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Phosphorylation of Synaptojanin differentially regulates synaptic vesicle endocytosis of distinct vesicle pools

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Phosphorylation of Synaptojanin differentially regulates synaptic vesicle endocytosis of distinct vesicle pools

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Phosphorylation of Synaptojanin differentially regulates
synaptic vesicle endocytosis of distinct vesicle pools

By
Liping Wang

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
August 2016
Copyright 2016 Liping Wang
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ACKNOWLEDGEMENTS
I would like to express my sincere thanks and appreciation to my adviser Dr. Karen T. Chang.
I really appreciate that she provided me with the precious and valuable opportunity to work on this
wonderful project. Her constant encouragement and intellectual knowledge towards research and
writing of this thesis have been really inspiring and important for me. She always gave me
constructive advice, helping me to strengthen both my spirit and science while pursuing my
master’s degree. I would also like to thank other thesis defense committee members Dr. Zoltan A.
Tokes, Dr. Ralf Langen for your guiding comments and suggestions.
I would like to thank all members in Dr. Chang’s lab: Dr. Junhua Geng, Joo Yeun Lee, Chun-
Kan Chen and Syed Qadri for building up a supportive team. I would like to send my thanks to
Chun-Kan Chen and Syed Qadri for conducting preliminary experiments that inspired this project.
In addition, I want to thank Dr. Junhua Geng and Joo Yeun Lee for conducting all the biochemistry
experiments and part of electrophysiology experiments.
I am also grateful to Dr. Derek Sieburth and his lab members for providing helpful and
inspiring comments to move this project forward during lab meetings.
Finally, I would like to thank my friends and parents for sharing all the joys and tears with me
and supporting me to achieve as I pursue my master’s degree.
Thank you everyone for your support and help!
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Table of Contents
ACKNOWLEDGEMENTS ............................................................................................................. i
LIST OF FIGURES ....................................................................................................................... iv
ABBREVIATIONS ....................................................................................................................... vi
ABSTRACT .................................................................................................................................. vii
CHAPTER 1 - INTRODUCTION .................................................................................................. 1
1 Synaptic transmission ....................................................................................................... 1
2 Drosophila melanogaster ................................................................................................. 1
3 Drosophila melanogaster neuromuscular junction (NMJ) .............................................. 2
4 Synaptic vesicle pool ........................................................................................................ 3
5 Synaptojanin ..................................................................................................................... 5
6 Synaptojanin is a substrate of Minibrain kinase............................................................. 10
CHAPTER 2 - MATERIALS AND METHODS ......................................................................... 13
1 Fly stocks and antibody generation ................................................................................ 13
2 Mass Spectrometry ......................................................................................................... 14
3 Synaptojanin PRD construction ..................................................................................... 15
4 Immunochemistry........................................................................................................... 15
5 Image Quantification ...................................................................................................... 16
6 Immunoprecipitation and protein interaction ................................................................. 16
7 In vitro phosphorylation of Synaptojanin....................................................................... 16
8 Phosphatidylinositol 5’-phosphatase activity in vitro .................................................... 17
9 In vivo Phosphatidylinositol 5’-phosphatase activity determination.............................. 17
10 FM1-43 dye labeling ...................................................................................................... 18
11 Electrophysiology ........................................................................................................... 19
12 Statistics .......................................................................................................................... 19
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CHAPTER 3 - RESULTS ............................................................................................................. 20
1 Minibrain kinase phosphorylates Synaptojanin at S1029 in vitro and in vivo ............... 20
2 Phosphorylation of Synaptojanin at S1029 enhances Synaptojanin activity and alters
Synaptojanin interaction with Endophilin ............................................................................. 26
3 Phosphorylation of Synaptojanin at S1029 is required for endocytosis but is not
necessary for reliable neurotransmission at high stimulation frequency ............................... 30
4 Synaptojanin is required to maintain synaptic vesicle pool size .................................... 35
5 Phosphorylation status of Synaptojanin differentially affect the recycling of ECP and
RP ....................................................................................................................................... 41
CHAPTER 4 - DISCUSSION ...................................................................................................... 45
REFERENCE ................................................................................................................................ 50

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LIST OF FIGURES
Figure 1 Drosophila melanogaster neuromuscular junction (NMJ)
Figure 2 Synaptic vesicle pools.
Figure 3 Domains and classical model of Synaptojanin function in Clathrin-mediated
endocytosis.
Figure 4 Latest model of Synaptojanin function in Clathrin-mediated endocytosis.
Figure 5 Synaptojanin is a substrate of Minibrain, and phosphorylation of Synaptojanin by
Minibrain is required for synaptic vesicle endocytosis.
Figure 6 Minibrain phosphorylates Synaptojanin at S1029 in vitro.
Figure 7 Phosphorylation of Synaptojanin at S1029 in vivo.
Figure 8 Phosphorylation of Synaptojanin at S1029 enhances Synaptojanin phosphoinositide
phosphatase activity and alters its interaction with Endophilin.
Figure 9 Phosphorylation of Synaptojanin at S1029 is required for normal synaptic vesicle
endocytosis.
Figure 10 Expression of either phospho-null or phospho-mimetic Synaptojanin can rescue
neurotransmission defect of synj mutant at high stimulation frequency.

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Figure 11 Phosphorylation of Synaptojanin differentially affects the size of the ECP and RP.
Figure 12 Phosphorylation of Synaptojanin affects endocytosis of the RP.
Figure 13 Schematic model of different phosphorylation status of Synaptojanin
differentially regulates synaptic vesicle endocytosis of distinct vesicle pools.

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ABBREVIATIONS
DS: Down syndrome
Synj: Synaptojanin
Mnb: Minibrain
ECP: Exo-endo cycling pool
RP: Reserve pool
NMJ: Neuromuscular junction
RRP: Readily releasable pool
PRD: Proline rich domain
SAC1: Suppressor of actin 1
Endo: Endophilin
SH3: Src Homology 3
DYRK1A: Dual-specificity tyrosine phosphorylation-regulated kinase 1A
EPSP: Excitatory post-synaptic potential

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ABSTRACT
Impaired synaptic transmission is a pathological alternation commonly found in various
neurological disorders including Down syndrome (DS), Parkinson’s and Autism. The rapid
replenishment of synaptic vesicles through endocytosis during intense neuronal activity is essential
for sustained function of nervous system. Synaptojanin (Synj), a phosphoinositide phosphatase, is
known to play an important role in facilitating uncoating of Clathrin from coated vesicles
following synaptic vesicle uptake. Previous work in our lab has demonstrated that Synj is a
substrate of the Minibrain (Mnb) kinase, a fly homolog of the dual-specificity tyrosine
phosphorylation-regulated kinase 1A (DYRK1A); however, the functional impacts of Synj
phosphorylation by Mnb are not well studied. In this thesis, we identify that S1029 is the target
sequence on Synj phosphorylated by Mnb kinase in Drosophila. We find that phosphorylation on
S1029 of Synj enhances Synj phosphoinositide phosphatase activity but reduces interaction with
Endophilin. In addition, it facilitates more robust endocytosis of the active cycling vesicle pool
(also designated as exo-endo cycling pool, ECP) but decreases endocytosis of the reserve pool
(RP). On the other hand, dephosphorylated Synj has deficiency in the endocytosis of the ECP,
however promotes RP vesicle endocytosis to rescue total vesicle pool size and further sustains
synaptic transmission during high-frequency stimulation. Together, our findings reveal novel roles
for Synj in modulating ECP and RP vesicle endocytosis and further provide valuable insights into
mechanisms affecting impaired synaptic communication found in neurological disorders.
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CHAPTER 1
INTRODUCTION
1 Synaptic transmission
During neuronal communication, synapses are key regulator of information flow that permit
a neuron to pass a chemical or an electrical signal to connecting neurons. In chemical synapses,
the chemical signal (neurotransmitters) are incorporated in synaptic vesicles. Synaptic vesicles are
replenished by endocytosis of the lipid membrane and protein components in pre-synaptic regions,
which enables neurons to maintain sufficient numbers of vesicles without depleting them. The
numbers of synaptic vesicles released during activity range from a few hundred to nearly a million,
depending on the stimulus intensity (Rizzoli & Betz, 2005). Both hypo- and hyper- synaptic
vesicle release have been linked to neurological disorders including Down syndrome (DS),
Parkinson’s disease, and Autism (Gross, 2006; Lotharius & Brundin, 2002; Yang, Sykora, Wilson,
Mattson, & Bohr, 2011). Hence, understanding how synapses maintain proper amount of
functional synaptic vesicles may provide valuable insights into mechanisms underlying these
diseases.
2 Drosophila melanogaster
Drosophila melanogaster (Fruit fly) is an extensively studied invertebrate model organism in
genetics, developmental biology, behavioral science and neuroscience. Drosophila melanogaster
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have several technical advantages: powerful genetic tools, short life cycle, and highly
evolutionarily conserved signaling pathways and biochemical mechanisms. The Gal4/UAS system
is a powerful tool that allows expression of gene constructs in Drosophila with spatial and temporal
specificity, thus allowing researchers to investigate the effects of gain-of-function and loss-of-
function mutations to understand cellular and molecular mechanism in neurological disorder
diseases. In addition to genetic techniques, immunofluorescence staining and electrophysiology
enable morphological and physiological assessments of larval neuromuscular junction in
Drosophila, which also offers better ways to further understanding mechanism of synaptic
transmission.
3 Drosophila melanogaster neuromuscular junction (NMJ)
Aside from advantages mentioned above, a vast number of studies on mechanisms underlying
synaptic transmission have been done using the larval neuromuscular junction synapse in
Drosophila (Figure 1A and 1B). Like most of central excitatory synapses in vertebrate, Drosophila
NMJ synapses are glutamatergic, and express many proteins homologous to human including
endocytic proteins in pre-synaptic regions and post-synaptic receptors (DiAntonio, Petersen,
Heckmann, & Goodman, 1999). In addition, distinct structure of Drosophila NMJ synapses
provide us a good way to monitor synaptic vesicles endocytosis by using styryl FM1-43 dye. FM1-
43 dye is incorporated into synaptic vesicles through endocytosis and released during exocytosis,
which enables studies about synaptic activity in real time during electrical stimulation or high K
+

depolarization (Kidokoro et al., 2004). Furthermore, distinct pools of synaptic vesicles also can be
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labeled by FM1-43 dye using different stimulation conditions (Verstreken et al., 2005; Verstreken,
Ohyama, & Bellen, 2008). Together, Drosophila NMJ synapses provide valuable insights of
synaptic transmission and further enable precise understanding in human neurological disorders.

Figure 1 Drosophila melanogaster neuromuscular junction. (A) Drosophila melanogaster
neuromuscular junction (B) Diagram of the Drosophila melanogaster neuromuscular junction.
The synapse consists of varicose functional units called boutons harboring active zones that are
essential for neurotransmitter release. Each muscle is a single muti-nucleate cellular post-synaptic
structure. Taken from (http://biochemistry2.ucsf.edu/labs/davis/projects/index.htm).
4 Synaptic vesicle pool
Rapid replenishment of synaptic vesicles through endocytosis maintains robust synaptic
transmission in neurons across a wide range of stimulation frequency without depleting their
supply of synaptic vesicles (Saheki & De Camilli, 2012; Soykan, Maritzen, & Haucke, 2016). At
A B
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least two distinct vesicle pools harbor at the Drosophila neuromuscular junction (NMJ) - the exo-
endo cycling pool (ECP) and reserve pool (RP) (Delgado, Maureira, Oliva, Kidokoro, & Labarca,
2000; Kuromi & Kidokoro, 1998; Rizzoli & Betz, 2005; Sudhof, 2004). Numerous researches have
worked on these pools respectively (Akbergenova & Bykhovskaia, 2009; Dulcis & Spitzer, 2012;
Kuromi & Kidokoro, 2000, 2002; McMahon & Boucrot, 2011).
The ECP vesicles, making up 10% to 15% of total synaptic vesicles, including readily
releasable vesicles and the recycling vesicles, are retrieved rapidly during low frequency
stimulation or high K
+
depolarization (Kuromi & Kidokoro, 1999; Rizzoli & Betz, 2005;
Verstreken et al., 2005). The RP, accounting for 80% to 90% of total synaptic vesicles, only can
be triggered during high frequency nerve stimulation in a compensative manner and is thought to
be refilled slowly after cessation of synaptic stimulation (Akbergenova & Bykhovskaia, 2009;
Kuromi & Kidokoro, 2002; Verstreken et al., 2005) (Figure2A and 2B). Studies have demonstrated
that both the ECP and RP are required for normal synaptic transmission; and the coordination
between ECP and RP makes it difficult to distinguish these two vesicle pools as they mix slowly
during high frequency nerve stimulation (Kuromi & Kidokoro, 1998, 2000, 2002; Rizzoli & Betz,
2005; Verstreken et al., 2005). A vast arrange of proteins including kinases, phosphatase, and
various scaffolding and endocytic proteins have been identified to participate and coordinate
synaptic retrieval through a series of precisely controlled endocytosis. However, whether they
differentially affect the ECP, RP, or both, are less understood.
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Figure 2 Synaptic vesicle pools. (A) At least two distinct vesicle pools harbor in synaptic
terminals. The exo-endo cycling pool (ECP) includes readily releasable pool (RRP) and recycling
pool, making up 10% to 15% of the total pool; the reserve pool (RP) accounts for 80% to 90% of
the total pool. Take from (Rizzoli & Betz, 2005) (B) Schematic diagram of conditions triggering
distinct synaptic vesicle pools. Taken from (Kidokoro et al., 2004).
5 Synaptojanin
Synaptojanin (Synj), a phosphoinositide phosphatase, is shown to play an important role in
Clathrin-mediated endocytosis and required for coated vesicles to release the Clathrin-adaptor
complex during uncoating (Cremona et al., 1999; Dickman, Horne, Meinertzhagen, & Schwarz,
2005; Haffner, Di Paolo, Rosenthal, & de Camilli, 2000; McPherson et al., 1996; Verstreken et al.,
2003). It has been reported that mutation in Synj is involved in Parkinson’s disease and Down
syndrome (Arai, Ijuin, Takenawa, Becker, & Takashima, 2002; Drouet & Lesage, 2014). In
A
B
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addition, mutations in Synj also lead to significant reduced synaptic vesicles and exhibit densely
Clathrin-coated vesicles accumulating in synaptic terminals in both vertebrates and invertebrates,
indicating Synj plays an important role in uncoating of Clathrin during Clathrin-mediated
endocytosis (Cremona et al., 1999; Dickman et al., 2005; Haffner et al., 2000; Harris, Hartwieg,
Horvitz, & Jorgensen, 2000; Mani et al., 2007; Verstreken et al., 2003).
Synj has two phosphoinositide phosphatase domains – the central 5-phosphatase domain and
N-terminal suppressor of actin 1 (SAC1) domain. In terms of enzymatic function, both of these
two domains are involved in dephosphorylating of phosphatidylinositol 4,5-bisphosphate
[PI(4,5)P2] that concentrates at plasma membrane (McPherson et al., 1996) (Figure 3A). During
Clathrin-mediated endocytosis, proteins including Clathrin adaptors and endocytic accessory
proteins bind to PI(4,5)P2 located at the inner leaflet of plasma (Di Paolo & De Camilli, 2006;
Ehlers, 2008). Dephosphorylation of PI(4,5)P2 to PI(4)P or to PI by Synj allows Clathrin adaptors
and endocytic accessory proteins to be released from Clathrin coated vesicles following
subsequent vesicle trafficking (Ehlers, 2008). The central 5’-phosphatase domain
dephosphorylates the 5 position of PI(4,5)P2 and converts it to PI(4)P, whereas N-terminal SAC1
domain further dephosphorylates PI(4)P to produce PI (McPherson et al., 1996) (Figure 3B). All
together it reveals a crucial role of Synj in phosphoinositide metabolism and further essential
function in uncoating Clathrin during Clathrin-mediated endocytosis.
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Figure 3 Domains and classical model of Synaptojanin function in Clathrin-mediated
endocytosis. (A and B) domain structure of Synj, from N terminus to C terminus Synj contains a
SAC1 domain that dephosphorylates 4 position of PI(4)P to PI, a central 5’-phosphatase domain
dephosphorylates 5 position of PI(4,5)P2 and converts it to PI(4)P, and a proline-rich domain (PRD)
that interacts with SH3 domain of endocytic proteins including Endophilin (Endo). Taken from
(Ehlers, 2008; Milosevic et al., 2011).

At C-terminal of Synj, there is another important domain – proline rich domain (PRD) which
mainly interacts with proteins containing a Src Homology 3 (SH3) domain, such as Endophilin
(Endo) and Amphiphysin (Cestra et al., 1999; McPherson et al., 1996; Ringstad, Nemoto, & De
Camilli, 1997; Schuske et al., 2003; Verstreken et al., 2003). Endophilin, known as a binding
partner of Synj, has two fundamental function during synaptic vesicle endocytosis: that N-BAR
A
B
C
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domain at N-terminal curves membrane in order to convert shallow invaginating coated pits to
deeply invaginated pits (Ambroso, Hegde, & Langen, 2014; Gallop et al., 2006), and that it recruits
Synj and stabilizes it to Clathrin coated pits during uncoating of Clathrin (Schuske et al., 2003;
Verstreken et al., 2003) (Figure 3C). However, a recent study suggested that the SAC1 domain of
Synj may also sufficiently recruit Synj to the Clathrin coated pits without interaction between PRD
of Synj and SH3 domain of Endophilin (Dong, Gou, Li, Liu, & Bai, 2015) (Figure 4). In addition,
the interaction between Endophilin and Synj is suggested to be involved in activity-dependent bulk
endocytosis (Mani et al., 2007). Together, these results shown that interaction between PRD of
Synj and protein containing SH3 domain is crucial for Clathrin-mediated endocytosis and might
be associated with activity-dependent bulk endocytosis.

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Figure 4 Latest model of Synj fuction in clathrin-mediated endocytosis. Aside from
endophilin-Synj interaction promoting synaptic vesicle endocytosis, SAC1 domain of Synj itself
appears sufficient to target Synj to endocytic membranes where endophlin resides in abscence of
endophilin-Synj interaciton, and in turn facilitates synaptic vesicle endocytosis. Taken from (Dong
et al., 2015).

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6 Synaptojanin is a substrate of Minibrain kinase
Previous study in our lab has indicated that the Minibrain (Mnb) kinase, a fly homolog of the
dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) (Tejedor et al., 1995),
phosphorylates Synj (Chen et al., 2014). mnb
1
mutant shows reduced phospho-Synj level but
normal total Synj level both in vivo and in vitro, suggesting that Synj is a substrate of the Mnb
kinase (Figure 5A and 5B) (Chen et al., 2014). Furthermore, endocytosis function also decreases
in mnb
1
mutant, indicating phosphorylation of Synj by Mnb might play an important role in
synaptic vesicle endocytosis (Figure 5C) (Chen et al., 2014).

Figure 5 Synaptojanin is a substrate of Minibrain, and phosphorylation of Synaptojanin by
Minibrain is required for synaptic vesicle endocytosis. (A) Western block of
A B
C
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immunoprecipitated Synj in the presence and absence of Alkaline Phosphatase (AP) and Mnb. The
phosphorylation levels of Synj increase in presence of Mnb. (B) immunofluorescence staining of
Synj in the NMJs of third instar larvae by using p-Synj and Synj-1 antibodies for indicated
genotypes. (C) Images of NMJs of third instar larvae after FM1-43 loading and unloading
stimulated by high K
+
depolarization. Taken from (Chen et al., 2014).

However, the site on Synj phosphorylated by Mnb has not been identified, and the precise
functional impacts of Mnb-dependent phosphorylation of Synj in regulating synaptic vesicle
recycling remain to be elucidated. Interestingly, both Mnb and Synj are overexpressed in Down
syndrome (Arai et al., 2002; Dowjat et al., 2007; Guimera, Casas, Estivill, & Pritchard, 1999), and
Synj and Mnb mutations have been linked to Parkinson’s disease and Autism (Iossifov et al., 2012;
Krebs et al., 2013; O'Roak et al., 2012; van Bon et al., 2016), respectively. An understanding of
Mnb and Synj functional interactions may thus shed light on mechanisms underlying these
neurological disorders.
In the present study, we demonstrate that the Mnb kinase phosphorylates Synj at S1029 in
vivo. Phosphorylation of S1029 by Mnb decreases Synj-Endophilin interactions, and enhances
Synj phosphoinositide phosphatase activity. FM1-43 labeling experiments revealed that
phosphorylation of Synj at S1029 is necessary for the endocytosis of the ECP, but surprisingly is
not required to maintain stable synaptic transmission during high frequency stimulation. We find
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non-phosphorylated Synj can still maintain RP endocytosis to compensate for defects in the
endocytosis of the ECP, whereas phosphorylation of Synj at S1029 promotes endocytosis of the
ECP at the expense of RP endocytosis. Together, these data reveal that Synj participates in both
the recycling of the ECP and RP vesicles, and that dynamic phosphorylation and
dephosphorylation of Synj regulate endocytosis of distinct vesicle pools at the Drosophila NMJ to
maintain stable synaptic function across a wide range of stimulation frequencies.

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CHAPTER 2
MATERIALS AND METHODS
1 Fly stocks and antibody generation
Flies were cultured at 25°C on standard cornmeal, yeast, sugar, and agar medium under a 12-
hour light and 12-hour dark cycle. The following fly lines were used: synj1 and synj2 (From Dr.
Hugo Bellen), mnb
1
(from Dr. Martin Heisenberg), UAS- PL C δ 1-PH-GFP (Bloomington stock
center # 39693), and UAS-synj
wt
(Chen et al., 2014). synj
S1029A
and synj
S1029E
transgene constructs
were generated using site-directed mutagenesis and were cloned into the pINDY6 vector, which
contains the HA tag. Transgenic flies were generated by standard transformation method. To drive
neuronal expression, n-synaptobrevin-Gal4 (nSynb-Gal4) (Pauli et al., 2008) was used (gift from
Julie Simpson). nSynb-Gal4, UAS- PL C δ 1-PH-GFP was generated by recombination. All other
stocks and standard balancers were obtained from Bloomington Stock Center (Bloomington, IN).
Polyclonal phospho-Synj
S1029
antibody was generated by immunizing rabbits with synthetic
peptide phosphorylated at serine corresponding to Synj
S1029
:
1027
PMSPKNSPRHLP
1038

(PrimmBiotech, Inc.). Phospho-specific antibody was purified by non-phospho-peptide depletion
column, followed by affinity purification using phospho-peptide affinity column. Specificity
against phosphorylated antigen was confirmed by dot assays using phosphorylated antigen and
non-phosphorylated antigen.

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2 Mass Spectrometry
His-Synj was purified and treated with alkaline phosphatase with or without Mnb as described
previously and below (Chen et al., 2014). Mass spectrophotometry was performed as described
earlier (Chen et al., 2014). Samples were analyzed using an LC/MS system consisting of an
Eksigent NanoLC Ultra 2D (Dublin, CA) and Thermo Fisher Scientific LTQ Orbitrap XL (San
Jose, CA). Proteome Discoverer 1.4 (Thermo Fisher Scientific) was used for protein identification
using Sequest algorithms. The following criteria were followed. For MS/MS spectra, variable
modifications were selected to include N,Q deamination, M oxidation and C
carbamidomethylation with a maximum of four modifications. Searches were conducted against
Uniprot or in-house customer database. Oxidized methionines and phosphorylation of tyrosine,
serine and threonine were set as variable modifications. For the proteolytic enzyme up to two
missed cleavages were allowed. Up to two missed cleavages were allowed for protease digestion
and peptide had to be fully tryptic. MS1 tolerance was 10 ppm and MS2 tolerance was set at 0.8
Da. Peptides reported via search engine were accepted only if they met the false discovery rate of
1%. There is no fixed cutoff score threshold, but instead spectra are accepted until the 1% FDR
rate is reached. Only peptides with a minimum of six amino acid lengths were considered for
identification. We also validated the identifications by manual inspection of the mass spectra.

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3 Synaptojanin PRD construction
Synj PRD constructs were cloned into PET15b vector (Novagen) between NdeI and XhoI sites.
PRD-Full contains the full PRD of Synj, which covers aa 977-1218 of Synj. PRD-1 contains aa
977-1042, PRD-2 covers aa 1043-1091, PRD-3 contains aa 1092-1139, PRD-4 contains 1140-
1218 of Synj. BL21(DE3) competent E.coli. strain containing the expression plasmids were grown
at 37°C until A600 of the culture reached 0.6-0.8. Expression of the proteins was induced by the
addition of isopropyl β−D-thiogalactopyranoside (IPTG) to a final concentration of 1.0 mM. After
growth at 30 °C for 4 hours, cells were harvested and stored at -80°C until purification. Ni-NTA
Purification System (Invitrogen) was used to purify His-PRD proteins.
4 Immunochemistry
Third-instar larvae were dissected in Ca
2+
free dissection buffer: 128 mM NaCl, 2 mM KCl,
4.1 mM MgCl2, 35.5 mM sucrose, 5 mM HEPES, 1 mM EGTA. Motor nerves were cut and
dissected preparations were fixed in 4% paraformaldehyde solution for 20 min at room temperature
(RT). Fixed samples were then washed with 0.1% triton X-100 in PBS (PBST) and blocked with
5% normal goat serum in PBST. Dissected preps were incubated with primary antibodies diluted
in blocking solution overnight at 4 °C. Antibodies were diluted as following: rabbit anti-Synj-1,
1:200; rabbit anti-p-Synj, 1:2,000; mouse anti-Dlg (4F3), 1:500 (developmental Studies
Hybridoma Bank); Cy3-conjugated anti-HRP, 1:100 (Jackson ImmunoResearch). Secondary
antibodies used were Alexa-488 or 405 conjugated, 1:250 (Invitrogen)
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5 Image Quantification
Images of synaptic terminals from NMJ 6/7 in segment A2 and A3 were captured by using a
Zeiss LSM5 confocal microscope with a × 63 1.6 numerical aperture oil-immersion objective.
Staining intensities were measured by normalizing the fluorescence intensity to bouton area
outlined by HRP using Image J. Exposure time was kept consistent for all genotypes per
experiment within same day while comparing intensity across genotypes. All values were
normalized to control done within each experimental set.
6 Immunoprecipitation and protein interaction
Fly heads (200) were homogenized in lysis buffer (10 mM HEPES, 100 mM NaCl, 10 mM
EDTA, 1% NP-40, 1 mM Na3VO4, 50 mM NaF, 250 nM cyclosporin A, and protease inhibitor)
and centrifuged to remove debris. Anti-HA-Agarose (Sigma) (20 μl) were added to the extracts
and rotated at 4°C overnight. After three washes in Lysis buffer, the immunocomplexes were
eluted with SDS sample buffer, and all of the eluates were used for Western blotting. Primary
antibodies were diluted in blocking solution as following: rabbit anti-HA, 1:200; guinea pig anti-
Endo (GP60), 1:5,000. All values were normalized to total Synj within the same experimental set.
Intensity of each band was quantified using Image J.
7 In vitro phosphorylation of Synaptojanin
Synj were immunoprecipitated using HA agarose beads from fly heads as described above,
and then dephosphorylated with Alkaline Phosphatase in CutSmart Buffer (New England BioLabs)
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at 37℃ for 1 h. Dephosphorylated Synj were then washed with kinase buffer (20 mM HEPES, pH
7.4, 10 mM MgCl2, 1 mM DTT and 2 mM Na3VO4) four times to remove CIP. For Mnb re-
phosphorylation, samples were subsequently treated with purified Mnb (0.5 μg) in kinase buffer
at 37 ℃ for 1 h and then washed with PBS for four times to remove Mnb. Synj were eluted with
SDS sample buffer, and used for Western blotting.
8 Phosphatidylinositol 5’-phosphatase activity in vitro
Determination of Synj Phosphotidylinositol 5’-phosphatase activity was previously
described(Chen et al., 2014). Adult fly heads were homogenized in Lysis buffer and Synj
immunoprecipitated using HA agarose beads. Purified Synj was incubated with labeled PI(4,5)P2
(GloPIPs BODIPY FL-PI(4,5)P2, Echelon) in inositol phosphatase activity assay buffer (30 mM
HEPES, pH 7.4, 100 mM KCl, 1 mM EGTA and 2 mM MgCl2) at room temperature for 5–10 min.
Lipid products were separated by TLC and visualized under ultraviolet radiation. Synj was eluted
with SDS sample buffer and Synj level was determined by western blotting. Phosphotidylinositol
5-phosphatase activity was normalized to the level of Synj.
9 In vivo Phosphatidylinositol 5’-phosphatase activity determination
Third instar larvae were dissected in Ca2+ free dissection buffer with motor nerves intact.
Dissected preps were fixed for 20 min in 4% Polyformaldehyde, washed with PBS, and then
incubated with HRP-Cy3 for 15 minutes. Images were taken right away using a Zeiss LSM5
confocal microscope. PL C δ-PH-eGFP fluorescence intensity was analyzed by outlining the
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bouton area using HRP in image J. All values were normalized to control done within each
experimental set.
10 FM1-43 dye labeling
Third-instar larvae were dissected in modified HL-3 Ca2+ free solution: 70 mM NaCl, 5 mM
KCl, 10 mM MgCl2, 10 mM NaHCO3, 5 mM Trehalose, 115 mM sucrose, 5 mM HEPES, 2.5 mM
EGTA. For high potassium stimulation, dissected samples were incubated with 4µM FM1-43 dye
(Invitrogen) in modified HL-3 solution containing 60mM KCl and 2mM CaCl2 for 5 min, then
washed with modified HL-3 Ca
2+
free solution. For reserve vesicle pool loading, dissected samples
were bathed in 4µM FM1-43 dye (Invitrogen) in modified HL-3 solution containing 2mM CaCl2,
and segmental motor nerves were stimulated using a suction electrode at 10Hz for 10 min plus an
additional 5 min wait without stimulation. Samples were washed with modified HL-3 Ca
2+
free
solution. For unloading experiment in both high potassium and electrical stimulation, larvae were
incubated with modified HL-3 solution containing 60mM KCl and 2mM CaCl2 for 1 min and 5
min respectively. Images were taken using Zeiss LSM5 confocal microscope with a 40x water-
dipping objective. Fluorescence intensities were calculated using Image J and normalized to the
average loading fluorescence intensity in controls within the same experimental set. The ratio of
unloaded/loaded was calculated by subtracting the fluorescence intensity remained after unloading
from the loading intensity, then divided by loaded fluorescence intensity: (Fload - Funload)/Fload. The
ratio of reserve vesicle pool was calculated by fluorescence intensity remaining after unloading
divided by loaded fluorescence intensity: Funload/Fload.
19 | P a g e

11 Electrophysiology
Third-instar larvae were dissected in modified HL-3 Ca
2+
free solution. Dissected larvae were
bathed in modified HL-3 solution containing 0.4 mM or 2mM Ca
2+
as indicated. Current-clamp
recordings were carried out on muscles 6 in abdominal segments A2 or A3, and severed ventral
nerves were stimulated with suction electrodes at 3 mSec stimulus duration. The recording
electrode with resistance between 20-40MΩ was filled with 3M KCl. Recordings were discarded
if Vm changed by > 10% and also were rejected in which the stimulated nerve did not function
fully throughout the recording which was determined by abrupt drops in EPSP amplitude. Data
was acquired using an Axopatch 200B amplifier, digitized using a Digidata 1440A, and controlled
using pClamp 10.3 software (Molecular Devices, Sunnyvale, CA). Electrophysiological sweeps
were sampled at a rate of 10 kHz and filtered at 400 Hz. Data was analyzed using MiniAnalysis
(Synaptosoft), SigmaPlot (Systat Software), and Microsoft Excel. Nonlinear summation was used
to correct the average EPSP amplitude. Bafilomycin A1 (Adipogen) and 1-(5-iodonaph-thalene-1
sulfonly1)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7) (Sigma) were used at 2μM and
1μM, respectively. For drug treatment, dissected preps were incubated with drug containing
modified HL-3 solution (2 mM Ca
2+
) for 30 minutes prior to recording.
12 Statistics
For paired samples, Student’s T-test was used. For multiple samples, ANOVA test followed
by Bonferroni posthoc test was used to determine statistical significance.
20 | P a g e

CHAPTER 3
RESULTS
1 Minibrain kinase phosphorylates Synaptojanin at S1029 in vitro and in
vivo
Mnb is a proline-directed kinase previously shown to phosphorylate Synj and regulate
synaptic vesicle endocytosis (Adayev, Chen-Hwang, Murakami, Wang, & Hwang, 2006; Chen et
al., 2014; Himpel et al., 2000). To further delineate the effects of Synj phosphorylation by Mnb,
we set out to identify the site(s) on Synj phosphorylated by Mnb. To this end, we purified HA-
tagged Synaptojanin (Synj-HA) from bacteria, treated it with alkaline phosphatase to remove the
existing phosphorylation, and then incubated Synj with or without purified Mnb kinase. LC-
MS/MS was then performed to identify phosphorylation of Synj by Mnb kinase. Serine 1029
(S1029) was the only Ser/Thr-Pro site differentially phosphorylated by Mnb with greater than 75%
probability (Figure 6A). Note that treatment of purified Synj with alkaline phosphatase is
necessary since it was shown that the majority of Synj purified from bacteria is phosphorylated
(Chen et al., 2014). Figure 6A further highlights that although the proline rich domain (PRD) of
Synj is not well conserved between fly and human, this potential Ser-Pro phosphorylation site is
conserved.
To further confirm that Mnb kinase can indeed phosphorylate Synj at S1029, we generated
transgenic flies with HA-tagged Synj phospho-null mutation (synj
S1029A
) and HA-tagged Synj
21 | P a g e

phospho-mimetic mutation (synj
S1029E
). First, we took an in vitro approach to test whether Mnb
kinase does indeed phosphorylate Synj at S1029. Using an antibody generated against
phosphorylated Synj at S1029 (p-SynjS1029), we confirmed that Mnb can phosphorylate HA-
tagged wildtype Synj (Synj
wt
) immunoprecipitated from fly heads (Figure 6B). However, Mnb
failed to phosphorylate Synj
S1029A
or Synj
S1029E
, confirming that Mnb does indeed act on S1029
(Figure 6B). Similar results were obtained using an antibody previously shown to be sensitive to
Mnb-dependent phosphorylation of Synj (p-Synj; Figure 6B), indicating p-Synj antibody is also
sensitive to phosphorylation at Ser 1029 (Chen et al., 2014). To further determine if Mnb acts on
other sites in addition to Ser 1029, we performed the experiment using an antibody specific for
phosphorylated Ser/Thr-Pro. Figure 5C shows that incubation of Mnb with Synj increased the p-
Ser/Thr-Pro signal detected for Synj
wt
, but not for Synj
S1029A
or Synj
S1029E
. Together, these results
demonstrate that Mnb phosphorylates Synj at S1029 and does not act on other Ser/Thr-Pro sites
with high efficiency.

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Figure 6 Minibrain phosphorylates Synaptojanin at S1029 in vitro. (A) Protein sequence
alignment of partial Drosophila Synj-1 PRD domain with mouse and human Synj-1 PRD domain
(Clustal Omega). Phosphorylation of Ser1029 by Mnb precedes a Proline residue, consistent with
Mnb being a proline-directed kinase (highlighted in yellow). (B), (C) Validation that Mnb
phosphorylates Synj at Ser1029 using the indicated antibodies. HA-tagged Synj
wt
, Synj
S1029A
,
Synj
S1029E
were expressed in flies, immunoprecipitated using HA-agarose beads, and incubated
with or without purified Mnb kinase following alkaline phosphatase (AP) treatment. Mutation at
S1029 abolished the increased phosphorylation by Mnb. (These results were obtained by Chun-
Kan Chen and Dr. Junhua Geng).
A
DROME|SYNJ_PRD 1006 TPELPQRPKQ PPTRPPARPP MPMSPKNSPR 1035
HUMAN|SYNJ_PRD 1061 VPSLPIRPSR APSRTPGPPS AQSSPIDAQP 1090
MOUSE|SYNJ_PRD 1061 APSLPIRPSR APSRTPGPPS SQGSPVDTQP 1090
.*.** **.: *:* *. * ** :：
C B
+ + + + + + + + AP
- + - + - + - + MNB
+
p-Synj
170kD
130kD
HA
170kD
130kD
p-Synj
S1029

170kD
130kD
IP: HA
nSynb-Gal4
+
+ + + + + + + + AP
- + - + - + - + MNB
170kD
130kD
p-Ser/Thr-Pro
HA
170kD
130kD
IP: HA
nSynb-Gal4
23 | P a g e

To investigate if phosphorylation of Synj at S1029 indeed occurs in vivo, we performed
immunostaining at the NMJ using phospho-Synj antibodies. Unfortunately, our newly generated
phospho-Synj
S1029
antibody did not work for immunostaining, we therefore used the p-Synj
antibody described previously (Chen et al., 2014). We found that expression of Synj constructs led
to an increase in the levels of total Synj and phospho-Synj staining compared to control (Figure
7A and 7B). After normalizing the phospho-Synj signal to total Synj signal, we find that only
Synj
wt
expression showed a significant increase in relative phospho-Synj level, whereas Synj
S1029A

and Synj
S1029E
both did not (Figure 7B). The fact that we observed an increase in phospho-Synj
signal when S1029 is mutated suggests that this antibody may recognize another phosphorylation
site other than S1029. We thus performed epitope mapping of the phospho-Synj antibody using
purified truncated proline-rich region of Synj. Figure 7C confirms that phospho-Synj antibody
indeed recognizes and is sensitive to phosphorylation of two different regions of Synj: PRD-1,
which covers S1029, as well as PRD-4 (see methods for information on different PRD constructs).
Since we did not detect differential phosphorylation of Synj by Mnb at another site using mass
spec or when we mutated S1029 (Figure 6), phosphorylation of PRD-4 region is independent of
Mnb. Furthermore, these results suggest that the relative phospho-Synj to total Synj level better
reflects the extent of Synj phosphorylation at S1029.
To further confirm that phosphorylation of Synj at S1029 is dependent on Mnb in vivo, we
examined phospho-Synj signal in mnb
1
mutant background. While mnb null mutants are lethal,
mnb
1
mutant harbors a point mutation that impairs kinase function (Chen et al., 2014; Ori-
24 | P a g e

McKenney et al., 2016; Tejedor et al., 1995), leading to an overall decrease in phospho-Synj signal
(Figure 7D and 7E). Indeed, expression of Synj
S1029A
and Synj
S1029E
constructs in mnb
1
mutant
background resulted in the same reduced phospho-Synj to total Synj ratio as observed for mnb
1

mutant. Although expression of Synj
wt
in mnb
1
mutant background did not decrease the ratio of
phospho-Synj to total Synj to that of mnb
1
level, it was nevertheless lower than expression in
wildtype background (Figure 7B). This result confirms that Mnb can phosphorylate Synj at S1029
in vivo. Note that the subtle increase in phospho-Synj to total Synj signal is likely caused by an
increase in the amount of available substrate, Synj, since mnb
1
impairs rather than completely
abolishes kinase activity (Chen et al., 2014; Tejedor et al., 1995). Together, our results indicate
that Mnb can phosphorylate Synj at S1029, and that phosphorylation of Synj at S1029 occurs in
vivo.

25 | P a g e

26 | P a g e

Figure 7 Phosphorylation of Synaptojanin at S1029 in vivo. (A) Staining of Synj in the third
intar NMJ using p-Synj and Synj-1 antibodies for the indicated genotypes in wild type background.
Scale bar = 5µm. (B) Quantification of relative staining intensity for p-Synj and Synj signals
normalized to control (top graph), and the relative p-Synj/Synj signals. Error propagation was
used to calculate fold change and standard error. n > 12 NMJ per genotype. (C) p-Synj antibody
detects RPD-1 and PRD-4 sequences (left panel). Upper blots show western blots detected with
pSynj antibody. Lower panels show direct blue 71(DB71) staining of the nitrocellulose membrane,
showing the amount of protein loaded in each lane. Right panel shows p-Synj does not detect
signals well following alkaline phosphatase (AP) treatment, confirming the antibody is sensitive
to phosphorylation status of Synj. (D) Staining of p-Synj and Synj-1 antibodies of the 3rd instar
NMJ for the indicated genotypes in mnb
1
mutant background. Scale bar = 5µm. (F) quantification
of relative staining intensity for p-Synj/Synj signals. n = 11 NMJ per genotype. All values
represent mean ± SEM, and * indicates p < 0.05 as compared to control. (These experiments were
done in collaboration with Syed Qadri).

2 Phosphorylation of Synaptojanin at S1029 enhances Synaptojanin
activity and alters Synaptojanin interaction with Endophilin
Previous studies have shown that Synaptojanin is a phosphoinositide phosphatase capable of
regulating PI(4,5)P2 levels (Lee, Wenk, Kim, Nairn, & De Camilli, 2004; McPherson et al., 1996).
27 | P a g e

To determine if phosphorylation of Synj at S1029 regulates its enzymatic activity, we measured
the 5’-phosphatase activity of immunoprecipitated Synj protein as assayed by conversion of
BODIPY-labeled PI(4,5)P2 to BODIPY-PI(4)P using TLC (Figure 8A). Consistent with previous
results, addition of Mnb enhanced Synj
wt
activity, while it failed to enhance the 5’-phosphatase
activity of Synj
S1029A
or Synj
S1029E
constructs (Figure 8A and 8B). Interestingly,
immunoprecipitated Synj
S1029E
displayed higher 5’-phosphatase activity compared to Synj
wt
construct, suggesting that phosphorylation at this site is necessary and sufficient to enhance Synj
enzymatic activity. Furthermore, we tested the importance of Synj phosphorylation at S1029 in
regulating its interaction with Endophilin, an endocytic protein thought to recruit Synj to the
synapse (Figure 8C and 8D) (Schuske et al., 2003; Verstreken et al., 2003). We found that the
phospho-null Synj
S1029A
construct displayed increased interaction with Endophilin, whereas the
phospho-mimetic Synj
S1029E
construct showed reduced interaction (Figure 8C and 8D). These
results are consistent with our previous findings that mnb
1
mutants show elevated Synj-Endophilin
interaction and decreased Synj activity (Chen et al., 2014). Together, these results reveal that
phosphorylation of Synj at S1029 is sufficient to alter both Synj phosphatase activity and regulate
Synj interaction with Endophilin.
To confirm if phosphorylation of Synj is sufficient to alter Synj activity locally at the
synapse in vivo, the EGFP fusion protein containing the phospholipase Cδ1 pleckstrin homology
domain (PLCδ1-PH-GFP) was used (Khuong, Habets, Slabbaert, & Verstreken, 2010). PLCδ1-
PH-GFP has been shown to specifically bind to PI(4,5)P2, so that the fluorescence intensity in the
28 | P a g e

synapse directly reflects the level of PI(4,5)P2. Indeed, PLCδ1-PH-GFP signal was elevated in
synj1/synj2 mutant (here after referred to as synj mutant), confirming reduced Synj activity (Figure
8E and 8F). Expression of synj
wt
in synj mutant restored PLCδ1-PH-GFP signal to control level
while expression of Synj
S1029A
did not. This lack of rescue by synj
S1029A
construct is not due to
differential Synj expression, since the level of total Synj is the same in different transgenic lines
in synj mutant background (p > 0.17 when comparing total Synj level in different transgenic lines).
Furthermore, expression of phospho-mimetic Synj
S1029E
in synj mutant led to reduced PLCδ1-PH-
GFP signal locally within the synapse when compared to the control, consistent with our
biochemical data that phosphorylation of Synj at S1029 is sufficient to elevate Synj activity.

29 | P a g e

30 | P a g e

Figure 8 Phosphorylation of Synaptojanin at S1029 enhances Synaptojanin phosphoinositide
phosphatase activity and alters its interaction with Endophilin. (A) TLC showing conversion
of BODIPY-PIP2 to BODIPY-PIP by Synj constructs. Lower panels show levels of total Synj. (B)
Quantification of relative PIP to total phospholipid (PIP+PIP2) level. n = 8 independent
experiments per genotype per condition. (C) Immunoprecipitation (IP) experiment using flies
overexpressing Synj tagged with HA reveals that Synj
S1029A
has strong interaction with Endophilin.
* marks a background band due to IgG. (D) Quantification of relative levels of Endophilin-Synj
interaction. n = 7 independent experiments. Values have been normalized to total Synj and then
fold change calculated relative to control. (E) PIP2 levels in the NMJ measured by PLCδ-PH-GFP
for the indicated genotypes. (F) Quantification of PIP2 levels in the synapse. n > 12 NMJ per
genotype. All values are mean ± SEM. * indicates P ≤ 0.05. (These experiments were done in
collaboration with Dr. Junhua Geng).

3 Phosphorylation of Synaptojanin at S1029 is required for endocytosis
but is not necessary for reliable neurotransmission at high stimulation
frequency
Synaptojanin is known to play an important role in synaptic vesicle recycling (Cremona et al.,
1999; Dickman et al., 2005; Mani et al., 2007; Schuske et al., 2003; Verstreken et al., 2003). To
further understand the impact of Synj phosphorylation in regulating synaptic vesicle endocytosis,
31 | P a g e

we measured the ability of wildtype, phospho-null, and phospho-mimetic Synj constructs in
rescuing the endocytosis defects of synj mutant. To this end, we used FM1-43 dye labeling, a
lipophilic dye that can incorporate into synaptic vesicles during endocytosis. Compared to control,
stimulation of synj mutant for 5 min using high extracellular potassium (60 mM) resulted in a
significantly reduced FM1-43 loading (Figure 9A and 9B). Expression of Synj
wt
and synj
S1029E

construct in synj mutant rescued FM1-43 load but the phospho-null Synj
S1029A
construct did not.
Because FM1-43 uptake is a function of both endocytosis and exocytosis, we further determined
if phospho-defective Synj
S1029A
influences exocytosis by calculating the amount of FM1-43 that
was released following subsequent stimulation, and then normalized it to the total load. As seen in
Figure 9B, there was no difference in FM1-43 ΔF/Fload across genotypes, suggesting that
phosphorylation of Synj does not influence synaptic vesicle exocytosis but is important for active
endocytosis of synaptic vesicles. Furthermore, this result indicates that dephosphorylated
Synj
S1029A
is defective in the endocytosis of the active recycling pool.

32 | P a g e

Figure 9 Phosphorylation of Synaptojanin at S1029 is required for normal synaptic vesicle
endocytosis. (A) Representative images of the NMJs after FM1-43 loading and unloading for the
indicated genotypes in Synj1/Synj2 background. 60 mM K
+
stimulation for 5 min was used to load
the dye, and 1 min stimulation with 60 mM K
+
was used to unload FM1-43. Scale bar = 5µm. (B)
Quantification of relative load and unload intensity for FM1-43 dye with normalization to loading
intensity of the control. (C) Quantification of FM1-43 signal removed during unloading
normalized to amount of FM1-43 loading. n ≥ 7 NMJ per genotype. All values represent mean ±
SEM, and * indicates p < 0.05 as compared to control of the same condition.

Robust synaptic vesicle endocytosis is particularly important for maintaining synaptic
transmission during high stimulation frequencies. We therefore used electrophysiological
33 | P a g e

recordings to assay if phosphorylation of Synj differentially affects sustained synaptic
communication. Electrophysiology recordings indicate that the normal evoked EPSP amplitude
was comparable across genotypes (Figure 10A), consistent with normal exocytosis in different
genotypes. Stimulation of synj mutant at high frequency (10 Hz) for a prolonged period (10 min)
caused a fast rundown of EPSP, characteristic of endocytic mutants (Figure 10B and 10C).
Expression of synj
wt
and synj
S1029E
in synj mutant background rescued the fast rundown phenotype
of synj mutant, consistent with the FM1-43 loading data. Surprisingly, despite a substantial defect
in FM1-43 load, expression of synj
S1029A
in synj mutant background also restored synaptic
transmission to normal (Figure 10C).

34 | P a g e

Figure 10 Expression of either phospho-null or phospho-mimetic Synaptojanin can rescue
neurotransmission defect of synj mutant at high stimulation frequency. (A) Average evoked
EPSP recorded using HL-3 containing 0.4 mM Ca
2+
. n = 6 per genotype. (B) Representative
EPSP recordings during 10 Hz stimulation for 10 min in HL-3 containing 2 mM Ca
2+
. (C) Relative
EPSP amplitude plotted over time for the indicated genotypes. n > 6 for each genotype. * indicates
p < 0.05 compared to control. All values represent mean ± SEM. (These results were obtained by
Joo Yeun Lee).
35 | P a g e

4 Synaptojanin is required to maintain synaptic vesicle pool size
The observation that synaptic transmission is maintained at high stimulation frequency despite
a substantial defect in endocytosis as revealed by FM1-43 loading is intriguing and unexpected for
Drosophila mutants with problems in synaptic vesicle endocytosis. There are at least two
functionally distinct synaptic vesicle pools at the Drosophila NMJ: the ECP that is activated by
high potassium or low frequency stimulation; the RP that is poorly accessed at low frequency but
efficiently recruited by high frequency stimulation at 10 Hz or above (S. M. Kim, Kumar, Lin,
Karunanithi, & Ramaswami, 2009; Kuromi & Kidokoro, 1998, 2002). Defects in maintaining
either pool size via endocytosis or in recruitment of these vesicle pools can affect synaptic
transmission (Kuromi & Kidokoro, 1998, 2002; Verstreken et al., 2005). Since our FM1-43
experiment done using high potassium loading condition revealed that phosphorylation of Synj at
S1029 is required for endocytosis of the ECP vesicles, the fact that we did not observe a
neurotransmission problem in the phospho-null Synj
S1029A
NMJ during 10 Hz electrical stimulation
further suggests that compared to synj mutant, the dephosphorylated Synj may 1) harbor a larger
ECP and/or RP vesicle pool size, and/or 2) enhance mobilization of vesicles from the RP to
compensate for defect in ECP endocytosis. First, we asked if phospho-null Synj
S1029A
maintains
stable synaptic transmission in synj mutant by increasing the size of the ECP vesicles. We used
electrophysiology to address this question since previous studies suggested that the ECP and RP
are intermixed in the synapse and that standard EM and FM1-43 cannot readily differentiate the
distinct pools based on location of the vesicles and staining pattern (Akbergenova & Bykhovskaia,
36 | P a g e

2009; Denker, Krohnert, & Rizzoli, 2009), respectively. We used bafilomycin A1, a drug that
blocks the refilling of vesicles with neurotransmitter (Roseth, Fykse, & Fonnum, 1995), to estimate
the size of the ECP using low frequency synaptic stimulation similar to a protocol described earlier
(S. M. Kim et al., 2009). Electrical stimulation at 3 Hz in the presence of bafilomycin A1 treatment
resulted in depletion of the ECP at this low stimulation frequency (Figure 11A). The initial phase
of the decay in EPSP amplitude represents the release of the ECP vesicles, whereas the slower
decay phase results from a slow mixing of vesicles between the RP and ECP. We find that
expression of the phospho-null Synj
S1029A
in synj mutant background showed a slower decline in
relative EPSP amplitude compared to synj mutant, suggesting that it has a larger ECP (Figure 11A).
To estimate the size of the ECP, we plotted the cumulative quantal content and used linear
regression to determine the size of the ECP (Figure 11A and 11B) (Delgado et al., 2000; S. M.
Kim et al., 2009). We find that compared to control, synj mutant showed a substantially reduced
ECP, revealing Synj is crucial for the maintenance of the size of the ECP (Figure 11C). Expression
of synj
wt
, phospho-null synj
S1029A
in synj mutant background all restored the size of the ECP.
Interestingly, expression of the phospho-mimetic synj
S1029E
in synj mutant background instead
enlarged the estimated ECP size (Figure 11C). Together, these results thus imply that the phospho-
null Synj
S1029A
rescues synaptic transmission of synj mutant by restoring ECP size to compensate
for endocytic defect, and that the phospho-mimetic Synj
S1029E
may enhance endocytosis of the ECP
vesicle.
37 | P a g e

We also determined the total vesicle pool size by depleting synaptic vesicles at 10 Hz
stimulation frequency in the presence of bafilomycin A1 and measured the cumulative quantal
content. We found that synj mutants showed a substantially reduced total synaptic vesicle pool
size compared to control (Figure 11D). This suggests that Synj is required to maintain both the
ECP and RP vesicle pool size, presumably through its ability to affect vesicle endocytosis. This
result is consistent with previous EM studies demonstrating depletion of synaptic vesicles in the
synaptic boutons of synj animals (Dickman et al., 2005; Verstreken et al., 2003). We also found
that expression of different synj constructs all rescued the total number of synaptic vesicle pools
to normal (Figure 11D), suggesting that the phosphorylation status of Synj does not regulate total
synaptic vesicle pool size. Interestingly, since the total pool size is a sum of the ECP and RP, the
larger ECP size observed for phospho-mimetic Synj
S1029E
further indicates that it has a reduced RP
size.
Next, we tested if altered mobilization of vesicles from the RP contributes to the rescue of
synj mutant by phospho-null synj
S1029A
expression. To this end, we blocked RP mobilization by
inhibiting the myosin light chain kinase (MLCK) activity (Mochida et al., 1994; Ryan, 1999).
Incubating control NMJ with ML-7, which has previously been shown to block MLCK and RP
mobilization (Kim et al., 2009; Verstreken et al., 2005), caused a gradual decline in evoked EPSP
during high frequency stimulation. We reasoned that if Synj
S1029A
does indeed rescue synaptic
transmission of synj mutant by facilitating RP mobilization, inhibiting RP recruitment would
abolish its ability to restore synaptic transmission during high frequency stimulation to become
38 | P a g e

synj mutant phenotype. We found that inhibiting RP mobilization in synj
S1029A
flies still rescued
synaptic transmission to control level during early phase of stimulation, but caused a more rapid
decline in EPSP amplitude so that it no longer rescued synj mutant phenotype toward the later
phase of stimulation (Figure 11E). Since the RP is thought to be recruited following depletion of
the readily releasable pool and the recycling pool (ECP), our results support the claim that
synj
S1029A
can rescue ECP size in synj mutant, and further indicate that they rely on mobilization
of the RP during persistent stimulation conditions to maintain stable synaptic transmission in the
presence ECP recycling defects. Interestingly, consistent with the observation that synj
S1029E
has a
larger ECP size, synj
S1029E
maintained synaptic transmission a little better than control for a short
duration in the absence of RP mobilization (Figure 11E).

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40 | P a g e

Figure 11 Phosphorylation of Synaptojanin differentially affects the size of the ECP and RP.
(A) Relative EPSP amplitude plotted over time for indicated genotypes in the presence of 2µM
bafilomycin A1 with 3Hz stimulation. Expression of the phospho-null Synj
S1029A
restores the size
of the ECP. Values represent mean ± SEM. * indicates p < 0.05 when comparing synj1/synj2 to
phospho-null synj
S1029A
. (B) Cumulative quantal content plot. Linear regression analysis was used
to back extrapolate from points between stimulus pulses 2,500 and 4,200 for indicated genotypes.
ECP estimates were observed from the y-intercepts. (C) Box plot showing ECP estimates obtained
from linear regression analysis of cumulative quantal content plot for the indicated genotypes. The
red dot highlights the mean value. * indicates p < 0.05 as compared to control, ** means p < 0.05
for the indicated genotypes when compared to synj1/synj2. For (A)-(C), n = 6 for each genotypes.
(D) Total synaptic vesicle pool size estimates for the indicated genotypes. synj1/synj2 has a
reduced total pool size. n = 4 per genotype. Values represent mean ± SEM. * indicates p < 0.05
as compared to control. (E) Relative EPSP amplitude in the presence of ML-7 treatment to inhibit
mobilization of the RP vesicles. n > 6 per genotype. Values are mean ± SEM. * (red) indicates p
< 0.05 when comparing synj
S1029A
expression in synj mutant background to synj mutant. * (blue)
indicates p < 0.05 when comparing control or synj
S1029E
expression in synj mutant background to
synj mutant. The bracket and ** highlights p < 0.05 when comparing control to synj
S1029E

expression in synj mutant background. Nonlinear summation correction was used to determine
quantal content. (These experiments were done in collaboration with Joo Yeun Lee).
41 | P a g e

5 Phosphorylation status of Synaptojanin differentially affect the
recycling of ECP and RP
Our data indicates that Synj is required to maintain the vesicle pool size, but how does
phospho-null synj
S1029A
, which is defective in ECP endocytosis, rescue the vesicle pool size? We
hypothesized that dephosphorylated Synj may still retrieve vesicles through a slower pathway such
as endocytosis of the RP that has been shown to occur at the cessation of stimulation, so that
vesicles could eventually replenish both the RP and the ECP. To this end, we took advantage of
an established FM1-43 labeling paradigm to label both the ECP and RP by including FM1-43 both
during 10 minutes of 10 Hz electrical stimulation and 5 minutes post stimulation (Verstreken et
al., 2008). Relative RP vesicle endocytosis was then determined by unloading the ECP with high
KCl. FM1-43 load thus represents the amount of both ECP + RP endocytosis, whereas FM1-43
signal remaining after unload with high KCl represents endocytosis of the RP. We found that
compared to control, synj mutant showed a substantial decrease in both FM1-43 load and FM1-43
retention following ECP unloading, suggesting Synj protein is crucial for ECP and RP endocytosis
(Figure 12A). This claim is supported by observation that Synj
wt
expression rescued both the
endocytosis of the ECP and RP in synj mutant to normal (Figure 12A and 12B). On the other hand,
expression of phospho-null Synj
S1029A
showed a reduced FM1-43 load but normal FM1-43
retention following ECP unload, suggesting that it has normal RP endocytosis but reduced ECP
endocytosis. More intriguingly, expression of phospho-mimetic Synj
S1029E
restored FM1-43 load
while displaying reduced FM1-43 dye retention following subsequent high potassium stimulation,
42 | P a g e

indicating enhanced ECP endocytosis but reduced RP endocytosis. This result is consistent with a
larger ECP but smaller RP size as determined electrophysiologically for Synj
S1029E
. We also
calculated the relative amount of RP endocytosis by normalizing FM1-43 staining remaining after
ECP unloading to the total FM1-43 uptake (Figure 12C). This calculation represents the proportion
of vesicle retrieval that went through the RP route since they were not readily released by KCl
depolarization. We found that phospho-mimetic Synj
S1029E
flies have reduced RP endocytosis,
whereas synj mutant did not alter the proportion that went through the RP pathway, likely because
it was deficient in both RP and ECP endocytosis. Together these results suggest that the
phosphorylation status of Synj differentially regulates the endocytosis of different vesicle pools,
in which phosphorylated Synj preferentially participates in the endocytosis of the ECP at the
expense of RP endocytosis, while dephosphorylated Synj has defective ECP endocytosis but
maintains the endocytosis of the RP.

43 | P a g e

Figure 12 Phosphorylation of Synaptojanin affects endocytosis of the RP. (A) Representative
images of the NMJ after FM1-43 loading and unloading. 10 Hz electrical stimulation for 10 min
plus an additional 5 min waiting was used to load both the ECP and RP. A subsequent 5 min
stimulation using 60 mM K
+
was used to unload the ECP, revealing the amount of RP endocytosis.
Scale bar = 5 µm. (B) Relative FM1-43 loading and unloading intensities for indicated genotypes
with normalization to the loading intensity of control. Values represent mean ± SEM, and *
indicates p < 0.05 as compared to control of the same loading/unloading condition. (C) Box plot
showing the relative amount of RP endocytosis by normalizing FM1-43 dye remaining after ECP
44 | P a g e

unloading to the total FM1-43 dye loading. Red dot highlights the mean. n > 6 per genotype. *
indicates p < 0.05 as compared to control. (These experiments were done in collaboration with Dr.
Junhua Geng).

45 | P a g e

CHAPTER 4
DISCUSSION
In this study, we demonstrate that Mnb phosphorylates Synj at S1029 in vivo, and that the
phosphorylation status of Synj differentially regulates endocytosis of distinct functional synaptic
vesicle pools at the Drosophila NMJ. We report that phosphorylation of Synj at S1029 enhances
Synj 5’-phosphatase activity, reduces interaction between Synj and Endophilin, and preferentially
promotes the recycling of the ECP vesicles at the expense of RP recycling. On the other hand,
dephosphorylated Synj maintains RP vesicle endocytosis but is defective in ECP vesicle
endocytosis. Our data reveal a novel role for Synj in both ECP and RP vesicle endocytosis and in
maintaining vesicle pool size, and suggests that coordinated phosphorylation and
dephosphorylation of Synj during synaptic activity optimizes endocytosis of different functional
synaptic vesicle pools. Together with our previous data showing acute phosphorylation of Synj by
the Mnb kinase during synaptic activity (Chen et al., 2014), we propose a simplified model in
which synaptic activity triggers Mnb-dependent phosphorylation of Synj at S1029, which acts to
selectively enhance ECP endocytosis during stimulation. Dephosphorylation of Synj at S1029 then
promotes RP vesicle endocytosis (Figure 13A and 13B). This precisely controlled
phosphorylation-dephosphorylation cycle delicately controls the proper timing of the ECP and RP
endocytosis to sustain communication across a wide range of synaptic activities.
46 | P a g e

Figure 13 schematic model of different phosphorylation status of Synaptojanin differentially
regulates synaptic vesicle endocytosis of distinct vesicle pools. (A and B) Model depicting
recycling of distinct functional vesicle pools modulated by phosphorylated-Synj or
dephosphorylated Synj.

How does Synj modulate endocytosis of both the ECP and RP vesicles? While some studies
propose that newly reformed vesicles are recaptured randomly by different vesicle pools
(Schikorski & Stevens, 2001), others suggest that the ECP and RP vesicles are retrieved through
different mechanisms (Koenig & Ikeda, 1996; Kuromi & Kidokoro, 2002). Our findings that the
phosphorylation status of Synj differentially affected distinct synaptic vesicle pool recycling
support the model that the ECP and RP are recycled through specific pathways. It is thought that
A B
47 | P a g e

the recycling of the ECP vesicles, which participate in synaptic vesicle release during moderate
synaptic activity, is rapidly replenished by Clathrin-mediated endocytosis (Cheung, Jupp, &
Cousin, 2010; Rizzoli & Betz, 2005). Synj has an established role in Clathrin-mediated
endocytosis. synj mutations in vertebrates and invertebrates lead to an elevation in PI(4,5)P2 levels,
accumulation of densely coated vesicles, and a delay in endocytic vesicle reavailability, suggesting
that Synj functions in Clathrin uncoating through dephosphorylation of PI(4,5)P2 (Cremona et al.,
1999; Dickman et al., 2005; Harris et al., 2000; W. T. Kim et al., 2002; Verstreken et al., 2003).
Our observations that the phospho-mimetic Synj
S1029E
rescued ECP endocytosis of synj mutant and
enhanced the size of the ECP (while the dephosphorylated Synj did not) thus imply that
phosphorylation of Synj selectively participates in Clathrin-mediated endocytosis. In addition, our
findings that phosphorylated Synj has reduced interaction with Endophilin but enhanced PI(4,5)P2
phosphatase activity are consistent with reports that Synj-Endophilin interaction is dispensable,
whereas the 5’-phosphatase activity of Synj is obligatory for Clathrin-mediated endocytosis during
mild activity (Dong et al., 2015; Granseth, Odermatt, Royle, & Lagnado, 2006; Mani et al., 2007).
The RP vesicles, which are recruited only during intense neuronal activity, are thought to be
selectively replenished by a bulk endocytosis pathway (Cheung et al., 2010; Clayton & Cousin,
2009; Delgado et al., 2000; Rizzoli & Betz, 2005). The role that Synj plays in this pathway is not
well understood, although recordings from hippocampal neurons of synj-/- mutant suggest that
Synj is required for all modes of synaptic retrieval, including bulk endocytosis (Mani et al., 2007).
Using FM1-43 loading and unloading conditions that permit preferential labeling of RP vesicles,
48 | P a g e

we confirmed that in addition to ECP endocytosis, synj is also important for normal RP endocytosis.
This is further supported by our electrophysiological recordings demonstrating a smaller total
vesicle pool size in synj mutant, as well as previous work showing an overall depletion of synaptic
vesicles at the synapse as revealed by EM (Dickman et al., 2005; Verstreken et al., 2003). Although
the molecular mechanisms differentially regulating ECP and RP endocytosis are not well
understood, we anticipate that Synj interaction with Endophilin will likely play an important role.
Synj-Endophilin interaction has been proposed to be particularly important for bulk or RP
endocytosis since expression of Synj that cannot interact with Endophilin display selective
endocytic defects during intense but not mild neuronal activity (Clayton & Cousin, 2009; Mani et
al., 2007). Interestingly, we also find that phosphorylated Synj, which shows reduced interaction
with Endophilin, displays decreased RP endocytosis. Conversely, dephosphorylated Synj, which
display increased interaction with Endo, maintains RP endocytosis while having a reduced 5’-
phosphatase activity. We envision a scenario in which dephosphorylated Synj is preferentially
recruited by Endophilin. Instead of targeting and stabilizing Synj to Clathrin coated pits,
interaction between dephosphorylated Synj and Endo leads to modification of the membrane
phospholipids at a slower rate due to the lower enzymatic activity of the dephosphorylated Synj,
thus providing a distinct temporal and spatial profile required for different synaptic vesicle
retrieval modes. RP vesicles can then be regenerated from bulk endosomes (Cheung & Cousin,
2013; Cheung et al., 2010; Clayton & Cousin, 2009; Richards, Guatimosim, & Betz, 2000; Rizzoli
& Betz, 2005; Wu et al., 2014), and mobilization of the RP can replenish the ECP vesicles as well
49 | P a g e

as restore the size of vesicle pools to sustain synaptic transmission during high frequency
stimulation (Kidokoro et al., 2004; Kuromi & Kidokoro, 2000; Richards et al., 2000). In addition,
Synj-Endophilin interaction is essential for prolonged stimulation but for mild stimulation, and
both phosphatase domains of Synj are required for efficient synaptic vesicle endocytosis to retrieve
membrane, which SAC1 appears to play an important role in milder stimulation (Mani et al., 2007).
It is possible that phosphorylation of Synj on S1029 by Mnb enhanced functions of both
phosphatase domains including SCA1 domain, which recently has shown that is sufficient to target
and stabilize Synj to Clathrin coated pits during Clathrin-mediated endocytosis (Dong et al., 2015).
If so, it might give a rational explanation that phospho-mimetic Synj
S1029E
shows decreased
interaction with Endo but normal ECP endocytosis. In line with this, we find that phospho-null
Synj
S1029A
becomes dependent on mobilization of RP during prolonged high frequency stimulation.
The exact role of Synj in RP/bulk endocytosis and RP formation/mobilization, as well as the
molecular mechanisms differentially regulating Clathrin-mediated and bulk endocytosis, remain
interesting areas to investigate in the future. In addition, as perturbations in Synj and Mnb have
been linked to neurological disorders including Parkinson’s, Autism, and Down syndrome (Arai
et al., 2002; Dowjat et al., 2007; Guimera et al., 1999; Krebs et al., 2013; O'Roak et al., 2012),
understanding mechanisms modulating Mnb-dependent phosphorylation of Synj and identifying
phosphatase(s) that trigger dephosphorylation of Synj will be important topics to study in the future.

50 | P a g e

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Abstract (if available)

Abstract Impaired synaptic transmission is a pathological alternation commonly found in various neurological disorders including Down syndrome (DS), Parkinson’s and Autism. The rapid replenishment of synaptic vesicles through endocytosis during intense neuronal activity is essential for sustained function of nervous system. Synaptojanin (Synj), a phosphoinositide phosphatase, is known to play an important role in facilitating uncoating of Clathrin from coated vesicles following synaptic vesicle uptake. Previous work in our lab has demonstrated that Synj is a substrate of the Minibrain (Mnb) kinase, a fly homolog of the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A)

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Asset Metadata

Creator Wang, Liping (author)

Core Title Phosphorylation of Synaptojanin differentially regulates synaptic vesicle endocytosis of distinct vesicle pools

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 06/30/2016

Defense Date 06/01/2016

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

Tag Drosophila,endocytosis,OAI-PMH Harvest,synapses,synaptic vesicle pools,Synaptojanin

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Chang, Karen T. (committee chair), Langen, Ralf (committee member), Tokes, Zoltan A. (committee member)

Creator Email lipingw@usc.edu,nickyjuesi@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-259642

Unique identifier UC11281203

Identifier etd-WangLiping-4489.pdf (filename),usctheses-c40-259642 (legacy record id)

Legacy Identifier etd-WangLiping-4489.pdf

Dmrecord 259642

Document Type Thesis

Format application/pdf (imt)

Rights Wang, Liping

Type texts

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

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

Repository Name University of Southern California Digital Library

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