(4.441)
249.0176
(29.998)
1.669
(0.291)
5.833
(0.405)
-3.342
(0.306)
12
<0.0001 (****),
0.5096 (ns),
0.0071 (**),
0.0137 (*)

62
Figure Label Genotype PhTx
GluRIIA intensity
(% wild type)
GluRIID intensity
(% wild type)
n
P value (significance:
GluRIIA, GluRIID)
3a,b wild type w
1118
- 100.000 (5.262) 100.000 (3.948) 10 -
3a,b inc
kk3
inc
kk3
- 93.777 (6.088) 103.001 (5.910) 13 0.4643 (ns), 0.6968 (ns)
not shown inc
kk4
inc
kk4
- 100.528 (7.178) 111.965 (7.701) 15 0.9576 (ns), 0.2446 (ns)

Figure Label Genotype PhTx
pCaMKII intensity
(% wild type)
BRP intensity
(% wild type)
n
(pCamKII,
BRP)
P value (significance:
pCaMKII, BRP)
3c,d wild type w
1118
- 100.000 (4.585) 100.000 (9.1939) 27, 11 -
3c,d wild type + PhTx w
1118
+ 38.220 (3.565) 149.283 (6.613) 14, 11
<0.0001 (****), <0.0001
(****)
3c,d inc
kk3
inc
kk3
- 115.120 (8.656) 96.930 (10.671) 16, 15 0.2828 (ns), 0.8408 (ns)
3c,d inc
kk3
+ PhTx inc
kk3
+ 53.920 (3.883) 101.273 (13.716) 19, 10 <0.0001 (****), 0.9394 (ns)
not shown inc
kk4
inc
kk4
- 113.300 (5.848) 100.253 (6.882) 17, 11 0.3953 (ns), 0.98193 (ns)
not shown inc
kk4
+ PhTx inc
kk4
+ 55.940 (3.1777) 94.549 (14.093) 18, 10 <0.0001 (****), 0.7399 (ns)

Figure Label Genotype PhTx Inc-smFP intensity (% baseline) n P value (significance)
4a,c inc-smFP inc-smFP - 100.000 (14.639) 10 -
4a,c inc-smFP + PhTx inc-smFP + 205.199 (19.719) 11 0.0005 (***)

Figure Label Genotype PhTx
Ubiquitin
intensity (%
wild type)
Ubiquitin
intensity (%
baseline)
n P value (significance)
4d,e wild type w
1118
- 100.000 (6.245) 100.000 (6.245) 10 -
4d,e wild type + PhTx w
1118
+ 142.158 (8.187) 142.158 (8.187) 10 0.0007 (***)
4d,e inc
kk3
inc
kk3
- 132.900 (7.039) 100.000 (5.297) 12 -
4d,e inc
kk3
+ PhTx inc
kk3
+ 123.596 (5.309) 93.001 (3.995) 10 0.3200 (ns)
not shown inc
kk4
inc
kk4
- 148.920 (10.412) 100.000 (6.992) 13 -
not shown inc
kk4
+ PhTx inc
kk4
+ 147.941 (7.730) 99.343 (5.191) 10 0.9438 (ns)

Figure Label Genotype PhTx
mEPSC
amplitude
(nA)
EPSC
amplitude
(nA)
QC
mEPSC
frequency
(Hz)
Input
Resistance
(MΩ)
Leak
Current
(nA)
n
P Value
(significance:
mEPSC amp,
EPSC, QC,
mEPSC freq)
S1b-f
wild
type
w
1118
-
0.416
(0.011)
47.538
(6.205)
116.894
(15.715)
2.538
(0.297)
19.000
(2.154)
4.403
(0.483)
17 -
S1b-f inc
kk3
inc
kk3
-
0.377
(0.019)
40.591
(3.403)
109.991
(10.179)
1.765
(0.216)
14.188
(0.532)
3.930
(0.532)
16
0.0797 (ns),
0.3422 (ns),
0.7185 (ns),
0.0460 (ns)
S1b-f inc
2
inc
2
-
0.416
(0.013)
42.522
(4.934)
101.760
(11.103)
0.814
(0.198)
10.143
(0.389)
3.516
(0.389)
7
0.9848 (ns),
0.6299 (ns),
0.5628 (ns),
0.0018 (**)
S1b-f inc
2/Df
inc
2
/inc
Df
-
0.394
(0.012)
41.214
(5.634)
103.545
(13.177)
1.599
(0.270)
6.667
(0.450)
2.796
(0.450)
12
0.1840 (ns),
0.4778 (ns),
0.5451 (ns),
0.0474 (ns)
S1b-f inc
2/1
inc
2
/inc
1
-
0.365
(0.009)
25.988
(2.730)
72.830
(8.496)
0.987
(0.167)
8.286
(0.635)
3.534
(0.635)
14
0.0015 (**),
0.0061 (**),
0.0276 (*),
0.0002 (***)

63
Figure Label Genotype Total daily sleep (min) n
P value
(significance)
S1g wild type w
1118
731.18 (25.15) 30 -
S1g inc
2/1
inc
2
/inc
1
501.98 (38.94) 28 p<0.01 (**)
S1g inc
kk3/1
inc
kk3
/inc
1
418.63 (35.93) 25 p<0.01 (**)
S1g inc
kk4/1
inc
kk4
/inc
1
334.18 (63.61) 10 p<0.01 (**)

Figure Label Genotype Total daily sleep (min) n
P value
(significance)
S1h wild type w
1118
996.16 (20.62) 27 -
S1h UAS-smFP-Inc/+ UAS-smFP-Inc/+ 934.51 (20.75 28 ns
S1h inc
1
, inc-Gal4/+ inc
1
, inc-Gal4/+ 480.94 (31.69) 16 p<0.01 (**)
S1h inc
1
, inc-Gal4/UAS-smFP-Inc inc
1
, inc-Gal4/UAS-smFP-Inc 952.90 (16.52) 43 ns

Figure Label Genotype PhTx
mEPSC
amplitude
(nA)
EPSC
amplitude
(nA)
QC
mEPSC
frequency
(Hz)
Input
resistance
(MΩ)
Leak
current
(nA)
n
P Value
(significance:
mEPSC amp,
EPSC, QC,
mEPSC freq)
S2a,b - w
1118
-
-0.416
(0.010)
-47.539
(6.205)
116.894
(15.715)
2.538
(0.297)
19.000
(2.154)
-4.403
(0.483)
17 -
S2a,b GluRIIA GluRIIA
SP16
-
-0.172
(0.009)
-31.717
(2.531)
185.189
(12.437)
0.717
(0.222)
12.272
(0.775)
-3.733
(0.238)
11
<0.0001 (****),
0.0655 (ns),
0.0080 (**),
0.0003 (***)
S2a,b - inc
kk3
-
-0.377
(0.018)
-40.591
(3.403)
109.991
(10.179)
1.765
(0.216)
14.188
(2.801)
-3.930
(0.532)
16 -
S2a,b
inc
kk3
,
GluRIIA
inc
kk3
; GluRIIA
SP16
+
-0.202
(0.011)
-20.0129
(3.003)
106.939
(19.961)
0.923
(0.547)
4.700
(0.456)
-3.530
(0.471)
10
<0.0001 (****),
0.0006 (***),
0.8959 (ns),
0.1672 (ns)
S2a,b - inc
kk3
/inc
Df
-
-0.327
(0.009)
-42.173
(6.412)
129.429
(20.323)
1.750
(0.159)
6.900
(1.14)
-1.236
(0.319)
10 -
S2a,b
inc
kk3/Df
,
GluRIIA
inckk3/incDf;
GluRIIA
SP16

+
-0.180
(0.007)
-21.987
(2.984)
118.852
(13.807)
1.610
(0.359)
6.000
(0.365)
-2.028
(0.393)
15
<0.0001 (****),
0.0042 (**),
0.6589 (ns),
0.7643 (ns)

Figure Label Genotype PhTx
mEPSC
amplitude
(nA)
EPSC
amplitude
(nA)
QC
mEPSC
frequency
(Hz)
Input
resistance
(MΩ)
Leak
current
(nA)
n
P Value
(significance:
mEPSC amp,
EPSC, QC,
mEPSC freq)
S3a-d wild type w
1118
-
0.419
(0.010)
47.656
(5.194)
114.300
(12.286)
2.869
(0.335)
8.125
(0.706)
2.686
(0.312)
16 -
S3a-d
neuronal
> smFP-
Inc
OK371-
Gal4/UAS-smFP-
Inc
-
0.387
(0.007)
44.644
(4.622)
116.406
(12.768)
1.633
(0.275)
5.666
(0.337)
1.917
(0.546)
9
0.038 (ns),
0.708 (ns),
0.915 (ns),
0.022 (*)
S3a-d
muscle >
smFP-Inc
MHC-Gal4/UAS-
smFP-Inc
-
0.397
(0.016)
41.769
(2.760)
107.062
(7.949)
3.610
(0.597)
6.929
(0.486)
2.643
(0.363)
14
0.238 (ns),
0.345 (ns),
0.636 (ns),
0.273 (ns)

64
Figure Label Genotype PhTx
mEPSC
amplitude
(nA)
EPSC
amplitude
(nA)
QC
mEPSC
frequency
(Hz)
Input
resistance
(MΩ)
Leak
current
(nA)
n
P Value
(significance:
mEPSC amp,
EPSC, QC,
mEPSC freq)
S3e,f wild type w
1118
-
0.423
(0.011)
44.171
(3.312)
106.268
(9.882)
2.413
(0.263)
8.100
(0.482)
4.734
(0.905)
10 -
S3e,f
wild type
+ PhTx
w
1118
+
0.179
(0.006)
37.309
(2.348)
210.0126
(14.121)
0.871
(0.101)
7.600
(0.542)
7.14
(0.515)
10
<0.0001 (****),
0.1082 (ns),
<0.0001 (****),
<0.0001 (****)
S3e,f inc-smFP inc-smFP -
0.380
(0.013)
40.186
(4.975)
108.034
(14.523)
0.914
(0.123)
5.818
(0.296)
2.345
(0.352)
11 -
S3e,f
inc-smFP
+ PhTx
inc-smFP +
0.238
(0.014)
48.286
(4.277)
206.972
(19.818)
1.050
(0.176)
5.900
(0.458)
2.917
(0.449)
10
<0.0001 (****),
0.2365 (ns),
0.0006 (***),
0.5271 (ns)

Figure Label Genotype PhTx
mEPSC
amplitude
(nA)
EPSC
amplitude
(nA)
QC
mEPSC
frequency
(Hz)
Input
resistance
(MΩ)
Leak
current
(nA)
n
P Value
(significance:
mEPSC amp,
EPSC, QC,
mEPSC freq)
S4a,b
neuronal
> cul3
RNAi
OK371-Gal4/UAS-
cul3RNAi
11861R
;
UAS-dcr2/+
-
0.342
(0.015)
36.546
(3.014)
107.969
(9.333)
2.363
(0.303)
7.500
(0.732)
1.275
(0.434)
10 -
S4a,b
neuronal
> cul3
RNAi
OK371-Gal4/UAS-
cul3RNAi
11861R
;
UAS-dcr2/+
+
0.176
(0.012)
29.324
(2.873)
165.346
(7.621)
1.144
(0.164)
8.000
(0.906)
2.210
(0.325)
8
<0.0001 (****),
0.1048 (ns),
0.0003 (***),
0.0033 (ns)
S4a,b
muscle >
cul3
RNAi
UAS-
cul3RNAi
11861R
/+;
MHC-Gal4/UAS-
dcr2
-
0.362
(0.012)
44.023
(4.485)
127.653
(16.999)
3.602
(0.696)
6.231
(0.257)
2.082
(0.361)
13 -
S4a,b
muscle >
cul3
RNAi
UAS-
cul3RNAi
11861R
/+;
MHC-Gal4/UAS-
dcr2
+
0.206
(0.012)
23.343
(2.183)
119.502
(13.153)
1.557
(0.153)
6.500
(0.294)
2.763
(0.373)
18
<0.0001 (****),
0.0001 (***),
0.7029 (ns),
0.0024 (**)

65
Chapter 3:
Extended Synaptotagmin localizes to
presynaptic ER and promotes
neurotransmission and synaptic growth
in Drosophila

66
3.1 Abstract
The endoplasmic reticulum (ER) is an extensive organelle in neurons with important
roles at synapses including the regulation of cytosolic Ca
2+
, neurotransmission, lipid
metabolism, and membrane trafficking. Despite intriguing evidence for these crucial
functions, how presynaptic ER influences synaptic physiology remains enigmatic. To
gain insight into this question, we have generated and characterized mutations in the
single Extended Synaptotagmin (Esyt) ortholog in Drosophila melanogaster. Esyts are
evolutionarily conserved ER proteins with Ca
2+
-sensing domains that have recently
been shown to orchestrate membrane tethering and lipid exchange between the ER and
plasma membrane. We first demonstrate that Esyt localizes to presynaptic ER
structures at the neuromuscular junction. Next, we show that synaptic growth, structure,
and homeostatic plasticity are surprisingly unperturbed at synapses lacking Esyt
expression. However, neurotransmission is reduced in Esyt mutants, consistent with a
presynaptic role in promoting neurotransmitter release. Finally, neuronal overexpression
of Esyt enhances synaptic growth and the sustainment of the vesicle pool during
intense activity, suggesting that increased Esyt levels may modulate the membrane
trafficking and/or resting calcium pathways that control synapse extension. Thus, we
identify Esyt as a presynaptic ER protein that can promote neurotransmission and
synaptic growth, revealing the first in vivo neuronal functions of this conserved gene
family.

67
3.2 Introduction
The endoplasmic reticulum (ER) is an essential intracellular organelle present across
metazoans with critical but enigmatic roles at synapses. The importance of synaptic ER
is underscored by its involvement in human disease, including hereditary spastic
paraplegias (Blackstone et al., 2011; Montenegro et al., 2012; Noreau et al., 2014),
amyotrophic lateral sclerosis (Teuling et al., 2007; Yang et al., 2009; Fasana et al.,
2010), and Alzheimer’s disease (Cheung et al., 2008; Zhang et al., 2009; Goussakov et
al., 2010). At presynaptic terminals, the ER has significant roles in both membrane
trafficking and the local regulation of Ca
2+
signaling (Verkhratsky, 2005; Chakroborty et
al., 2009; Renvoise and Blackstone, 2010; Deng et al., 2013; Kwon et al., 2016). In
particular, the ER can modulate constitutive membrane trafficking pathways to the
plasma membrane that are necessary for proper synaptic growth and maintenance
(Pfenninger, 2009; Ramirez and Couve, 2011; Deng et al., 2013; Deshpande and
Rodal, 2016). In addition, presynaptic ER tightly regulates local Ca
2+
dynamics by
orchestrating intracellular Ca
2+
release and sequestration (Bardo et al., 2006;
Chakroborty et al., 2009; Kwon et al., 2016; de Juan-Sanz et al., 2017). However, the
molecules and mechanisms through which presynaptic ER controls synaptic growth and
function remain obscure.
Extended Synaptotagmins (Esyts) are a family of ER-resident proteins that are
attractive candidates to function as modulators of synaptic growth and activity. Esyts are
defined by the presence of a hydrophobic stretch (HS) followed by a Synaptotagmin-like
Mitochondorial lipid binding Protein (SMP) domain and multiple Ca
2+
-binding C2
domains (Min et al., 2007; Giordano et al., 2013). Esyt is evolutionarily conserved from
68
yeast (Tricalbin, Tcb1-3) through mammals (Esyt1-3) (Min et al., 2007; Manford et al.,
2012; Herdman and Moss, 2016), suggesting this gene family performs important
functions that have been selected for and maintained throughout evolution. Studies in
yeast and mammalian cell culture have revealed that Esyts mediate tethering of ER-
plasma membrane (PM) contact sites to facilitate ER-PM lipid transfer (Giordano et al.,
2013; Saheki et al., 2016; Yu et al., 2016; Saheki and De Camilli, 2017), while other
functions for Esyts have also been proposed (Jean et al., 2010; Jean et al., 2012;
Tremblay et al., 2015). Interestingly, Esyt-dependent membrane tethering and lipid
transfer is activated only upon relatively high intracellular concentrations of Ca
2+
, such
as those achieved during store-operated Ca
2+
entry and neurotransmission (Idevall-
Hagren et al., 2015). This raises the intriguing possibility that Esyt may become active in
modulating lipid metabolism during synaptic activity. However, a recent study reported
no apparent changes in synaptic function in mutant mice lacking all three Esyt isoforms
(Sclip et al., 2016), leaving open questions about what functions, if any, Esyts may have
at synapses.
The fruit fly Drosophila melanogaster is a powerful model system to elucidate the
in vivo functions of Esyt. In contrast to mammals, there is a single, highly conserved
Esyt ortholog. Further, the fly neuromuscular junction (NMJ) enables an array of
imaging, electrophysiological, and genetic approaches to illuminate the fundamental
roles of genes at synapses. We have therefore generated and characterized the first
Esyt mutations in Drosophila to test the role of Esyt in synaptic growth, function, and
plasticity. Specifically, we examined synapses lacking and overexpressing Esyt at basal
69
states and under synaptic stress. These studies have defined a role for Esyt at
presynaptic ER in promoting neurotransmission and synaptic growth.

3.3 Results
Generation of null mutations in Drosophila Esyt
The yeast and rodent genomes encode three extended synaptotagmin isoforms (Esyt1,
Esyt2, and Esyt3; (Min et al., 2007; Manford et al., 2012)). In contrast, the Drosophila
genome encodes a single Esyt ortholog, closest and equally similar to the three mouse
Esyts by phylogeny analyses (Figure 1, A and B). RNA-seq data suggests Esyt is
expressed in embryonic stages after 10h through adults in all tissues examined (Daines
et al., 2011; Graveley et al., 2011; Berger et al., 2012), consistent with Esyt being
ubiquitously expressed. We generated a null mutation in the Drosophila Esyt gene,
Esyt
1
, using CRISPR/Cas-9 genome editing technology (Gratz et al., 2013b). Esyt
1

encodes a frameshift mutation, predicted to generate a stop codon at position 32,
truncating the Esyt protein before the hydrophobic stretch (Figure 1, C and D). In
addition, we obtained a separate Esyt mutation (Esyt
2
) containing a MiMIC transposon
insertion in the first coding intron. This transposon has a gene trap cassette (Venken et
al., 2011; Nagarkar-Jaiswal et al., 2015) that is predicted to truncate the Esyt transcript
by introducing a stop codon at the second amino acid (Figure 1, C and D). We
generated a polyclonal antibody against a C-terminal stretch of the Drosophila Esyt
protein, which recognized an immunoblot band at ~90 kDa (Figure 1, E and F),
consistent with the predicted molecular mass of Esyt. Using this antibody, we confirmed
that Esyt is expressed in the adult brain and larval CNS, and that Esyt
1
and Esyt
2
are
70
protein null mutations by immunoblot analysis (Figure 1, E and F). These Esyt mutants
are viable and fertile. Finally, we generated a series of transgenic Esyt constructs under
UAS control (Figure 1D). We engineered a full length Esyt transgene tagged with both
mCherry and 3xFlag tags (Esyt
mCh
), and a separate transgene tagged with only a
3xFlag tag (Esyt
Flag
). We also generated an Esyt transgene without the conserved
hydrophobic stretch (Esyt
ΔHS
), predicted to disrupt membrane targeting of Esyt
(Giordano et al., 2013)), and specifically mutated each C2 domain to render them
unable to bind to Ca
2+
(Esyt
D-N
; see methods). Using these reagents, we went on to
determine the presynaptic localization of Esyt.

The hydrophobic stretch anchors Esyt to axonal ER
In neurons, the ER is an extensive network present in the somatic perinucleus as well
as in distal dendrites and axons (Summerville et al., 2016; de Juan-Sanz et al., 2017).
Given that ER proteins can have uniform or heterogeneous localization in the elaborate
ER network (Chang and Liou, 2016), we sought to determine the subcellular localization
of Drosophila Esyt at presynaptic terminals. We were unable to obtain specific
immunolabeling against endogenous Esyt using the antibody we generated. We also
attempted to generate an endogenously tagged Esyt using RMCE with the MiMIC
insertion in the Esyt locus, but this effort was unsuccessful. Therefore, we expressed
tagged Esyt constructs in motor neurons. Esyt
mCh
trafficked to presynaptic terminals,
where it co-localized with an established ER marker, the ER retention signal KDEL
fused to GFP (GFP-KDEL; Figure 2A; (Okajima et al., 2008)).
71
Next, we tested whether Esyt localization and trafficking to ER was dependent on
the HS domain and on Ca
2+
binding to the C2 domains. Previous studies have shown
that the HS domain tethers Esyt to the ER, while deletion of the HS domain shifts Esyt
localization to the cytosol and plasma membrane (Min et al., 2007; Giordano et al.,
2013). We therefore expressed the mCherry-tagged Esyt construct lacking the HS
domain in neurons (Esyt
∆HS
; see Figure 1D and methods). As expected, the Esyt
∆HS

signal was no longer restricted to the axonal ER, and instead filled the presynaptic
terminal, indicating a shift to cytosolic localization (Figure 2B). We then tested the
requirement of Ca
2+
binding for Esyt trafficking and localization by expressing Esyt
D-N
,
which lacks the negatively charged amino acids in each C2 domain required for Ca
2+

binding. Interestingly, we were unable to detect any Esyt
D-N
signal at synaptic terminals
(Figure 2, C and D). Instead, most of the Esyt
D-N
signal was restricted to the cell body
(data not shown). This indicates that trafficking of Esyt to synaptic terminals requires the
ability to bind Ca
2+
, although we cannot exclude the possibility that the D-N mutations
might have resulted in misfolding of the protein, potentially disrupting trafficking or
stability. We also found that expression of Esyt
D-N
led to embryonic lethality when driven
pan-neuronally or in muscle. This is not unexpected, as others have observed that
Synaptotagmin expression with similar mutations to C2 domains acquired a lethal
toxicity (Mackler et al., 2002). Together, these data demonstrate that Drosophila Esyt is
localized to presynaptic ER structures through the HS domain and that Ca
2+
binding to
Esyt appears to be required for Esyt trafficking in neurons.
Given that Esyt localizes to axonal ER, we examined the morphology of the ER
network at synaptic terminals with loss or overexpression of Esyt. Expression of GFP-
72
KDEL alone in motor neurons labeled an extensive presynaptic network throughout
boutons, as observed by others (data not shown; (Summerville et al., 2016)). This
network did not appear to be perturbed in Esyt mutants, nor with overexpression (data
not shown). These results suggest that ER structure is not dependent on Esyt
expression. Lastly, we found that the ER network labeled by Esyt is localized near, but
distinct from, other synaptic compartments including active zones, synaptic vesicle
pools, periactive zone regions, and the neuronal plasma membrane (Figure 2, E and F).
Thus, Esyt is localized to the presynaptic ER network and present near areas of
synaptic vesicle fusion and recycling at presynaptic terminals where it could, in
principle, modulate synaptic structure and function.

Synaptic phospholipid balance does not require Esyt
Given that Esyt has been implicated in phospholipid transfer and homeostasis in non-
neuronal cells, we next sought to determine whether the level and distribution of
phospholipids at presynaptic terminals was altered in Esyt mutants and/or with
overexpression of Esyt in motor neurons (Esyt-OE). The phospholipid
phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) plays crucial roles at presynaptic
terminals, regulating synaptic protein-protein interactions, ion channel biophysics,
neurotransmission, and synaptic vesicle trafficking (Ueda, 2014; Lauwers et al., 2016).
To measure synaptic levels of PI(4,5)P2, we expressed a fluorescently tagged pleckstrin
homology domain of phospholipase C-δ1 (PLCδ1-PH-GFP) that specifically labels
PI(4,5)P2 (Verstreken et al., 2009). Expression of this transgene in motor neurons
revealed specific labeling of the plasma membrane at presynaptic terminals, consistent
73
with the expected distribution of PI(4,5)P2 (Figure 3A; (Chen et al., 2014)). When
PLCδ1-PH-GFP was expressed in either Esyt mutants or Esyt-OE animals, we were
unable to detect any difference in GFP intensity or distribution (Figure 3, A and B).
Thus, we find no evidence that PI(4,5)P2 levels or distribution is altered at synapses
with gain or loss of Esyt expression.
In addition to the plasma membrane, the ER also associates with early and late
endosomal structures, contributing to synaptic growth and vesicle trafficking (Rowland
et al., 2014; Raiborg et al., 2015). Thus, we considered the possibility that Esyt may
regulate lipid transfer or otherwise influence endosomes at synapses. We focused on
early endosomes known to be involved in synaptic vesicle trafficking and recycling.
These early endosomes are specifically labeled by the small GTPase Rab5 and are
enriched in the phospholipid phosphatidylinositol-3-phosphate (PI(3)P) (Wucherpfennig
et al., 2003). The FYVE finger domain of the Rab5 effectors EEA1 and Rabenosyin-5
bind specifically to PI(3)P, which is an established marker for early endosomes
(Wucherpfennig et al., 2003). To test if PI(3)P levels and/or distribution are dependent
on Esyt expression, we expressed a GFP-fused FYVE domain transgene (GPF-myc-
2xFYVE) in Esyt mutants and Esyt-OE. GFP-myc-2xFYVE expression in controls
labeled punctate structures in presynaptic boutons, as expected (Figure 3C). However,
we observed no differences in the intensity of GFP-myc-2xFYVE in Esyt mutants and
Esyt-OE compared to controls (Figure 3, C and D). Thus, we find no evidence that Esyt
is involved in phospholipid balance, transfer, or distribution at presynaptic terminals.

74
Presynaptic overexpression of Esyt promotes synaptic growth
Axonal ER plays a critical role in synaptic growth and neurotransmission in mammals
and Drosophila (Wong et al., 2014; Summerville et al., 2016; de Juan-Sanz et al.,
2017). We therefore sought to determine to what extent Esyt expression contributes to
synaptic development and neurotransmission. First, we quantified synaptic growth by
immunostaining NMJs with antibodies that recognize neuronal membrane (HRP), active
zones (BRP), and postsynaptic glutamate receptors (DGluRIII). Esyt mutants exhibited
no significant differences in the number of synaptic boutons, nor in the number or
density of active zones or glutamate receptor clusters at the NMJ (Figure 4, A-E).
However, overexpression of the Esyt
Flag
transgene in motor neurons revealed a ~40%
increase in synaptic growth, including increased neuronal membrane surface area and
total number of active zones per NMJ (Figure 4, A-D), and similar results were observed
when Esyt
mCh
was overexpressed (Table S1). Thus, while loss of Esyt has no apparent
impact on synaptic growth or structure, elevated levels of Esyt in motor neurons
promotes synaptic growth at the NMJ.

Esyt has a role in facilitating presynaptic neurotransmitter release
Axonal ER is known to modulate presynaptic function (Wong et al., 2014; de Juan-Sanz
et al., 2017), and we next assessed the impact on neurotransmission with loss or gain
of Esyt expression. We recorded mEPSCs and EPSCs in low (0.4 mM) and high (3 mM)
extracellular Ca
2+
concentrations using a two-electrode voltage clamp configuration. We
found no significant difference in mEPSC frequency or amplitude in Esyt mutants
(Figure 5, A and B; Table S1), consistent with no changes in the number of active zones
or postsynaptic receptor clusters (Figure 4D). However, EPSC amplitude was reduced
75
in Esyt mutants by over 50% at low extracellular Ca
2+
, with a concomitant decrease in
quantal content compared with wild type (Figure 5, A-D). This reduction in EPSC
amplitude and quantal content in Esyt mutants was also observed in high extracellular
Ca
2+
conditions, and rescued by expression of Esyt in motor neurons (Figure 5D and
Table S1), demonstrating that presynaptic Esyt is necessary to promote
neurotransmitter release. Indeed, there was an apparent shift in the Ca
2+
sensitivity, but
not cooperativity, of neurotransmission between wild type and Esyt mutants, when
quantal content was assessed across a range of lowered extracellular Ca
2+

concentrations (Figure 5D and Table S1). Thus, while Esyt mutants have no obvious
defects in synaptic growth, Esyt is required for proper neurotransmission across a range
of extracellular Ca
2+
concentrations.
In contrast, we observed no significant differences in synaptic physiology at both
low and elevated extracellular Ca
2+
levels in Esyt-OE animals compared with controls
(Figure 5, A-D, and Table S1). Given the enhanced synaptic growth and active zone
number in Esyt-OE, one may have expected a similarly enhanced degree of EPSC
amplitude and quantal content, assuming no changes in release probability per active
zone. This stable level of synaptic strength in Esyt-OE implies a reduction in release
probability per active zone in Esyt-OE, an apparent homeostatic adaption (Davis and
Muller 2015).
Finally, we probed short term synaptic plasticity in Esyt mutants and Esyt-OE by
evoking four stimuli at 60 Hz in low and high extracellular Ca
2+
. Given that Esyt is a
putative Ca
2+
sensor localized to axonal ER, this protocol would test a role for Esyt
during rapid changes in presynaptic Ca
2+
levels (Muller et al., 2011; Genc et al., 2017).
76
At 0.4 mM extracellular Ca
2+
, both wild type and Esyt-OE animals showed moderate
facilitation, with EPSC amplitudes finishing at ~150% of the starting EPSC amplitude
(Figure 5, E and F). In contrast, Esyt mutants showed enhanced facilitation, with the last
EPSC finishing at ~270% compared to the initial EPSC (Figure 5, E and F), consistent
with reduced release probability in this condition. Similarly, at 3 mM extracellular Ca
2+
,
wild type and Esyt-OE NMJs exhibited synaptic depression, with the fourth EPSC
finishing at ~60% of the starting EPSC amplitude (Figure 5, G and H). Consistent with
reduced release probability in this condition, Esyt mutants showed reduced depression,
finishing at ~90% of the starting EPSC amplitude, which was rescued by presynaptic
Esyt expression (Figure 5, G and H). Together, this data is consistent with a function for
Esyt at axonal ER in promoting synaptic vesicle release during evoked activity.

Esyt-OE synapses resist depletion during high frequency stimulation
Axonal ER is involved in calcium homeostasis at presynaptic terminals (Wong et al.,
2014), and we considered that during sustained levels of high activity, a role for Esyt in
potentially regulating this process may be revealed. We therefore assessed synaptic
transmission during elevated periods of activity in Esyt mutants and Esyt-OE. During
high frequency synaptic stimulation, endocytosis rates must be increased to sustain the
elevated level of exocytosis, and any imbalance in this coupling will deplete the
functional vesicle pool (Haucke et al., 2011). We utilized a previously established
protocol, in which we subject the fly NMJ to 10 Hz stimulation at elevated extracellular
Ca
2+
for 10 min, followed by recovery for an additional 10 min, taking a test pulse at 0.2
Hz (Verstreken et al., 2002; Dickman et al., 2005). This analysis revealed that wild-type
77
and Esyt-mutant synapses exhibited a similar rate of vesicle pool depletion and
recovery, finishing at ~30% of the original EPSP amplitude, followed by a recovery to
~60% of the initial value (Figure 6A). In both genotypes, a similar number of total quanta
was released (Figure 6B). In contrast, Esyt-OE conferred a resistance to depletion of
the functional synaptic vesicle pool as well as enhanced recovery. 10 Hz stimulation of
Esyt-OE NMJs revealed a slower rate of rundown and faster recovery of the depleted
vesicle pool (Figure 6A), finishing at ~60% and recovering to ~90% of starting EPSP
amplitudes. Indeed, more total quanta were released in Esyt-OE compared to both wild
type and Esyt mutants during this sustained period of activity (Figure 6B). Together, this
demonstrates that while the loss of Esyt does not significantly impact synaptic growth,
structure, or vesicle recycling, overexpression of Esyt in neurons promotes synaptic
growth which, while not affecting baseline transmission, appears to sustain the vesicle
pool during prolonged activity.
The slowed rate of synaptic vesicle pool depletion in Esyt-OE could, in principle,
be due to an increase in the number of synaptic vesicles participating in exocytosis and
recycling at individual boutons. Alternatively, synaptic vesicle recycling at each bouton
may be the same, and the increased maintenance of the vesicle pool may be due to
reduced release probability per bouton coupled with the increased number of boutons in
Esyt-OE, in effect serving as a reservoir of additional vesicles available for release. We
therefore examined NMJ ultrastructure to determine whether a change in the density of
synaptic vesicles in each bouton was apparent that may suggest an increased starting
vesicle pool in Esyt-OE. We did not observe any significant change in the density or
distribution of synaptic vesicles within NMJ boutons or near active zones in Esyt-OE
78
compared with wild type and Esyt mutants (Figure 7, A-C). More generally, active zone
length, T-bar morphology, and membrane compartments appear similar in all three
genotypes (Figure 7, A-E). Thus, there is no evidence that Esyt-OE results in increased
numbers or altered distribution of synaptic vesicles within NMJ boutons.
Despite there being no change in the number of synaptic vesicles per bouton in
Esyt-OE NMJs, it is possible that more synaptic vesicles participate in exo-endocytosis
during activity which, in turn, could account for the increased maintenance of the
functional vesicle pool. We therefore measured the pool of vesicles participating in
endocytosis during high activity using the lipophilic dye FM1-43, which is absorbed by
newly endocytosed synaptic vesicles from the plasma membrane following exocytosis
and is a measure of the number of vesicles participating in release at each bouton
(Dickman et al., 2005; Verstreken et al., 2008; Chen et al., 2014)). Following
stimulation, we observed a trend of reduced intensity of the vesicle pool labeled by
FM1-43 in Esyt-OE compared to wild type and Esyt mutants (Figure 7, F and G),
perhaps suggesting a reduction in the number of vesicles participating in exocytosis per
bouton in Esyt-OE, consistent with reduced release probability per bouton. Thus, the
increase in the total number of synaptic boutons, perhaps coupled with less vesicle
release per bouton, likely accounts for the resistance to depletion of the vesicle pool in
Esyt-OE during elevated activity.

Esyt is not required for presynaptic homeostatic potentiation
Thus far, we have found that Esyt has no apparent role in synaptic growth and
structure, but is required to promote synaptic vesicle release across a range of
79
extracellular Ca
2+
concentrations. Interestingly, elevated Ca
2+
influx at presynaptic
terminals at the Drosophila NMJ has been demonstrated to drive an adaptive form of
synaptic plasticity referred to presynaptic homeostatic potentiation (PHP) (Muller and
Davis, 2012; Davis and Muller, 2015). At this synapse, pharmacological or genetic
perturbations to postsynaptic glutamate receptors triggers a retrograde signal resulting
in a precise increase in presynaptic neurotransmitter release that compensates for
reduced receptor functionality, restoring synaptic strength to baseline levels (Frank,
2014). Intriguingly, a recent study identified a multiple C2 domain protein, called MCTP,
to be anchored to axonal ER and necessary for PHP at the Drosophila NMJ (Genc et
al., 2017). MCTP is structurally similar to Esyt, and we hypothesized that Esyt may also
be an axonal ER C2 domain protein that promotes vesicle release in response to
homeostatic signaling. Application of the glutamate receptor antagonist philanthotoxin-
433 (PhTx; (Frank et al., 2006)) to wild-type NMJs led to the expected ~50% reduction
in mEPSP amplitude but normal EPSP amplitude because of a homeostatic increase in
quantal content (presynaptic release) (Figure 8, A and B). Similarly, PhTx reduced
mEPSP amplitudes in both Esyt mutants and Esyt-OE, and both genotypes exhibited a
robust increase in quantal content (Figure 8, A and B). Thus, loss or increased
expression of Esyt has no consequence for the acute induction or expression of PHP.

3.4 Discussion
We have generated the first mutations in the single Drosophila Esyt ortholog and
characterized the presynaptic localization and functions of this gene at the NMJ. We
demonstrate that Drosophila Esyt is localized to an extensive axonal ER network.
80
Although Esyt was previously shown to mediate ER-PM tethering and to promote lipid
exchange between the two membranes in non-neuronal cells, we find no evidence that
lipid balance is altered at Esyt mutant synapses. Although there is no apparent change
in synaptic growth or structure in Esyt mutants, we find that Esyt is required to facilitate
presynaptic release across a range of extracellular Ca
2+
. Interestingly, presynaptic
overexpression of Esyt promotes synaptic growth and, in turn, resistance to synaptic
depression during elevated activity. Together, our study establishes Esyt as a
conserved ER-localized protein that regulates neurotransmission and synaptic growth
when overexpressed.

Esyt localizes to axonal ER and promotes neurotransmission
Given the high conservation of the Esyt family throughout evolution and its function in
Ca
2+
-dependent lipid transfer, Esyt was an attractive candidate to play a role in
modulating lipid metabolism at synapses. Accordingly, we find that Esyt is localized to
the axonal ER. However, we find that Esyt is not required to maintain lipid homeostasis
at synapses, at least for the major phospholipids PI(4,5)P2 and PI(3)P. This
demonstrates that lipid balance and membrane homeostasis can be maintained during
the extreme demands of regulated membrane trafficking and exchange at presynaptic
terminals in the absence of Esyt. In retrospect, this may not be surprising, as a lipid
cycle nested within the synaptic vesicle cycle has long been known to exist at synapses,
supported by key synaptic proteins such as Synaptojanin, Rab5, Minibrain
kinase/Dyrk1A, and Sac1 (De Camilli et al., 1996; Nemoto et al., 2000; Wenk and De
Camilli, 2004; Chen et al., 2014). Importantly, there is no known involvement or
81
requirement for acute lipid transfer from the ER in synaptic vesicle recycling (De Camilli
et al., 1996; Wenk and De Camilli, 2004). Accordingly, lipid homeostasis during synaptic
vesicle trafficking, like protein homeostasis, may be sufficiently embedded and coupled
in membrane trafficking itself so as not to lead to imbalances, even during rapid
synaptic vesicle exo- and endo-cytosis.
Despite no apparent changes in synaptic lipid metabolism in Esyt mutants, these
animals demonstrated a significant reduction in EPSC amplitude across a range of
extracellular Ca
2+
conditions. This finding suggests that Esyt may promote presynaptic
function by coupling local Ca
2+
dynamics to axonal ER Ca
2+
release. Indeed, axonal ER
has emerged as a crucial organelle that can sense and dynamically respond to changes
in cytosolic Ca
2+
to modulate presynaptic function and plasticity (Verkhratsky, 2005;
Bardo et al., 2006; Kwon et al., 2016; de Juan-Sanz et al., 2017). For example, Ca
2+

influx from the extracellular space can induce additional Ca
2+
release from the ER via
ryanodine receptors, a process referred as Ca
2+
-induced Ca
2+
-release (CICR), which
can be activated during single action potentials or trains of stimuli (Verkhratsky, 2005;
Bardo et al., 2006; Kwon et al., 2016; de Juan-Sanz et al., 2017). An intriguing
possibility is that Esyt may work in conjunction with ryanodine receptors as Ca
2+

sensors that promotes CICR at the presynaptic terminal in response to activity. In this
model, Esyt may respond to elevated Ca
2+
at synapses during single action potentials,
leading to a supplemental source of presynaptic Ca
2+
, perhaps through release of
intracellular ER stores (Scott et al., 2008).

82
Esyt, axonal ER, and synaptic growth
Perhaps the most striking and unexpected finding was that elevated expression of Esyt
in motor neurons led to increased synaptic growth and enhanced resistance to synaptic
depression. To our knowledge, genes encoding proteins with multiple C2 domains and a
hydrophobic stretch, such as Esyt, MCTP, and Ferlins, have not been associated with
synaptic growth (Lek et al., 2012; Genc et al., 2017). We consider two possible
mechanisms for how overexpression of Esyt may promote synaptic growth. First, Esyt
may modulate intracellular Ca
2+
levels at synapses, which have been shown to play a
role in regulating synaptic growth at the Drosophila NMJ. Indeed, the TRPV channel
Inactive (Iav) is present on axonal ER and modulates synaptic growth by regulating
Ca
2+
release from ER stores, which signals through calcineurin to stabilize microtubules
(Wong et al., 2014). Although loss of Esyt does not appear to impact synaptic growth,
further studies will be necessary to determine whether elevated levels of Esyt alters ER
Ca
2+
release or resting Ca
2+
levels, perhaps by interacting with factors such as Inactive.
Another attractive possibility is that elevated Esyt expression at axonal ER may
promote increased membrane trafficking from the ER to the plasma membrane at
synapses through the constitutive pathway. Indeed, the membrane necessary for
synaptic growth is delivered through this pathway, as synapses are established and
continue to grow and elaborate even when toxins are expressed that block or inhibit
synaptic vesicle fusion (Broadie et al., 1995; Sweeney et al., 1995; Dickman et al.,
2006; Choi et al., 2014). Facilitated delivery of synaptic proteins and membrane to the
presynaptic terminal, in turn, may lead to excess membrane and fuel increased synaptic
growth. Further, inhibition of axonal ER export during early developmental stages
results in defective axon growth in mouse hippocampal neurons (Aridor and Fish, 2009).
83
Thus, the unanticipated finding that increased Esyt expression promotes synaptic
growth raises the intriguing possibility that synaptic proteins and membrane derived
from axonal ER may be a rate-limiting step in driving synapse expansion.
We have established Esyt as an axonal ER protein that promotes presynaptic function
and that may also have unanticipated roles in regulating synaptic growth. Future studies
using approaches such as Ca
2+
imaging will be necessary to elucidate the molecular
mechanisms of how Esyt modulates axonal ER activity to ultimately promote
neurotransmission. Further genetic experiments will likely reveal additional insights into
how excess Esyt levels at synapses enhances synaptic growth, perhaps through
interactions with other axonal ER proteins that control Ca
2+
release from ER stores,
such as Inactive. Although axonal ER was first observed over 40 years ago (Tsukita and
Ishikawa, 1976; Ramirez and Couve, 2011), the functions of this complex organelle
have remained enigmatic. Recent studies have begun to reveal how axonal ER sculpts
presynaptic Ca
2+
dynamics and modulates presynaptic function and plasticity (de Juan-
Sanz et al., 2017), roles that seem certain to contribute to a variety of neurological
diseases.

3.5 Materials and Methods
Fly stocks
All Drosophila stocks were raised at 25° on standard molasses food. The w
1118
strain
was used as the wild-type control unless otherwise noted, as this was the genetic
background into which all genotypes and transgenic lines were crossed. The Drosophila
stocks used in this study were following: OK6-Gal4 (Aberle et al., 2002), BG57-Gal4
84
(Budnik et al., 1996), UAS-GFP-KDEL (Dong et al., 2013; Nandi et al., 2014), UAS-
GFP-myc-2xFYVE (Wucherpfennig et al., 2003), UAS-PLCδ1-PH-GFP (Verstreken et
al., 2009; Khuong et al., 2010). The Esyt
2
mutant (Mi{ET1}Esyt2
MB029221
), the deficiency
(Df(3R)Exel7357), and all other stocks were obtained from the Bloomington Drosophila
Stock Center unless otherwise noted. Female larvae were used unless otherwise
specified. Phylogenetic analysis was performed using PhyML 3.0 software (Guindon et
al., 2010), and visualized using FigTree software
(http://tree.bio.ed.ac.uk/software/figtree/).

Molecular biology
There are four predicted Esyt isoforms in Drosophila based on expressed sequence
tags (www.flybase.org). However, one transcript, Esyt2-RB, appears to be the major
isoform based on expression profiling. Therefore, we generated transgenic Esyt
constructs using the Esyt2-RB transcript (RE26910; BDGP). Full-length Esyt cDNA was
PCR amplified and subcloned into the pACU2 vector (Han et al., 2011b). An mCherry or
3xFlag tag was inserted in-frame into the C-terminal end of the pACU2-Esyt construct.
To generate Esyt
∆HS
, the Drosophila Esyt hydrophobic stretch was identified by the
SMART domain online tool (http://smart.embl-heidelberg.de/). Esyt coding DNA before
and after the identified hydrophobic stretch were separately PCR amplified and ligated
into the pACU2-mCherry vector using the Gibson Assembly Cloning Kit (New England
Biolabs Inc., E5510S). Finally, to generate pACU2-Esyt
D-N
, the conserved aspartates in
each C2 domain were identified and mutated into asparagine (D364N, D374N, D421N,
D423N, E429Q for C2A; D517N, D564N for C2B; D746N, D752N for C2C). All constructs
85
were inserted into the VK18 recombination site on the second chromosome by
BestGene, Inc (Chino Hill, CA).
Esyt
1
mutants were generated using a CRISPR/Cas9 genome editing strategy as
described (Gratz et al., 2013b). Briefly, a target Cas-9 cleavage site in Esyt was chosen
in earliest target of the first common exon shared by all putative Esyt isoforms without
obvious off target sequences in the Drosophila genome (sgRNA target sequence:
5’GACAAATGGAAACTCAATTGTGG3’, PAM underscored). DNA sequences covering
this target sequence were synthesized and subcloned into the pU6-BbsI-chiRNA
plasmid (Addgene 45946). To generate the sgRNA, pU6-BbsI-chiRNA was PCR
amplified and cloned into the pattB vector (Bischof et al., 2007). This construct was
injected and inserted into the VK18 target sequence and balanced. Screening of 20
lines with active CRISPR mutagenesis led to 17 independent deletions or insertions with
predicted frameshift mutations in the Esyt open reading frame. The line which produced
the earliest stop codon (K32stop) was chosen for further analyses and named the Esyt
1

allele.

Immunohistochemistry
Wandering third-instar larvae were dissected in ice cold 0 Ca
2+
modified HL3 saline
(Stewart et al., 1994; Dickman et al., 2005) containing (in mM): 70 NaCl, 5 KCl, 10
MgCl2, 10 NaHCO3, 115 Sucrose, 5 Trehelose, 5 HEPES, pH 7.2, and immunostained
as described (Chen et al., 2017). Briefly, larvae were washed three times with modified
HL3 saline, and fixed in either Bouin’s fixative (Sigma, HT10132-1L) or 4%
paraformaldehyde in PBS (Sigma, F8775). Larvae were washed with PBS containing
86
0.1% Triton X-100 (PBST) and incubated in primary antibodies at 4° overnight. The
larvae were then washed in PBST and incubated in secondary antibodies at room
temperature for two hours. Samples were transferred in VectaShield (Vector
Laboratories) and mounted on glass cover slides. The following antibodies were used:
mouse anti-Bruchpilot (BRP; nc82; 1:100; Developmental Studies Hybridoma Bank;
DSHB); affinity-purified rabbit anti-GluRIII (1:2000; (Marrus et al., 2004; Chen et al.,
2017))), mouse anti-Flag (1:500; F1804; Sigma-Aldrich), guinea pig anti-vGlut (1:2000;
(Chen et al., 2017))), mouse anti-FasII (1:20; 1D4; DSHB), mouse anti-GFP (1:1000;
3e6; Invitrogen). Alexa Fluor 647-conjugated goat anti-HRP (Jackson
ImmunoResearch) was used at 1:200. Donkey anti-mouse, anti-rabbit, and anti-guinea
pig Alexa Fluor 488-, Cy3, and Rhodamine Red X secondary antibodies (Jackson
ImmunoResearch) were used at 1:400.

Confocal imaging and analysis
Larval muscle 4 of abdominal segments A2 and A3 were imaged on a Nikon A1R
resonant scanning confocal microscope using a 100x APO 1.4NA oil immersion
objective with NIS Elements software as described (Chen et al., 2017). The
fluorescence signals were excited by separate channels with laser lines of 488 nm, 561
nm, and 637 nm. Images were acquired using identical settings optimized for signal
detection without saturation for all genotypes within an experiment. The general
analysis toolkit in the NIS Elements software was used to quantify bouton number, BRP
and GluRIII puncta number, and density by applying intensity thresholds on each of the
three channels. For live imaging of Esyt
mCh
, third-instar larvae were dissected, washed,
87
and incubated in Alexa Fluor 647-conjugated goat anti-HRP in 0 Ca
2+
modified HL3 at
1:200 for 5 min. The samples were then washed in 0 Ca
2+
saline and mounted on glass
cover slides. Images were acquired and analyzed as described above.
FM1-43 experiments were performed as described (Dickman et al., 2005).
Briefly, larvae were dissected in ice-cold 0 Ca
2+
modified HL3 and washed, then
stimulated for 10 min with a modified HL3 solution containing 90 mM KCl and 10 µM
FM1-43 (Molecular Probes, Eugene, Oregon). Larvae were then washed in 0 Ca
2+

saline before imaging. Images were acquired using a Nikon A1R confocal microscope
using a 60x APO 1.0NA water immersion objective and imaged as described above.
The general analysis toolkit in the NIS Elements software was used to quantify the
mean intensity by applying intensity thresholds.

Western blotting
Third-instar larval CNS extracts (50 animals of each genotype) and adult heads (7 of
each genotype) were homogenized in ice cold lysis buffer (10 mM HEPES + 150 mM
NaCl, pH 7.4), mixed with an EDTA-free protease inhibitor cocktail (Roche), and run on
4-12% Bis Tris Plus gels (Invitrogen). After blotting onto PVDF membrane (Novex) and
incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5%
Tween 20) for 60 min, the membrane was washed once with TBST and incubated with
anti-Esyt (1:2000) and anti-actin (1:2000; JLA20, DSHB) antibodies overnight at 4°C.
Membranes were washed and incubated with a 1:5000 dilution of horseradish
peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) for 1 hr. Blots
were washed with TBST and developed with the ECL Plus Western Blotting system
88
(HyGLO). To generate Esyt polyclonal antibodies, a peptide antigen was synthesized
consisting of amino acids 799-816 of Esyt (CTQTGLNSWFDLQPEIRHE). This peptide
was conjugated to KLH and injected into two rabbits (Cocalico Inc, Pennsylvania). The
rabbit immunosera was affinity purified and used at 1:2000.

Electron microscopy
EM analysis was performed as described (Atwood et al., 1993). Wandering third-instar
larvae were dissected in Ca
2+
-free HL3, then fixed in 2.5% glutaraldehyde/0.1M
cacodylate buffer at 4°. Larvae were then washed in 0.1M cacodylate buffer. The whole
mount body wall musculature was placed in 1% osmium tetroxide/0.1M cacodylate
buffer at room temperature for 1hr. After washing, larvae were then dehydrated in
ethanol. Samples were cleared in propylene oxide and infiltrated with 50% Eponate 12
in propylene oxide overnight. The following day, samples were embedded in fresh
Eponate 12. Electron micrographs were obtained on a Morgagni 268 transmission
electron microscope (FEI, Hillsboro, OR). The junctional region was serially sectioned at
a thickness of 60-70 nm. Sections were stained in 2% uranyl acetate for 3 minutes,
washed briefly 3x in distilled water, stained in Reynolds lead citrate for 1 minute,
washed briefly 3x in distilled water, and dried. Sections were mounted on Formvar
coated single slot grids. Larval muscles 6/7 of abdominal segments were viewed at
23,000x magnification and acquired with a Megaview II CCD camera. Images were
analyzed blind to genotype using the general analysis toolkit in the NIS Elements
software.

89
Electrophysiology
All dissections and recordings were performed in modified HL3 saline at room
temperature with 0.4 mM CaCl2 (unless otherwise specified). Sharp electrode two-
electrode voltage clamp recordings (electrode resistance between 10-35 MΩ) were
performed on muscles 6 of abdominal segments A2 and A3 as described (Kiragasi et
al., 2017). Briefly, recordings were acquired using an Axoclamp 900A amplifier, Digidata
1440A acquisition system and pClamp 10.5 software (Molecular Devices).
Electrophysiological sweeps were digitized at 10 kHz, and filtered at 1 kHz. Miniature
excitatory postsynaptic currents (mEPSCs) were recorded for one min in the absence of
any stimulation. Excitatory postsynaptic currents (EPSCs) were recorded while cut
motor axons were stimulated with 0.5 msec duration using an ISO-Flex stimulus isolator
(A.M.P.I.). Intensity was adjusted for each cell, set to consistently elicit responses from
both neurons innervating the muscle segment, but avoiding overstimulation. In all
voltage clamp recordings, muscles were clamped at -70 mV. Recordings were rejected
when muscle input resistance (Rin) was less than 3 MΩ, or leak current larger than 10
nA. Data were analyzed using Clampfit (Molecular devices), MiniAnalysis (Synaptosoft),
Excel (Microsoft), and SigmaPlot (Systat) software. Average mEPSC, EPSC, and
quantal content were calculated for each genotype. Larvae with intact motor nerves
were dissected and incubated with or without philanthotoxin-433 (PhTx; Sigma; 20 μM)
for 10 min to block postsynaptic glutamate receptors as described (Frank et al., 2006;
Dickman and Davis, 2009).

90
Statistical Analysis
All data are presented as mean +/-SEM. Data were compared using either a one-way
ANOVA and tested for significance using a 2-tailed Bonferroni post-hoc test, or using a
Student’s t-test (where specified) with Graphpad Prism or Microsoft Excel software, and
with varying levels of significance assessed as p<0.05 (*), p<0.01 (**), p<0.001 (***),
ns=not significant. Quantal content was calculated for each individual recording using
the equation QC = EPSC/mEPSC. Full statistical details and information can be found in
Table S1.

Data availability
Fly stocks are available upon request and can be obtained from the Bloomington Stock
Center. Table S1 lists genotypes used and full statistical details for each figure.

91

Figure 3-1: Genetic analysis and generation of null mutations in Drosophila Esyt.
(A) Phylogenic analysis of the single D. melanogaster Esyt ortholog and the three Esyt
genes encoded in M. musculus (Esyt1-3) and S. cerevisiae (Tcb1-3). (B) Schematic of
the three Esyt protein structures aligned with the Drosophila homolog. The Esyt proteins
contain a hydrophobic stretch (HS) and a Synaptotagmin-like-Mitochondrial lipid-binding
E
(kD)
Adult CNS
Esyt-OE
wild
type
Esyt
2
Esyt
1
*
C
Esyt2
F
Esyt
Esyt
2
Esyt
1
100 aa
HS C2A C2B C2C SMP
HS C2A C2B C2C SMP
B
1 kb
ATG
Esyt
1
K32STOP
Esyt
2
(kD)
36 –
Larval CNS
K32STOP
N2STOP
HS C2A C2B C2E C2D C2C SMP
HS C2B C2C C2A SMP
Esyt1
Esyt3
Mouse
Drosophila
Drosophila Esyt locus
Esyt
∆HS
Esyt
D-N
HS C2A C2B C2C SMP * * *
HS C2A C2B C2C SMP Esyt
mCh
mCherry 3xFlag
HS C2A C2B C2C SMP Esyt
Flag
3xFlag
C2A C2B C2C SMP mCherry 3xFlag
3xFlag
36 –
Esyt-OE
wild
type
Esyt
2
Esyt
1
M. musculus
Esyt1
Esyt2
Esyt3
Esyt
Tcb1
Tcb2
Tcb3
D. melanogaster
S. cererisiae
A
D
98 – 98 –
92
Protein (SMP), followed by multiple C2 domains (C2). Red line indicates the antigen the
antibody was raised against. (C) Schematic of the Drosophila Esyt locus. The CRISPR-
induced early stop codon in Esyt
1
(red asterisk) and the MiMIC transposon insertion site
of Esyt
2
(red triangle) are shown. Esyt
1
and Esyt
2
mutations are predicted to each
truncate the open reading frame before the HS domain. (D) Schematic of Esyt
mutations and engineered transgenes. Immunoblot analysis of Esyt expression in adult
head lysates (E) and larval CNS lysates (F) demonstrates that Esyt is expressed in the
nervous system and confirms both Esyt
1
and Esyt
2
alleles are protein nulls. The arrow
indicates the expected molecular mass of Esyt. Neuronal overexpression of Esyt (Esyt-
OE: c155-Gal4;UAS-Esyt
Flag
) results in elevated levels and increased molecular mass
for the tagged transgene, as expected. Anti-Actin was used as loading control.

93

Figure 3-2: The hydrophobic stretch is necessary to localize Esyt to axonal ER.
(A) Representative images of third-instar larval NMJ with motor neuron expression of an
mCherry-tagged Esyt transgene (Esyt
mCh
: red) and a GFP-tagged ER marker (KDEL;
green) (w;OK6-Gal4/UAS-Esyt
mCh
;UAS-GFP-KDEL), immunolabeled with the neuronal
membrane marker HRP (white). Esyt
mCh
co-localizes with axonal ER. (B) Deletion of the
hydrophobic stretch (Esyt
ΔHS
: w;OK6-Gal4/UAS-Esyt
ΔHS
;UAS-GFP-KDEL) results in a
failure to localize to the ER, instead acquiring a cytosolic distribution. (C)
Representative NMJ images of the Flag-tagged Esyt transgene (Esyt
Flag
: anti-Flag; red)
HRP
C
HRP
A
KDEL
HRP
D
B
FasII BRP
Esyt
Flag
vGlut
F
Esyt
Flag
HRP
2 μm
Esyt
Flag
vGlut
E
Esyt
Flag
HRP
KDEL
BRP FasII
Esyt
mCh
Esyt
∆HS
Esyt
mCh
KDEL
Esyt
Flag
Esyt
Flag
HRP Esyt
D-N
HRP
Esyt
D-N
HRP
Esyt
∆HS
KDEL
94
expressed in motor neurons (w;OK6-Gal4/UAS-Esyt
Flag
) co-stained with HRP
(white/blue). Esyt-Flag traffics to the presynaptic terminal and appears similar in
distribution to Esyt
mCh
. (D) Mutations in the Esyt
Flag
transgene that prevent Ca
2+
binding
to C2 domains (Esyt
D-N
: w;OK6-Gal4/UAS-Esyt
D-N
) no longer traffics to presynaptic
terminals. (E) Axonal ER structures labeled with Esyt-Flag (anti-Flag; red) is shown
relative to active zones (BRP; green) and synaptic vesicle structures (vGlut; white/blue).
(F) Axonal ER labeled by Esyt-Flag (anti-Flag; red) is shown co-labeled with a peri-
active zone marker (FasII; green) and a neuronal membrane marker (HRP; white/blue).

95

Figure 3-3: PI(4,5)P2 and PI(3)P phospholipid levels are unchanged at presynaptic
terminals in Esyt mutants and Esyt-OE.
(A) Representative NMJ images of PI(4,5)P2 labeled with PLCδ-PH-GFP (GFP; green)
and HRP (white/blue) in control (w;OK6-Gal4/+;UAS-PLCδ-PH-GFP/+), Esyt mutants

(w;OK6-Gal4/+;Esyt
1
/Esyt
2
,UAS-PLCδ-PH-GFP), and Esyt-OE (w;OK6-Gal4/+; UAS-
PLCδ1-PH-GFP/UAS-Esyt
Flag
). (B) Quantification of mean GFP intensity levels of the
indicated genotypes. (C) Representative images of PI(3)P distribution at the NMJ
labeled by GFP-myc-2xFYVE in control (w;OK6-Gal4/UAS-GFP-myc-2xFYVE), Esyt

mutants (w;OK6-Gal4/UAS-GFP-myc-2xFYVE;Esyt
1
/Esyt
2
), and Esyt-OE (w;OK6-
Gal4/UAS-GFP-myc-2xFYVE;UAS-Esyt
Flag
/+). (D) Quantification of mean GFP intensity
levels of the indicated genotypes. Error bars indicate ±SEM. One-way analysis of
variance (ANOVA) test was performed, followed by a Tukey’s multiple-comparison test.
NS=not significant, p>0.05. Detailed statistical information for represented data (mean
values, SEM, n, p) is shown in Table S1.

D
Mean GFP Intensity
(% control)
0
50
150
100
GFP-myc-2xFYVE HRP
5 μm
Esyt-OE Esyt control
A
C
HRP
Plcδ1-PH-GFP
HRP
GFP-myc-2xFYVE
HRP
Esyt-OE Esyt control
NS
B
Mean GFP Intensity
(% control)
0
50
150
100
NS
Plcδ1-PH-GFP
96

Figure 3-4: Presynaptic overexpression of Esyt promotes synaptic growth.

(A) Representative NMJ images of wild type (w
1118
), Esyt

mutants (Esyt: w;Esyt
1
/Esyt
2
),
and Esyt-OE (w;OK6-Gal4/UAS-Esyt
Flag
) immunostained for anti-BRP (green), anti-
GluRIII (red), and anti-HRP (white; insert). Bottom panels: BRP and GluRIII images at
higher magnification. Quantification of bouton number per NMJ (B), neuronal membrane
surface area (C), total BRP puncta number per NMJ (D), BRP density (E), and BRP
area (F) of the indicated genotypes. Note that Esyt-OE results in increased bouton
number and a corresponding increase in membrane and BRP number. Error bars
indicate ±SEM. One-way ANOVA test was performed, followed by a Tukey’s multiple-
comparison test. NS=not significant, p>0.05; **p ≤0.01; ***p ≤0.001. Detailed statistical
information for represented data (mean values, SEM, n, p) is shown in Table S1.

wild type
BRP Density (#/µm
2
)
BRP Area (µm
2
)
0.1
0.2
0
0.5
1.0
0
Bouton #/NMJ
0
20
40
**
BRP #/NMJ
400
200
0
***
Neuronal Membrane
Surface Area (µm
2
)
500
250
0
***
BRP
GluRIII
BRP
GluRIII
B C D E F
600 1.5 60
A
10 μm
HRP
5 μm
Esyt Esyt-OE
NS NS
NS
NS
NS
97

Figure 3-5: Esyt promotes presynaptic neurotransmitter release.

(A) Representative electrophysiological EPSC and mEPSC traces for wild type, Esyt
mutants and Esyt-OE recorded in 0.4 mM extracellular Ca
2+
. Quantification of mEPSC
amplitude (B), EPSC amplitude (C), and quantal content plotted as a function of
extracellular Ca
2+
concentration on logarithmic scales (D) of the indicated genotypes.
Esyt mutants exhibit significantly reduced synaptic transmission compared to wild type
and Esyt-OE. No significant difference was observed in the slope of the best fit lines
used to determine the apparent Ca
2+
cooperativity. Representative EPSC traces
following four pulses of 60 Hz stimulation in wild type, Esyt

mutants, Esyt

neuronal
rescue (w;OK6-Gal4/UAS-Esyt
Flag
; Esyt
1
/Esyt
2
), and Esyt-OE in 0.4 mM (E) and 3 mM
(G) extracellular Ca
2+
. Quantification of average EPSC ratio (% 4
th
EPSC/1
st
EPSC) for
the indicated genotypes in 0.4 mM (F) and 3 mM (H) extracellular Ca
2+
. Esyt mutants
show reduced neurotransmission and short-term synaptic plasticity. Error bars indicate
±SEM. One-way ANOVA test was performed, followed by a Tukey’s multiple-
200 ms
0
A
400
B C
200
600
mEPSC Amplitude (pA)
wild type Esyt Esyt-OE
0.4 mM Ca
2+
20 nA
F E
PPR (% EPSC4/EPSC1)
0
EPSC (nA)
60
0
wild type Esyt
Esyt +
Neuronal rescue
*
Esyt-OE
0.4 mM Ca
2+
10 ms
50 nA
100
H G
PPR (% EPSC4/EPSC1)
0
50
wild type Esyt
Esyt +
neuronal rescue
**
Esyt-OE
3 mM Ca
2+
10 ms
40
20
300
200
100
D
Esyt +
neuronal
rescue
500 pA
10 nA
10 ms
Quantal Content
External [Ca
2+
] (mM)
1
10
100
3.0 0.4
wild type
Esyt
Esyt-OE
1000
1.5
NS
*
NS
NS
NS
NS
NS
NS
*
NS
98
comparison test. NS=not significant, p>0.05; *p≤0.05; **p 0.01. Detailed statistical
information for represented data (mean values, SEM, n, p) is shown in Table S1.

99

Figure 3-6: Depletion of the synaptic vesicle pool in Esyt mutants and Esyt-OE.

(A) Depletion and recovery of the functional vesicle pool in the indicated genotypes.
NMJs were stimulated at 10 Hz for 10 min in 10 mM extracellular Ca
2+
, then allowed to
recover for 10 mins while monitoring this recovery with stimulation at 0.2 Hz. EPSP
amplitudes were averaged, normalized to pre-stimulus amplitudes, and plotted as a
function of time. (B) Quantification of total quanta released during the 10 mins of 10 Hz
stimulation for the indicated genotypes. Error bars indicate ±SEM. One-way ANOVA
test was performed, followed by a Tukey’s multiple-comparison test. NS=not significant,
p>0.05; ***p ≤0.001. Detailed statistical information for represented data (mean values,
SEM, n, p) is shown in Table S1.

Esyt
wild type
10Hz
0.2Hz
% Starting EPSP
A
25
50
75
0
100
0 200 400 600 800 1000 1200
Esyt-OE
wild type Esyt Esyt-OE
B
Total Quanta Released
(x 10
3
)
0
200
300
***
Time (s)
100
NS
100

Figure 3-7: Synaptic vesicle density and endocytic pools are unchanged in Esyt mutants
and Esyt-OE.

(A) Representative electron micrograph images of NMJs for wild type, Esyt mutants,
and Esyt-OE. Quantification of synaptic vesicle density (B), synaptic vesicles within 300
nm of the active zone (C), T-bar length (D), and active zone length (E) in the indicated
genotypes. No significant differences were observed. (F) Representative images of
FM1-43 dye loading of the indicated genotypes. (G) Quantification of mean intensity of
the FM1-43 signal in the indicated genotypes. Error bars indicate ±SEM. One-way
ANOVA test was performed, followed by a Tukey’s multiple-comparison test. NS=not
significant, p>0.05. Detailed statistical information for represented data (mean values,
SEM, n, p) is shown in Table S1.

Esyt
Synaptic Vesicle
Density (#/µm
2
)
0
50
100
150
B
Synaptic Vesicle Number
within 300nm of AZ
0
C
300
100
200
wild type Esyt-OE
A
500nm
T Bar Length (nm)
D
0
200
100
F
wild type
Esyt
Esyt-OE
wild type
Esyt
Esyt-OE
5µm
G
Mean GFP Intensity
(% wild type)
0
50
100
FM1-43
NS NS
Active Zone Length (nm)
E
0
150
100
50
NS NS
NS
101

Figure 3-8: Esyt is dispensable for presynaptic homeostatic plasticity.

(A) Representative EPSC and mEPSC traces for wild type, Esyt mutants, and Esyt-OE
before (baseline) and following PhTx application. Note that while mEPSC amplitudes
are reduced following PhTx application, EPSC amplitudes recover to baseline levels
because of a homeostatic increase in presynaptic release (quantal content). (B)
Quantification of mEPSC and quantal content values after PhTx application normalized
to baseline values. No significant differences were observed. Error bars indicate ±SEM.
One-way ANOVA test was performed, followed by a Tukey’s multiple-comparison test.
Detailed statistical information for represented data (mean values, SEM, n, p) is shown
in Table S1.

A
% Baseline (-PhTx)
wild type
+ PhTx
Esyt
+ PhTx
B mEPSP quantal
content
150
100
0
50
200
Esyt-OE
+ PhTx
0.4 mM Ca
2+
10 nA
10 ms
wild type
baseline
Esyt
baseline
wild type
+ PhTx
Esyt
+ PhTx
Esyt-OE
baseline
Esyt-OE
+PhTx
200 ms
500 pA
102
Table 3-S1: Absolute and additional values for normalized and presented data.

Figure Label Genotype
Plcδ1-PH-GFP Intensity
(% wild type)
n
P Value
(significance)
3A,B Control
w
1118
; OK6-Gal4/+; UAS-PLCδ-PH-
GFP/+
100
(5.121)
11
3A,B Esyt
w
1118
; OK6-Gal4/+; Esyt
1
/Esyt
2
,
UAS-PLCδ-PH-GFP
119.211
(8.986)
11 0.0780 (ns)
3A,B Esyt-OE
w
1118
; OK6-Gal4/+; UAS-PLCδ1-
PH-GFP/UAS-Esyt
Flag
;
137.201
(17.707)
12 0.0658 (ns)
Figure Label Genotype
GFP-myc-2xFYVE
Intensity
(% wild type)
n
P Value
(significance)
3C,D Control
w
1118
; OK6-Gal4/UAS-GFP-myc-
2xFYVE
100
(3.618)
10
3C,D Esyt
w
1118
; OK6-Gal4/UAS-GFP-myc-
2xFYVE; Esyt
1
/Esyt
2

100.167
(5.491)
11 0.9804 (ns)
3C,D Esyt-OE
w
1118
; OK6-Gal4/UAS-GFP-myc-
2xFYVE; UAS-Esyt
Flag
/+
109.861
(4.552)
13 0.1209 (ns)

Figure Label Genotype
Bouton
Number
Neuronal
Membrane
Surface
Area (μm
2
)
BRP
#/NMJ
BRP
Density
(/µm
2
)
BRP Area
(μm
2
)
n
P Value
(significance:
Bouton #,
Membrane
Surface Area,
BRP #/NMJ,
BRP Density,
BRP Area)
4A-F wild type w
1118

31.750
(2.972)
280.067
(25.205)
337.500
(26.839)
1.234
(0.054)
59.578
(4.766)
12
4A-F Esyt w
1118
;; Esyt
1
/Esyt
2

32.818
(2.423)
313.736
(24.891)
338.091
(20.662)
1.137
(0.094)
63.103
(4.353)
11
0.7856 (ns),
0.3539 (ns),
0.9864 (ns),
0.3764 (ns),
0.7695 (ns)
4A-F Esyt-OE
w
1118
; OK6-
Gal4/UAS-Esyt
Flag

46.000
(2.799)
445.615
(32.373)
486.167
(26.754)
1.110
(0.044)
80.273
(6.931)
12
0.0021 (**),
0.0006 (***),
0.0007 (***),
0.0912 (ns),
0.3622 (ns)
Not shown NA
w
1118
; OK6-
Gal4/UAS-Esyt
mCh

49.700
(3.114)
473.291
(37.492)
512.800
(38.086)
1.102
(0.051)
86.131
(6.687)
10
0.0005 (***),
0.0003 (***),
0.0010 (***),
0.0956 (ns),
0.5455 (ns)

Figure Label Genotype [Ca
2+
]
mEPSC
amplitude
(pA)
mEPSC
frequenc
y (Hz)
EPSC
(nA)
QC
Input
Resista
nce
(MΩ)
Leak
Current
(nA)
n
P Value
(significance:
mEPSC amp,
mEPSC freq,
EPSC, QC)
5A-D
wild
type
w
1118
0.4
-521.184
(34.236)
2.740
(0.314)
-48.786
(13.471)
96.867
(28.767)
9.375
(0.375)
-3.033
(0.493)
7
5A-D Esyt w
1118
;; Esyt
1
/Esyt
2
0.4
-539.886
(19.112)
2.877
(0.228)
-21.445
(5.505)
40.805
(10.995)
7.455
(0.638)
-2.646
(0.711)
9
0.6120 (ns),
0.7274 (ns),
0.0458 (*),
0.0489 (*)
5A-D
Esyt
recue
w
1118
; OK6-
Gal4/UAS-Esyt
Flag
;
Esyt
1
/Esyt
2

0.4
-436.698
(9.558)
2.667
(0.553)
-41.852
(5.251)
96.388
(12.850)
8.286
(0.787)
-4.482
(2.851)
9
0.0105 (*),
0.9053 (ns),
0.5761 (ns),
0.9862 (ns)
5A-D
Esyt-
OE
w
1118
; OK6-
Gal4/UAS-Esyt
Flag

0.4
-453.664
(16.148)
2.019
(0.180)
-45.744
(7.691)
93.523
(18.061)
8.667
(0.575)
-1.760
(0.309)
15
0.0518 (ns),
0.0450 (*),
0.8458 (ns),
0.9233 (ns)

103

Figure Label Genotype [Ca
2+
]
mEPSC
amplitude
(pA)
EPSC
(nA)
QC
Input
Resistan
ce
(MΩ)
Leak
Current
(nA)
n
P Value
(significance:
QC)
5D
wild
type
w
1118
1.5
-450.666
(24.021)
-121.851
(13.781)
275.466
(33.501)
10.286
(1.728)
-3.471
(1.284)
7
5D Esyt w
1118
;; Esyt
1
/Esyt
2
1.5
-412.004
(9.141)
-74.990
(8.278)
187.452
(25.777)
11.375
(1.165)
-1.865
(0.997)
14 0.0212 (*)
5D
Esyt-
OE
w
1118
; OK6-Gal4/UAS-
Esyt
Flag

1.5
-424.308
(12.000)
-122.506
(7.006)
288.902
(15.404)
8.500
(1.000)
-4.233
(0.952)
8 0.7095 (ns)
5D
wild
type
w
1118
3.0
-468.450
(16.126)
-235.727
(12.185)
507.770
(29.561)
11.200
(1.526)
-3.682
(0.332)
10
5D Esyt w
1118
;; Esyt
1
/Esyt
2
3.0
-543.625
(21.252)
-175.172
(18.352)
323.801
(34.036)
7.375
(0.680)
-4.522
(0.577)
8 0.0008 (***)
5D
Esyt
recue
w
1118
; OK6-Gal4/UAS-
Esyt
Flag
; Esyt
1
/Esyt
2

3.0
-432.083
(9.491)
-232.206
(20.847)
537.128
(46.699)
7.625
(0.532)
-3.046
(0.282)
8 0.5882 (ns)
5D
Esyt-
OE
w
1118
; OK6-Gal4/UAS-
Esyt
Flag

3.0
-463.322
(27.241)
-243.366
(16.005)
541.470
(57.371)
7.625
(0.800)
-3.612
(0.502)
7 0.5780 (ns)

Figure Label Genotype [Ca
2+
]
EPSC 1
(nA)
EPSC 2
(nA)
EPSC 3
(nA)
EPSC 4
(nA)
PPR
(EPSC4/EPSC1)
n
P Value
(significance:
PPR)
5E,F wild type w
1118
0.4
-48.786
(13.471)
-58.458
(15.204)
-59.546
(15.006)
-64.168
(13.139)
1.494
(0.192)
8
5E,F Esyt w
1118
; Esyt
1
/Esyt
2
0.4
-21.445
(5.505)
-25.338
(4.967)
-33.088
(4.871)
-39.937
(4.998)
2.746
(0.495)
9 0.0405 (*)
5E,F
Esyt

+
neuronal
rescue
w
1118
; OK6-
Gal4/UAS-Esyt
Flag
;
Esyt
1
/Esyt
2

0.4
-41.852
(5.251)
-50.389
(5.596)
-53.090
(6.214)
-56.064
(6.297)
1.358
(0.050)
9 0.3932 (ns)
5E,F Esyt-OE
w
1118
; OK6-
Gal4/UAS-Esyt
Flag

0.4
-45.744
(7.692)
-53.576
(7.062)
-59.997
(6.989)
-67.920
(6.600)
1.915
(0.216)
15 0.3013 (ns)
5G,H wild type w
1118
3.0
-235.728
(12.186)
-176.158
(14.671)
-158.895
(13.545)
-145.829
(12.728)
0.613
(0.032)
10
5G,H Esyt w
1118
; Esyt
1
/Esyt
2
3.0
-175.173
(18.352)
-156.200
(14.530)
-157.531
(12.401)
-142.820
(10.817)
0.859
(0.076)
8 0.0053 (**)
5G,H
Esyt

+
neuronal
rescue
w
1118
; OK6-
Gal4/UAS-Esyt
Flag
;
Esyt
1
/Esyt
2

3.0
-232.206
(20.848)
-161.540
(14.482)
-150.038
(13.173)
-131.543
(11.281)
0.569
(0.013)
8 0.2539 (ns)
5G,H Esyt-OE
w
1118
; OK6-
Gal4/UAS-Esyt
Flag

3.0
-243.367
(16.005)
-196.454
(11.530)
-184.588
(12.270)
-169.124
(12.732)
0.691
(0.028)
7 0.1013 (ns)

Figure Label Genotype Total quanta released n
P Value
(significance)
6A,B wild type w
1118

170746.2
(13327.07)
7
6A,B Esyt w
1118
;;Esyt
1
/Esyt
2

185279.4
(20449.02)
6 0.5526 (ns)
6A,B Esyt-OE w
1118
;OK6-GAL4/UAS-Esyt
Flag

286885.4
(7323.839)
7 <0.0001 (***)

Figure Label Genotype
Synaptic
Vesicle
Density
(#/µm2)
Synaptic
Vesicle
Number
within
300nm of
AZ
T Bar
Length
(nm)
Active Zone
Length (nm)
n (SV
Density,
SV#, T bar
length, AZ
length)
P Value
(significance:
SV Density,
SV # within
300nm of AZ,
T Bar Length,
AZ Length)
7A-E wild type w
1118

120.095
(18.270)
267.100
(13.934)
178.061
(17.930)

618.601
(48.895)
11,18,14,21
7A-E Esyt w
1118
; Esyt
1
/Esyt
2

105.311
(17.145)
231.480
(12.717)
166.827
(12.947)
597.936
(30.003)
8,14,9,24
0.5769 (ns),
0.0762 (ns),
0.6548 (ns),
0.7131 (ns)
7A-E Esyt-OE
w
1118
; OK6-
Gal4/UAS-Esyt
Flag

94.309
(11.161)
238.215
(8.299)
183.306
(16.969)
507.527
(35.967)
10,30,14,31
0.2545 (ns),
0.0641 (ns),
0.8334 (ns),
0.0673 (ns)
104

Figure Label Genotype
FM1-43 Intensity
(% wild type)
n
P Value
(significance)
7F,G wild type w
1118

100
(5.584)
10
7F,G Esyt w
1118
;Esyt
1
/Esyt
2

102.545
(4.869)
11 0.7338 (ns)
7F,G Esyt-OE w
1118
;OK6-Gal4/UAS-Esyt
Flag

84.487
(4.990)
8 0.0606 (ns)

Figure Label Genotype [Ca
2+
] PhTx
mEPSC
(pA)
EPSC
(nA)
QC
Leak
Current (nA)
n
P Value
(significance:
mEPSC amp,
EPSC, QC)
8A,B wild type w
1118
0.4 -
-521.184
(34.236)
-48.786
(13.471)
96.867
(28.767)
-3.033
(0.493)
7
8A,B wild type w
1118
0.4 +
-247.505
(11.750)
-33.785
(1.855)
142.951
(11.613)
-6.737
(0.497)
17
8A,B Esyt w
1118
;; Esyt
1
/Esyt
2
0.4 -
-539.886
(19.112)
-21.445
(5.505)
40.805
(10.995)
-2.646
(0.711)
9
0.6120 (ns),
0.0458 (*),
0.0489 (*)
8A,B Esyt w
1118
;; Esyt
1
/Esyt
2
0.4 +
-294.331
(13.337)
-21.815
(3.173)
74.209
(10.868)
-2.716
(0.485)
11
8A,B Esyt-OE
w
1118
; OK6-
Gal4/UAS-Esyt
Flag

0.4 -
-453.664
(16.148)
-45.744
(7.691)
93.523
(18.061)
-1.760
(0.309)
15
0.0597 (ns),
0.8458 (ns),
0.9233 (ns)
8A,B Esyt-OE
w
1118
; OK6-
Gal4/UAS-Esyt
Flag

0.4 +
-240.749
(17.108)
-36.660
(3.297)
160.813
(17.770)
-1.719
(0.291)
12

The figure and panel, genotype, and conditions used are noted (external calcium concentration as well as whether PhTx
was applied or not). Average values with standard error values noted in parentheses are shown for all morphology data
such as neuronal membrane surface area, bouton number, BRP number, BRP density, BRP area as well as intensity values.
For electrophysiological experiments, all passive membrane properties (input resistance, leak current) and mEPSC, EPSC,
QC, number of data samples (n), p-values from statistical test are shown, with standard error values noted in parentheses.

105
Chapter 4: Conclusions

106
4.1 Insomniac
Here, screening of more than 300 synaptic targets of FMRP identified six novel
genes that promote the expression of presynaptic homeostatic potentiation (PHP). Of
six, five are required in the presynaptic neuron, while a single gene, insomniac (inc), is
required in the postsynaptic cell. There, Inc appears to act as a target-specifying
adaptor for Cul3 ubiquitin ligase, engaging in ubiquitination of a synaptic protein
specifically at the postsynaptic SSR to drive retrograde PHP signaling. This finding
contributes to the understanding of the enigmatic mechanism, PHP induction.
Future directions
The substrate of Cul3-Inc dependent ubiquitination is not known. We eliminated
the possibility of GluRs and pCamKII as a target of Cul3-Inc dependent ubiquitination,
and further narrowed down the potential targets to be synaptic proteins at the SSR.
Future studies that identifies Inc-interacting proteins, such as mass spectrometry, will be
useful to identify the substrate and thus provide insights into the mechanisms
delineating how Cul3-Inc dependent ubiquitination leads to the generation of retrograde
signaling.
Application to human diseases
inc mutants have deficits in sleep regulation. The current study, thus, raised an
interesting possibility that inc may regulate homeostatic synaptic plasticity in the
postsynaptic muscle, which may in turn regulate sleep. While sleep regulation has been
assumed to take place almost exclusively within the brain, a recent study demonstrated
that sleep is also regulated in the muscle (Ehlen et al., 2017), consistent with the
107
putative role of Inc regulating the induction of PHP and sleep in the postsynaptic
muscle.
In addition, the current study demonstrated that ubiquitination might be a key
regulator for synaptic homeostasis and sleep. Indeed, protein levels of key synaptic
markers in the Drosophila brain increase after a wake period, and decrease after sleep
(Gilestro et al., 2009), suggesting that protein degradation of these synaptic proteins
might be an active regulator of sleep. While proteasome-dependent protein degradation
is less likely to be involved in the postsynaptic induction mechanism of PHP (Wentzel et
al., 2018), it is possible that the synaptic proteins are degraded in other pathways such
as lysosomal dependent degradation or autophagy. Accordingly, ubiquitination has
been shown to mediate these protein degradation pathways as well. Together, these
studies indicate that inc may couple synaptic homeostasis and sleep regulation, and
suggest that inc can be a therapeutic target for the sleep deficits.

4.2 Extended Synaptotagmin
Characterization of Drosophila Esyt ortholog revealed that Esyt is localized to an
extensive axonal ER network in vivo. We find that Esyt is required to facilitate
presynaptic release across a range of extracellular Ca
2+
. Interestingly, presynaptic
overexpression of Esyt promotes synaptic growth and, in turn, resistance to synaptic
depression during elevated activity. Thus, our study establishes Esyt as a conserved
ER-localized protein that couples ER functions with neurotransmission and synaptic
growth.

108
Future directions
Currently, how Esyt may regulate ER functions to promote synaptic growth and
neurotransmission is not clear. We proposed a hypothetical model where Esyt
upregulates cytosolic Ca
2+
levels during activity and rest, which in turn leads to
potentiated neurotransmission and synaptic growth. It would be of great interest to
directly observe the change in the levels of local cytosolic Ca
2+
at synapses during rest
and activity in Esyt mutants to further gain insights into the mechanism linking the ER
functions to the synaptic functions.
Application to human diseases
Dysfucntion of Synaptic ER has been implicated in various neurological
diseases, including hereditary spastic paraplegias (Blackstone et al., 2011; Montenegro
et al., 2012; Noreau et al., 2014), amyotrophic lateral sclerosis (Teuling et al., 2007;
Yang et al., 2009; Fasana et al., 2010), and Alzheimer’s disease (Cheung et al., 2008;
Zhang et al., 2009; Goussakov et al., 2010). While it is unclear whether and how Esyt
may contribute to the etiology of these diseases, the current study implicated the
regulation of cytosolic Ca
2+
, which can be a therapeutic target for these diseases.
Strikingly, we also discovered that overexpression of Esyt results in synaptic
overgrowth. This suggests that overexpression of Esyt can potentially be used as a
therapeutic use for diseases with loss or reduction of synaptic activities, such as spinal
muscular atrophy (SMA).
At the end
Together, findings in the current studies provide insights into an extremely
complex mechanism of synaptic development and plasticity. Presynaptic
109
overexpression of Esyt seems to affect all synapses in the single neuron as observed in
the uniform overgrowth. However, insomniac mediated PHP seems to be spatially
confined at each individual synapse in the postsynaptic cell, suggesting a local
regulation of synaptic plasticity. These studies illustrate how intricate coordination of
pre- and postsynaptic cell contribute to the global and local regulation of synapses,
which coexist and intersect at the same synapse during development and plasticity.

110
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Abstract (if available)

Abstract At synapses, homeostatic plasticity maintains the synaptic activities in the physiologically appropriate range. Impaired synaptic homeostatic plasticity is associated with complex neurological diseases such as Schizophrenia. At the Drosophila neuromuscular junction (NMJ), postsynaptic glutamate receptor inhibition induces retrograde signaling, leading to a compensatory increase in presynaptic neurotransmitter release, thereby precisely restoring baseline levels of synaptic strength. Despite the recent advances in knowledge for presynaptic mechanisms involved in this process, mechanisms that drive the retrograde homeostatic signaling have remained elusive. Here, we conducted dual electrophysiology-based forward-genetic screens, and identified insomniac (inc) as an essential regulator for retrograde signaling. Upon the induction of homeostatic synaptic plasticity, insomniac is recruited to the NMJ, functions together with ubiquitin ligase Cul3 downstream of pCamKII activity. We conclude that ubiquitination is a key regulator for the homeostatic retrograde signaling. ❧ In the second project, I investigated how presynaptic endoplasmic reticulum influences synaptic physiology. We characterized mutations in Extended Synaptotagmin (Esyt), evolutionarily conserved ER proteins with Ca²⁺-sensing domains. We found that Esyt localizes to presynaptic ER structures. While synaptic structure, membrane lipid balance, and homeostatic plasticity are surprisingly unperturbed, neurotransmission is reduced in Esyt mutants. Surprisingly, neuronal overexpression of Esyt enhances synaptic growth and the sustainment of the vesicle pool during intense activity. Thus, we identify Esyt as a presynaptic ER protein that can promote neurotransmission and synaptic growth, revealing the first in vivo neuronal functions of this conserved gene family.

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Asset Metadata

Creator Kikuma, Koto (author)

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

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 08/13/2019

Defense Date 06/20/2018

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

Tag Drosophila,homeostasis,neuromuscular junction,OAI-PMH Harvest,retrograde signaling,synaptic plasticity,ubiquitination

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Ko, Chien-Ping (committee chair), Chang, Karen (committee member), Dickman, Dion (committee member)

Creator Email kikuma@usc.edu,koto.kikuma@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-67079

Unique identifier UC11671472

Identifier etd-KikumaKoto-6737.pdf (filename),usctheses-c89-67079 (legacy record id)

Legacy Identifier etd-KikumaKoto-6737.pdf

Dmrecord 67079

Document Type Dissertation

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Rights Kikuma, Koto

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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

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