drosophila nedd4-long reduces amphiphysin levels in ...€¦ · chapter 3: results ... c2 –...

Drosophila Nedd4-long reduces Amphiphysin levels in

muscles and leads to impaired T-tubule formation

by

Frozan Safi

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Biochemistry

University of Toronto

© Copyright by Frozan Safi 2015

ii

Drosophila Nedd4-long reduces Amphiphysin levels in

muscles and leads to impaired T-tubule formation

Frozan Safi

Master of Science

Department of Biochemistry

University of Toronto

2015

Abstract

Drosophila Nedd4 (dNedd4) is a HECT ubiquitin ligase with two main splice isoforms: dNedd4

short (dNedd4S) and long (dNedd4Lo). We previously showed that while dNedd4S promotes

neuromuscular synaptogenesis, dNedd4Lo inhibits it. To delineate the cause of this inhibition,

we searched for binding partners to the N-terminal unique region of dNedd4Lo in embryo

lysates using mass-spectrometry and identified Amphiphysin (dAmph). dAmph is a postsynaptic

protein containing SH3-BAR domains, which regulates muscle T-tubule formation in flies. We

validated the interaction by co-immunoprecipitation and showed direct binding between

dAmph-SH3 domain and dNedd4Lo-N-terminus. Accordingly, dNedd4Lo was co-expressed

with dAmph postsynaptically and at muscle T-tubules. Moreover, expression of dNedd4Lo in

the muscle during embryonic development led to disappearance of dAmph and to impaired T-

tubules formation, phenocopying amph null mutants. This effect was not seen in muscles

expressing dNedd4S or a catalytically-inactive dNedd4Lo(CA). We propose that dNedd4Lo

destabilizes dAmph in muscles, leading to impaired T-tubule formation.

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Acknowledgments

I would like to thank my supervisor, Dr. Daniela Rotin, for her guidance and encouragement

throughout my Master’s. I am grateful for your mentorship and support. I want to thank my

supervisory committee members, Dr. Gabrielle Boulianne and Dr. Bill Trimble for their

insightful suggestions and comments during the committee meetings.

Thank you to Dr. Alina Kotler for your unwavering guidance, support and mentorship

throughout this project. Thanks to Dr. Avinash Persaud for teaching me a lot of the lab

techniques and for the helpful suggestions throughout the project. A special thanks to Yunan

Zhong, who performed the mass spectrometry screen.

I’m especially grateful for the support of my fellow lab members. The past few years have been

memorable because I got to share them with: Avi, Chong, Chen, Anthony and Ruth, who made

my Master’s study an enjoyable and fun experience. I will miss our buffet lunches at Joe’s and

Mandarin!

My project involved a lot of fly genetics, which could not be done without the Boulianne lab,

who provided all the necessary reagents for my fly work. I want to thank Mike, David and

Oxana for sharing with me their knowledge in fly genetics. A special thanks to Brenda for

providing me with the Amph transgenic lines and antibody.

Thanks to my family and friends for their patience and support throughout my Master’s!

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Table of Contents

Abstract ...........................................................................................................................................ii

Acknowledgments ......................................................................................................................... iii

List of Tables .................................................................................................................................. vi

List of Figures ................................................................................................................................vii

Abbreviations .................................................................................................................................. ix

Chapter 1: Introduction ................................................................................................................ 1

1. Introduction ................................................................................................................................. 1

1.1 Drosophila Muscle Development ........................................................................................ 1

1.2 Ubiquitination ...................................................................................................................... 4

1.2.1 Role of Ubiquitination System in NM synaptogenesis............................................ 5

1.3 The Nedd4 family of E3 ubiquitin ligases ........................................................................... 6

1.3.1 Physiological Functions of Mammalian Nedd4 ...................................................... 9

1.3.2 Drosophila Nedd4 family members ...................................................................... 10

1.4 Muscle Contraction ............................................................................................................ 23

1.5 Amphiphysin ..................................................................................................................... 26

1.5.1 Mammalian Amphiphysin ..................................................................................... 26

1.5.2 Drosophila Amphiphysin ...................................................................................... 30

1.6 Rationale ............................................................................................................................ 33

Chapter 2: Materials and Methods ............................................................................................ 34

2. Methodology ............................................................................................................................. 34

2.1 Fly Stocks .......................................................................................................................... 34

2.2 Plasmid Constructs ............................................................................................................ 34

2.3 Mass Spectrometry ............................................................................................................ 38

v

2.4 In vitro binding assay between dAmph variants and dNedd4Lo N-terminus ................... 39

2.5 Co-immunoprecipitation of dNedd4Lo and dAmph in Drosophila Schneider 2 tissue culture cells ........................................................................................................................ 39

2.6 Third instar larval fillet preps and immunofluorescent staining of body wall muscles ..... 41

2.7 Western Blot analysis of muscle protein levels of dAmph in dNedd4 transgenic larvae.................................................................................................................................. 43

2.8 Statistical Analysis............................................................................................................. 43

Chapter 3: Results ....................................................................................................................... 45

3. Results ....................................................................................................................................... 45

3.1 dAmph interacts with dNedd4Lo....................................................................................... 45

3.2 dNedd4Lo N-terminus directly binds to the SH3 domain of dAmph ................................ 53

3.3 dAmph is enriched postsynaptically and co-localizes with dNedd4Lo ............................. 56

3.4 dAmph lacking its SH3 domain is mislocalized in muscles .............................................. 59

3.5 dNedd4Lo regulates the levels of dAmph in the postsynaptic region ............................... 62

3.6 dNedd4Lo regulates the organization of muscle T-tubules ............................................... 68

3.7 dAmph protein levels in the muscle are reduced in flies overexpressing dNedd4Lo ....... 72

Chapter 4: Discussion and Future Directions ........................................................................... 77

4. Discussion and Future Directions ............................................................................................. 77

4.1 Validating interaction of dNedd4Lo with other target proteins from the mass spectrometry screen ........................................................................................................... 81

5. Conclusion ................................................................................................................................ 83

References...................................................................................................................................... 84

vi

List of Tables

Table 1: Antibody Staining ........................................................................................................... 40

Table 2: Fly line crosses ............................................................................................................... 42

Table 3: Mass spectrometry identification of binding partners to Nedd4Lo unique N-terminus

and Middle regions. ....................................................................................................................... 46

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List of Figures

Figure 1: Neuromuscular system of the Drosophila melanogaster larva ....................................... 2

Figure 2: Phylogenetic relationship between Nedd4 and Nedd4-2 ubiquitin ligases among

different species. .............................................................................................................................. 7

Figure 3: Schematic representation of dNedd4S and dNedd4Lo unique regions and Akt

phosphorylation sites. .................................................................................................................... 12

Figure 4: dNedd4 regulates Comm endocytosis from the muscle membrane. ............................. 15

Figure 5: Neuromuscular innervation and locomotion defects in larvae overexpressing

dNedd4Lo in the muscle. ............................................................................................................... 18

Figure 6: The inhibitory role of dNedd4Lo in NM synaptogenesis is regulated by its N-terminus

and middle region, not by dAkt phosphorylation. ......................................................................... 20

Figure 7: T-tubules form junctional triads that are in close association with the sarcoplasmic

reticulum and mediate excitation-contraction (EC) coupling. ....................................................... 24

Figure 8: Domain structure of Mammalian Amphiphysin I and Drosophila Amphiphysin

(dAmph). ........................................................................................................................................ 27

Figure 9: Drosophila Amphiphysin is required for muscle T-tubule organization. ..................... 31

Figure 10: The GAL4/UAS system for targeted expression of dNedd4 in the muscles. ............. 36

Figure 11: dNedd4Lo co-immunoprecipitates with dAmph ......................................................... 51

Figure 12: Drosophila Amphiphysin directly binds the N-terminus of dNedd4Lo via its SH3

domain. .......................................................................................................................................... 54

viii

Figure 13: Drosophila Amph and dNedd4 are enriched postsynaptically at the neuromuscular

junctions and in muscle T-tubules. ................................................................................................ 57

Figure 14: dAmph(ΔSH3) mislocalizes in Drosophila muscles. ................................................ 60

Figure 15: Postsynaptic levels of dAmph are significantly reduced in Drosophila muscles

overexpressing dNedd4Lo. ............................................................................................................ 63

Figure 16: Muscle-specific overexpression of dNedd4Lo leads to decreased V5-dAmph and

Dlg levels in the postsynaptic region. ............................................................................................ 66

Figure 17: Overexpression of dNedd4Lo in muscles impairs the transverse tubule network. ..... 69

Figure 18: Muscle protein levels of dAmph are reduced in flies overexpressing dNedd4Lo. ..... 73

Figure 19: Muscle protein levels of V5-dAmph are reduced in flies overexpressing dNedd4Lo.75

ix

Abbreviations

BAR- bin–amphiphysin–rvs

BIN1- bridging integrator 1

BMP- bone morphogenetic protein

BSA- bovine serum albumin

C2 – conserved region 2; a Ca2+-binding domain first identified in Ca2+ responsive forms of

protein kinase C

Ca+2

- calcium

Cav1.2- L-type calcium channel

CMN2- centronuclear myopathy

Co-IP- co-immunoprecipitation

Comm- commissureless

dAkt- Drosophila Akt

dAmph- Drosophila Amphiphysin

dAmph SH3(W->A)- tryptophan -> alanine SH3 domain mutant of dAmph

dAmph ΔSH3- dAmph with SH3 domain deleted

DHPR- dihydropyridine receptors

DLG- discs large

dNedd4 – Drosophila Nedd4

dNedd4Lo – dNedd4 long isoform

x

dNedd4Lo S->A – dNedd4L (S39->A, S543->A)

dNedd4Lo (CA)-catalytically inactive dNedd4Lo

dNedd4Lo ∆Mid - dNedd4L with its unique middle 159 amino acid-long sequence deleted

dNedd4Lo Middle (Mid)- residues 304-473 of dNedd4Lo

dNedd4Lo N-terminus (Nterm)- residues 1-63

dNedd4Lo ΔNterm – dNedd4L with its unique N-terminal 63 amino acid-long sequence

dNedd4S – dNedd4 short isoform

dNedd4S S->A – dNedd4S (S653->A)

DPP- decapentaplegic

DS- donkey serum

E1 – ubiquitin activating enzyme

E2 – ubiquitin conjugating enzyme

E3 – ubiquitin ligase

EC- excitation-contraction

EDTA- ethylenediaminetetraacetic acid

ENaC- epithelial Na+ channel

ERAD- endoplasmic reticulum associated protein degradation

Faf- fat facets

FGFR1- Fibroblast growth factor receptor 1

FLAG-epitope DYKDDDDK

xi

GST- glutathione S-transferase

HA- hemagglutinin tag YPYDVPDYA

HECT- homologous to E6-associated protein C-terminus

HEPES- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

Hiw- highwire

His- poly-histidine tag

HRP- horseradish peroxidase

IFM- indirect flight muscles

ISN- intersegmental nerve branch

LUBAC- linear ubiquitin chain assembly complex

MAD- mothers against decapentaplegic

mEPP- miniature endplate potential

MS- mass spectrometry

Myc- epitope EQKLISEEDL

NGS- normal goat serum

NM- neuromuscular

NMJ- neuromuscular junction

PBS- phospho-buffered saline

PBT- 0.1% Tween-20 in 1x PBS

PCR- polymerase chain reaction

xii

PFA- paraformaldehyde

PMSF- phenylmethylsulfonyl fluoride

PY motif – the sequence L/PPxY that binds to WW domains

RBR- RING-between-RING

RING- really interesting new gene

RIPA- radioimmunoprecipitation assay buffer

RNAi- RNAi interference

Robo- roundabout

RT – room temperature (22-23oC)

RTK- receptor tyrosine kinase

RyR- ryanodine receptors

S2- Drosophila Schneider 2 cells

SDS-PAGE- sodium dodecyl sulfate polyacrylamide gel electrophoresis

SH3- src homology 3 domain

SH3 dAmph- SH3 domain of dAmph

SH3PX1- drosophila sorting nexin 9

SNX9- sorting nexin 9

SN- segmental nerve branch

SR- sarcoplasmic reticulum

SSR- subsynaptic reticulum

xiii

Su(dx)- suppressor of Deltex

SV- synaptic vesicle

SVE- synaptic vesicle endocytosis

TN- transverse nerve

TNF- tumor necrosis factor

Tris-HCl – tris hydrochloride

T-tubule- transverse-tubule

UAS- upstream activating sequence

UPS- ubiquitin proteasome system

V5- GKPIPNPLLGLDST epitope

WASP- wiskott–aldrich syndrome protein

WT- wildtype

1

Chapter 1: Introduction

1. Introduction

1.1 Drosophila Muscle Development

Muscle development is a complex process that in body wall muscles of Drosophila begins by

mesoderm specification of myoblasts, which is under the control of several transcription factors

and signalling molecules such as Wingless and decapentaplegic (DPP; a bone morphogenetic

protein 2 (BMP2) and BMP4 orthologue). Myoblast founder cells fuse to fusion competent

myoblasts to generate multinucleated muscle cells (Collins & DiAntonio, 2007). The muscle size

is determined by the number of such fusion events, while muscle identity is controlled by the

expression of a group of transcriptional regulators termed identity genes. These muscles then

attach to specialized tendon cells within the epidermis (Collins & DiAntonio, 2007) and are

innervated to allow them to respond to stimuli arriving from the CNS. Drosophila larvae contain

30 unique body wall muscles innervated by 32 well-characterized motor neurons (Fig. 1) by a

process called neuromuscular (NM) synaptogenesis, each adapting a defined innervation

pathway (Collins & DiAntonio, 2007). The growth cones of these motor neurons reach their

muscle targets during late embryogenesis, and then differentiate into neuromuscular junction

synapses. The site of interaction (synapse) between the axon and muscle is called a bouton.

While many proteins are involved in the regulation of NM synaptogenesis in flies (DiAntonio et

al., 2001), the role of the ubiquitin system in this process is not well characterized. It is known

that the deubiquitinating enzyme Fat facet (DiAntonio et al., 2001) and the RING-family

ubiquitin ligase Highwire (Wan et al., 2000) negatively regulate synapse formation and function.

2

Figure 1: Neuromuscular system of the Drosophila melanogaster larva (A) Schematic

representation of an abdominal muscle hemisegment showing all 30 muscles that are innervated

by one or more of the 5 nerve branches, ISN: intersegmental nerve branch, SNa-d: segmental

nerve branch a-d; TN: transverse nerve. (B) Confocal microscopy image of third instar larva

body wall muscles stained with rhodamine-phalloidin showing the symmetrical and segmental

organization of the muscles stained with an actin probe. (C) Zoomed-in image of (B) showing

morphological details of the typical muscle pattern present in each hemisegment (numbers

identify single muscles) (Adapted from Peron et al., 2009)

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4

1.2 Ubiquitination

Ubiquitination is the covalent attachment of the 76 amino acid protein ubiquitin to one or more

lysine residues in the substrate protein, which can signal proteins for degradation, alter their

localization, trafficking, or function. A covalent bond is formed between the carboxyl group of

the carboxy-terminal Gly residue of ubiquitin and the ε-amino group of an internal Lys in the

substrate (Hershko & Ciechanover, 1998). Substrate proteins can be mono-ubiquitinated, multi-

monoubiquitinated, or poly-ubiquitinated, and the type of ubiquitylation determines the fate of

the protein. Ubiquitin itself contains seven lysine residues that can form different ubiquitin

linkage types; for example poly-ubiquitination on Lys48 of ubiquitin targets substrates for

degradation by the 26S proteosome, while mono-ubiqutination or polyubiquitylation through

Lys63 are associated with endocytosis and vesicular sorting (Hicke & Dunn, 2003). There is also

another form of ubiquitin linkage called linear ubiquitination, which does not involve any of the

lysine residues in the ubiquitin molecule, but occurs between the amino-terminal methionine of

one ubiquitin and the carboxy-terminal glycine of the next in the chain (Kirisako et al., 2006).

Linear ubiquitin, chains also known as M1-linked chains, are generated by a novel type of E3

ubiquitin ligase called the linear ubiquitin chain assembly complex (LUBAC). Linear ubiquitin

chain linkages have been shown to be important in several innate and adaptive immune signaling

pathways, including the tumor necrosis factor (TNF) pathway (Gerlach et al., 2011).

Ubiquitination is an ATP dependent process and it is initiated by the E1-activating enzyme,

which binds ubiquitin and activates it by adding an ATP before transferring it to an E2-

conjugating enzyme. Next, the E2 binds directly to the substrate protein or to an E3-ligase that is

bound to the substrate protein. Finally, the E3 ligase protein removes the ubiquitin from E2 and

attaches it to the substrate protein (Hershko and Ciechanover, 1998; Deshaies and Joazeiro,

2009). Organisms contain only a few E1s, many E2s and a large number of E3s, which dictate

the specificity of the ubiquitination system (Li et al., 2008). E3s are responsible for substrate

recognition and transfer of ubiquitin onto them, either indirectly (e.g. RING and U-box E3

ligases) or directly (e.g. HECT E3 ligases (*Rotin & Kumar, 2009). RING (really interesting

new gene) and U-box (a modified RING motif without the full complement of Zn2+

-binding

5

ligands) function as scaffolds to bring the E2 and the substrate in close proximity and facilitate

the direct transfer of ubiquitin from the E2 to the substrate. In contrast, HECT (homologous to

E6-associated protein C-terminus) contain a conserved catalytic Cys residue that accepts

ubiquitin from E2s before directly ubiquitinating the substrate (Ardley & Robinson, 2005).

Recently, a third class of E3 ubiquitin ligases known as the RING-between-RINGs (RBRs) was

discovered. RBRs are a subclass of RING-containing E3 ligases and they use the hybrid

RING/HECT mechanism to transfer ubiquitin to substrates. The proposed mechanism for RBR

ubiquitination involves binding of the ubiquitin-conjugated E2 to the RING1 domain of the RBR

E3 ligase. The ubiquitin is then transferred from the E2 to the E3 RING2 domain from which it

is transferred to the substrate (Wenzel & Klevit, 2012).

1.2.1 Role of Ubiquitination System in NM synaptogenesis

The development of functional synapses requires a balance between protein synthesis and

degradation, which is usually mediated by the ubiquitin proteasome system (UPS) (Speese, 2003;

Kawabe and Brose, 2011). Ubiquitination of specific pre- and post-synaptic proteins and their

degradation by the UPS has been shown to be important for neuronal development (Haas and

Broadie, 2008). Highwire (hiw) is a putative ubiquitin ligase in flies that localizes at synaptic

terminals and regulates development of the Drosophila NMJ (DiAntonio et al., 2001). Hiw

functions as a negative regulator of synaptic growth, as loss of function Hiw mutants exhibit

overgrown synaptic terminals but weakened synaptic transmission (Wan et al., 2000). There is a

strong genetic interaction between the gene encoding a deubiquitinating enzyme fat facets (faf)

and hiw in Drosophila (DiAntonio et al., 2001). Neuronal overexpression of faf causes a large

increase in the number of synaptic boutons, an elaboration of the synaptic branching pattern and

defects in neurotransmitter release, which is similar to the hiw loss-of-function (DiAntonio et al.,

2001).

Other E3 ligase complexes that regulate synaptic development include the anaphase-promoting

complex/cyclosome which regulates synaptic size and transmission by controlling the

presynaptic levels of Liprin-alpha (van Roessel et al. 2004) and postsynaptic levels of glutamate

receptors (Juo & Kaplan, 2004), respectively, at the fly NMJs. In addition, work from our lab

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831508/#R26http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831508/#R26

6

showed that the short isoform of the fly HECT E3 ubiquitin ligase Nedd4 (dNedd4), regulates

NMJ formation by promoting ubiquitination and internalization of Commissureless (Comm)

(*Ing et al. 2007), while the long isoform of Nedd4 inhibits NM synaptogenesis (*Zhong et al.

2011) (See below for detail).

1.3 The Nedd4 family of E3 ubiquitin ligases

Nedd4 family members comprise a common C2-WW (n)-HECT domain architecture and belong

to the HECT family of ubiquitin ligases (Fig. 2). The C2 domain of Nedd4 targets proteins to

phospholipid membranes in a calcium dependent manner (*Plant et al., 1997; *Plant et al., 2000;

Dunn et al., 2004), and can also be involved in protein-protein interactions (Morrione et al.,

1999). Furthermore, the C2 domain is responsible for intramolecular regulatory interactions with

the HECT domain (Wiesner et al., 2007; Wang et al., 2010; *Persaud et al., 2014). The HECT

domain is the catalytic domain and is composed of ~350 amino acids located on the C-terminal

region of HECT E3 ligases (Huibregtse et al., 1995). This domain mediates the interaction with

E2s (Kar et al., 2012; Sheng et al., 2012) and forms a thioester complex with ubiquitin through a

cysteine residue (Scheffner & Staub, 2007; *Rotin & Kumar, 2009; Huibregtse et al., 1995;

Schwarz et al., 1998) to catalyze the attachment of ubiquitin onto substrate proteins. The WW

domains are protein-protein interaction modules, containing two conserved tryptophan (W)

residues, which interact primarily with proline rich PPxY (PY) motifs or phospho-

serine/threonine residues in substrates (Sudol et al., 1995). The number of WW domains in

Nedd4 proteins varies between family members and species; for instance, human Nedd4 proteins

contain four WW domains, whereas mouse Nedd4-1 and Drosophila contains three WW

domains (Yang and Kumar, 2010).

7

Figure 2: Phylogenetic relationship between Nedd4 and Nedd4-2 ubiquitin ligases among

different species. Phylogenetic tree representing the conserved modular protein domains of

Nedd4 and Nedd4-2 among species. The length of each box is proportional to the domain size. c:

chicken (Gallus gallus); d: fruitfly (Drosophila melanogaster); h: human (Homo sapiens); m:

mouse (Mus musculus); x: frog (Xenopus tropicalis); and z: zebrafish (Danio rerio) (Adapted

from Yang & Kumar, 2010).

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1.3.1 Physiological Functions of Mammalian Nedd4

There are nine Nedd4 family members in mammals including Nedd4 (also known as Nedd4-1)

and Nedd4L (also known as Nedd4-2). Nedd4 is known to regulate key signaling pathways, such

as cellular growth and proliferation that are important in animal development and cancer

(Reviewed by *Rotin & Kumar, 2009). A few examples are provided below to illustrate the

identified roles of Nedd4 proteins. Nedd4L regulates the cell surface stability of the epithelial

Na+ channel (ENaC) by interacting with the PY motifs of ENaC, promoting its endocytosis and

degradation by ubiquitination (*Staub et al., 1996; Kamynina et al., 2001; *Lu et al., 2007).

Regulation of ENaC by Nedd4L prevents Na+ overload in epithelial cells and is necessary for

salt and fluid balance in the body, which is perturbed in PY motif deletion or mutants of ENaC

that cause the hereditary hypertension disease known as Liddle syndrome (Lifton et al., 2001).

Nedd4L also regulates ENaC in the lung epithelia since knockout of Nedd4L in this region

causes mice to develop cystic fibrosis-like symptoms due to elevated ENaC abundance, which

can be rescued when treated with an ENaC inhibitor, amiloride (*Kimura et al., 2011).

Nedd4-1 regulates the signalling of the receptor tyrosine kinase (RTK) fibroblast growth factor

receptor 1 (FGFR1), which controls cellular proliferation and differentiation. FGFR1 contains a

novel site (VL***PSR) that binds the WW3 domain of Nedd4-1, leading to the ubiquitination

and down-regulation of FGFR1 at the cell surface, and terminating downstream signalling

(*Persaud et al., 2011). Accordingly, mutant FGFR1 lacking the recognition motif, FGFR1(Δ6),

cannot bind Nedd4-1 and thus is retained on the plasma membrane where it can constitutively

activate downstream signalling in response to FGF stimulation. Expression of FGFR1(Δ6) in

zebrafish embryos induces defects in brain development, similar to a constitutively active

FGFR1 (*Persaud et al., 2011). Activation of FGFR1 or EGFR leads to c-Src-mediated

phosophorylation of Nedd4-1 on several tyrosines, including one in the C2 domain and one in

the HECT domain, which dissociates the intramolecular inhibitory interaction between the

HECT and C2 domains, leading to enhanced ubiquitin ligase activity of Nedd4-1 and as a result

enhanced endocytosis and degradation of FGFR1 (*Persaud et al., 2014).

10

Nedd4-1 is also involved in the development of the mammalian nervous system, as mutant mice

deficient in Nedd4 show significant reduction in the skeletal muscle fiber sizes and motorneuron

numbers. Furthermore, Nedd4 mutant mice contain synapses that are composed of numerous

smaller nerve terminal profiles and motorneurons that defasciculate upon reaching their target

muscle. Accordingly, electrophysiological analyses reveal functional impairments of

neuromuscular (NM) synaptic activities (Liu et al., 2009). Nedd4 mutant NMJ impairments

include increased spontaneous miniature endplate potential (mEPP) frequency, decreased

response to membrane depolarization, increase in the number, but decrease in the diameter, of

pre-synaptic nerve terminal branches. These ultrastructural changes are consistent with

functional alternation of the NMJs in Nedd4 mutants (Liu et al., 2009). Interestingly, Nedd4-1

also plays an important role in the regulation of denervation-induced skeletal muscle atrophy in

animals. Nedd4-1 skeletal muscle-specific knockout mice demonstrate partial protection from

muscle atrophy, show heavier denervated muscle weights and large type II fibre cross sectional

areas (Nagpal et al., 2012).

Nedd4-1 also regulates dendrite development in mammalian neurons as Nedd4-1 deficient mice

show a decrease in dendrite length and complexity, causing a decrease in the number of

functional synapses and synaptic transmission (Kawabe et al., 2010). Nedd4-1 forms a ternary

complex with the serine/threonine kinase TNIK, and Rap2A, which leads to ubiquitination of

Rap2A by Nedd4-1. Nedd4-1-mediated ubiquitination of Rap2A inhibits its function, which

reduces the activity of Rap2 effector kinases of the TNIK family and promotes dendrite growth

(Kawabe et al., 2010).

1.3.2 Drosophila Nedd4 family members

Drosophila melanogaster contain three Nedd4 family members, which include Nedd4 (dNedd4),

suppressor of Deltex (Su(dx), also known as ITCH or AIP4), and Smurf (also known as Lack).

dNedd4 regulates neuromuscular synaptogenesis by targeting Commisureless for degradation

(see below). dNedd4 also antagonizes Notch signaling by promoting ligand-independent

endocytosis and degradation of Notch, as well as its positive regulator Deltex (Sakata et al.,

11

2004). Notch signaling is also negatively regulated by Su(dx), which is involved in sorting of

Notch to the late endosome for lysosomal degradation (Wilkin et al., 2004). Smurf negatively

regulates DPP signalling during embryonic dorsal–ventral patterning by mediating degradation

of DPP-activated MAD, a Smad protein that transduces the signal of DPP (Liang et al., 2003).

Moreover, mutations of Smurf are lethal and lead to defects in hindgut organogenesis (Podos et

al., 2001).

1.3.2.1 Drosophila Nedd4 (dNedd4)

In mammals, Nedd4 and Nedd4-2 are encoded by different genes (Kamynina et al., 2001), while

flies have a single dNedd4 gene, which undergoes alternative splicing to produce several splice

isoforms, including two prominent isoforms: dNedd4-short (dNedd4S) and dNedd4-long

(dNedd4Lo) (Fig.3). Differences between the two isoforms of dNedd4 include an alternate start

codon site resulting in a longer N-terminal region in dNedd4Lo, and an extra exon inserted

between the WW1 and WW3 domains (*Zhong et al., 2011).

12

Figure 3: Schematic representation of dNedd4S and dNedd4Lo unique regions and Akt

phosphorylation sites. Both dNedd4S and dNedd4Lo contain a C2 domain, three WW domains

and a HECT domain. dNedd4Lo contains two unique regions absent in dNedd4S, an N-terminal

region (Nterm) and an extra sequence between the WW1 and WW3 domains (Mid). Both

dNedd4S and dNedd4Lo contain putative Akt phosphorylation sites, however, only the N-

terminal unique sequence of dNedd4Lo is phosphorylated by dAkt (*Zhong et al., 2011).

14

1.3.2.2 dNedd4S regulation of Commisureless

dNedd4S was shown to be important for axon guidance at the midline by controlling the levels of

Commisureless (Comm), a regulator of Roundabout (Robo) (Keleman et al., 2002). Upon axon

crossing, the surface levels of Robo increase, which prohibits midline recrossing (Kidd et al.,

1999). The expression of Robo is regulated in part by Comm, which binds to and is regulated by

dNedd4. In accord, an intact dNedd4 binding site and ubiquitin acceptor sites within Comm are

required for Comm to regulate Robo (Myat et al., 2002). However, another study has found that

ubiquitination of Comm by dNedd4 is not involved in axon guidance at the midline (Keleman et

al., 2005). Therefore, the role of dNedd4 and ubiquitination in midline crossing remains

controversial.

In addition to being expressed in axons, all muscles of Drosophila embryos express Comm

during the period of motoneuron-muscle interactions (Wolf et al., 1998). However, Comm must

be internalized from the muscle surface prior to synaptogenesis (Wolf et al., 1998). Earlier work

from our lab showed that dNedd4S promotes endocytosis of Comm from the muscle surface, as

wildtype Comm is expressed in intracellular vesicles in the muscle, but Comm containing

mutations in the two PY motifs (L/PPXY) responsible for binding dNedd4 [Comm (2PYAY)],

or bearing LysArg mutations in all Lys residues that serve as ubiquitin acceptor sites [Comm

(10KR)], fail to endocytose and display aberrant muscle innervation (Fig. 4) (*Ing et al.,

2007). RNAi-mediated knockdown of dNedd4 expression or expression of catalytically inactive

dNedd4(CA), bearing a Cys-to-Ala mutation in the conserved Cys of the HECT domain,

results in the accumulation of Comm at the muscle surface and synaptic innervation defects,

similar to those observed in Comm (2PYAY) or the Comm (10KR) mutant. The role of

Nedd4 in regulating neuromuscular (NM) synaptic activities is conserved in mice, although

mammals do not have Comm.

15

Figure 4: dNedd4 regulates Comm endocytosis from the muscle membrane. (A) Motor

neurons innervating muscles 12 and 13 expressing Comm mutants: Comm (2PY->AY) or Comm

(10K->R) exhibit aberrant branching (indicated by yellow arrows). RNAi-mediated knockdown

of muscular dNedd4 expression yields synaptic mutant phenotypes similar to those observed in

the Comm(2PYAY) or the Comm(10KR) mutant. (B) Comm (WT) accumulates in

intracellular vesicles, while Comm mutants: Comm (2PY->AY) and Comm (10K->R)

accumulate at the plasma membrane of the muscle, where they co-localize with the plasma

membrane marker concanavalin A (ConA), shown in green. (C) Schematic representing dNedd4

mediated endocytosis of WT Comm in intracellular vesicles, Comm (2PY->AY) and Comm

(10K->R) fail to endocytose and thus accumulate on the plasma membrane (From *Ing et al.,

2007).

17

1.3.2.3 dNedd4 regulation of neuromuscular synaptogenesis

Recently, our lab showed that while dNedd4S is essential for NM synaptogenesis (*Ing et al.,

2007), the dNedd4Lo isoform inhibits it, resulting in impaired locomotion and larval lethality.

Specifically, muscle-specific overexpression of dNedd4Lo causes abnormal motor neuron

innervation along the SNb branch on body wall muscles 13->12, such as inappropriate backward

innervation from muscles 12->13 and increased number of nerve branches on muscle 12 (Fig. 5)

(*Zhong et al., 2011).

In order to determine the mechanism used by dNedd4Lo to inhibit NM synaptogenesis and

muscle function, our lab examined the unique N-terminal region of dNedd4Lo, which contains a

putative Akt phosphorylation site (S39) that is phosphorylated by Drosophila Akt (dAkt) (Fig.

6A). Mutating the dAkt phosphorylation site of dNedd4Lo did not alter the muscle innervation

defects (Fig. 6A) (*Zhong et al., 2011). Therefore, regulation of the negative role of dNedd4Lo

in NM synaptogenesis is not mediated by dAkt phosphorylation. In addition, the unique N-

terminal or middle regions of dNedd4Lo did not bind to the HECT domain, nor inhibit the

catalytic activity of dNedd4S in vitro, hence dNedd4Lo does not inhibit NM synaptogenesis by

inhibiting the catalytic activity of dNedd4S. Furthermore, overexpression of dNedd4Lo did not

affect internalization of Comm in S2 cells. Therefore, dNedd4Lo does not act by interfering with

the interaction of dNedd4S with its substrate, Comm.

The unique N-terminal and middle region, as well as the HECT domain of dNedd4Lo, regulate

the adverse function of dNedd4Lo in NM synaptogenesis. Removing either the N-terminal or the

middle region or creating an inactivating mutation in the HECT domain (Cys->Ala) rescued the

lethality and alleviated the muscle innervation defects (Fig6B). Therefore, the unique N terminal

18

Figure 5: Neuromuscular innervation and locomotion defects in larvae overexpressing

dNedd4Lo in the muscle. (A) dNedd4 null mutant lethality is partially rescued with dNedd4S,

but not dNedd4Lo, expression driven by Actin-GAL4 throughout the embryo. (B) Muscle-

specific overexpression of dNedd4Lo (dNedd4Lo/5-GAL4) leads to aberrant synaptic innervation

along the SNb branch from body wall muscles 13->12 of third instar larvae (motoreneurons

shown in red). (C) Overexpression of dNedd4Lo but not dNedd4S inhibits larval locomotion as

measured by total path length (From *Zhong et al., 2011).

20

Figure 6: The inhibitory role of dNedd4Lo in NM synaptogenesis is regulated by its N-

terminus and middle region, not by dAkt phosphorylation. (A) Schematic representation of

dNedd4Lo showing its consensus Akt phosphorylation sites that were mutated to alanine in the

phosphorylation mutant: dNedd4Lo (S39A,S645A). Mutation of the dAkt phosphorylation sites

in dNedd4Lo (S39A,S645A) fails to reduce the innervation defects. (B) Schematic representation

of dNedd4Lo mutants: dNedd4LoΔMiddle (Mid) and dNedd4LoΔN-terminus (Nterm) that has

its unique middle and N-terminal sequence deleted, respectively. Muscle innervation defects are

decreased in embryos/larvae expressing dNedd4Lo lacking its Nterm, Mid region or its catalytic

activity in the muscle (From *Zhong et al., 2011).

22

and middle regions of dNedd4Lo mediate its negative function on NM synaptogenesis and larval

locomotion, a process that also requires active dNedd4Lo. Thus, we suspect that the unique

regions of dNedd4Lo regulate NM synaptogenesis and muscle function by an unknown

mechanism, possibly by interacting with other proteins or substrates (*Zhong et al., 2011).

To identify such putative interacting partners, Yunan Zhong from our lab incubated embryo

lysates with purified (immobilized) unique regions of dNedd4Lo (N terminus and Middle

regions) and identified several interacting proteins by mass spectrometry (See Results section).

Three of these proteins comprised BAR-SH3 domain architecture: Syndapin, Sorting Nexin 9

(SH3PX1) and, importantly, Amphiphysin (dAmph), which was a binding partner of the unique

N-terminal region of dNedd4Lo. In Drosophila, dAmph regulates muscle architecture and

function, as detailed in the following sections.

23

1.4 Muscle Contraction

Muscle contraction occurs when an action potential propagates along the sarcolemma (muscle

fiber membrane) and triggers the release of calcium from intracellular calcium stores present in

the sarcoplasmic reticulum (SR) (Al-Qusairi & Laporte, 2011). This Excitation-Contraction

coupling is mediated by the transverse-tubule (T-tubule) network, which forms regularly-spaced

perpendicular invaginations toward the interior of the muscle fibre, where they make physical

contact with the SR in a region called the terminal cisternae, or triad (Fig. 7A). At the triad

junctions, voltage-dependent Ca2+

channels in the T-tubular membranes known as

dihydropyridine receptors (DHPRs) detect the depolarization and transduce it into a signal for

opening Ca2+

release channels (also called ryanodine receptors (RyRs)) on the closely opposed

SR membrane. RyR activation causes Ca2+

efflux from the SR into the cytosol, which diffuses

and binds to the regulatory subunit of troponin, thereby removing troponin’s inhibitory effect on

the contractile proteins, actin and myosin, and allowing muscle contraction (Fig. 7B) (Al-Qusairi

& Laporte, 2011). Several genes/proteins have been identified that are involved in T-tubule

biogenesis, including Amphiphysin 2 (BIN1), and its Drosophila orthologue dAmph (Al-Qusairi

& Laporte, 2011).

24

Figure 7: T-tubules form junctional triads that are in close association with the

sarcoplasmic reticulum and mediate excitation-contraction (EC) coupling. (A) Left:

Electron micrograph of a triad junction showing a T-tubule flanked on both sides by a

sarcoplasmic reticulum terminal cisternae element. Arrows indicate electron-dense regions

corresponding to the ryanodine receptor-dihydropyridine receptor complex. Right: Schematic

representing muscle sarcomere and surrounding membranes. T-tubules are shown in gray as

specialized invaginations of the muscle plasma membrane surrounded by the sarcoplasmic

reticulum network shown in blue (Al-Qusairi & Laporte, 2011). (B) A schematic of the skeletal

muscle fiber illustrating the sequence of EC coupling. An action potential originating from a

motor neuron propagates into the muscle fiber interior via a specialized system of tubular

membranes (called T-tubules), which are extensions of the sarcolemma that form specialized

intracellular junctions called triads. At the triad junctions, voltage-dependent Ca2+

channels in

the T-tubular membranes known as dihydropyridine receptors (DHPRs) detect the depolarization

and activate close by ryanodine receptors (RyRs) on the SR membrane, which causes an efflux

of Ca2+

into the cytosol where it can induce muscle contraction (Adapted from Judith A. Heiny).

26

1.5 Amphiphysin

Amphiphysins are members of the BAR-SH3 domain-containing family of proteins, encoded by

a single gene in flies (dAmph), two genes in mammals (Amph I & IIa/IIb (BIN1)) and two in

yeast (Rvs167/Rvs161) (Fig. 8) (Leventis et al., 2001). The N-terminal BAR (Bin-Amphiphysin-

Rvs) domain forms homodimers, binds negatively charged curved membranes and promotes

their further curvature and tubulation (Peter et al., 2004). The structure of the Drosophila

amphiphysin BAR domain has been solved and it is a crescent-shaped dimer that binds

preferentially to highly curved and negatively charged membranes. Amphiphysin belongs to the

N-BAR family of proteins because it contains an N-terminal amphipathic helix and BAR

domain, which allows it to drive membrane curvature in vitro and in vivo (Peter et al., 2004). The

C-terminal SH3 domain is a protein: protein interaction module that usually binds proline-rich

motifs (Stamenova et al., 2007). The SH3 domain of Amph can bind ubiquitin (Stamenova et al.,

2007), which is interesting since proteins with ubiquitin binding domains/motifs are usually

themselves targeted for ubiquitination, often by Nedd4 family members (Woelk et al., 2006).

1.5.1 Mammalian Amphiphysin

Amphiphysins were originally suggested to function in endocytosis, particularly in synaptic

vesicle (SV) recycling during neurotransmission, by interacting with clathrin/dynamin, a

function critical for recycling of SVs for successive rounds of neurotransmission (Lichte et al.,

1992). Amphs were shown to bind nerve termini (Ramjuan et al., 1997) and to bind to several

components of the endocytic machinery in mammalian cells (e.g. clathrin, endophilin, AP2,

27

Figure 8: Domain structure of Mammalian Amphiphysin I and Drosophila Amphiphysin

(dAmph). The central region on Amph I contains binding sites for endophilin, the AP-2, and

clathrin, while the SH3 domain of Amph I contains the dynamin/synaptojanin binding site. These

binding regions are absent in the Drosophila orthologue, dAmph (adapted from Craft et al.,

2008).

29

dynamin (Ramjuan et al., 1997). In accord, mice lacking Amph I exhibit decreased SV recycling

(Di Paolo et al., 2002), although Bin1 (splice isoform of Amph IIb) knockout mice are lethal but

did not show endocytic defects and lack the clathrin/AP-2 binding motifs (Fig. 8) (Muller et al.,

2003). Bin1 is highly expressed in the heart, where it localizes to a specialized membrane system

of the heart known as the transverse tubule (T-tubule) network that facilitates muscle contraction

(Muller et al., 2003).

1.5.1.1 Bridging integrator 1 (Bin1)

The BAR domain of the isoform of mammalian Amphiphysin 2, Bridging integrator 1 (Bin1), is

known to be involved in T-tubule formation in cardiac and skeletal muscles (Caldwell et al.,

2014; Lipsett et al., 2015). Bin1 induces tubular plasma membrane invaginations when expressed

in non-muscle cells and segregates in tubular portions of the T-tubule system in developing

myotubes (Lee et al., 2002). Bin1 is mutated in a subset of centronuclear myopathies, a disease

in which patients exhibit muscle weakness and mislocalized nuclei within muscle fibre (Bohm et

al., 2010). Patients with Bin1 mutation in these myopathies have severely disorganized T-tubule

networks (Toussaint et al., 2011).

Cardiac Bin1 has been implicated in calcium channel trafficking and formation of the inner

membrane folds of the cardiac T-tubules (Hong et al., 2014; Lipsett et al., 2015). Bin1 localizes

to cardiac T-tubules with the L-type calcium channel, Cav1.2, by tethering dynamic

microtubules to membrane scaffolds, allowing targeted delivery of Cav1.2 to cardiac T-tubules

(Hong et al., 2010). Knockdown of Bin1 reduces surface Cav1.2 and delays development of the

calcium transient (Hong et al., 2010). In cardiomyopathy, decrease in Bin1 alters T-tubule

morphology and can cause arrhythmia (Hong et al., 2014). Mice with cardiac Bin1 deletion show

decreased T-tubule folding, which leads to free diffusion of local extracellular ions, prolonging

action-potential duration and increasing susceptibility to arrhythmias (Hong et al., 2014).

Bin1 is also important for the maintenance of intact T-tubule structure and Ca²⁺ homeostasis in

adult skeletal muscle (Tjondrokoesoemo et al., 2011). Adult mouse skeletal muscles with Bin1

knockdown display swollen T-tubule structures, alterations to intracellular Ca²⁺ release and

30

compromised coupling between the voltage-gated calcium channel, DHPR, and the intracellular

calcium channel, RyR1 (Tjondrokoesoemo et al., 2011).

1.5.2 Drosophila Amphiphysin

DAmph was recently shown to participate in plasma membrane remodeling during cleavage

furrow ingression that is required for de novo formation of cells in Drosophila embryo (Su et al.,

2013). The BAR domain of dAmph is required for the formation of endocytic tubules that form

at the cleavage furrow tips. In accord, amphiphysin null embryos fail to form tubules, which

correlates with faster cleavage furrow ingression rates (Su et al., 2013).

Interestingly, studies in Drosophila have shown enrichment of dAmph postsynaptically at

neuromuscular junctions and in a reticular pattern in larval and adult indirect flight muscles,

where it colocalizes with the T-tubule marker, Discs Large (Leventis et al., 2001; Zelhof et al.,

2001; Razzaq et al., 2001). Moreover, dAmph is not involved in SVs recycling, since Amph

mutants lack obvious endocytic defects in SV recycling (Leventis et al., 2001; Zelhof et al.,

2001; Razzaq et al., 2001), most likely because (like its orthologue Bin1) it lacks the

clathrin/AP2 binding motifs present in Amph I. DAmph is, however, required for normal

localization of the postsynaptic proteins Discs large, lethal giant larvae and Scribble (Zelhof et

al., 2001), as well as the organization of the excitation-contraction coupling machinery of

muscles (Razzaq et al., 2001). Accordingly, dAmph mutant larvae and flies show severe

locomotor defects and flight impairments, indicative of defects in muscle function (Leventis et

al., 2001; Razzaq et al., 2001). Amph null flies show a disordered T-tubule network that is

largely longitudinal, with expanded tubules and few transverse elements (Fig. 9) (Razzaq et al.,

2001). RyR localization in the muscle is also disrupted in Amph null flies (Razzaq et al., 2001).

In accord, expression of wildtype Amph partially rescues the T-tubule network and locomotion

defects of Amph null flies (Razzaq et al., 2001).

31

Figure 9: Drosophila Amphiphysin is required for muscle T-tubule organization. (A)

Amphiphysin is localized on muscle T-tubules in larval body wall muscles and adult indirect

flight muscles (IFM), Amph (green) and actin (red). (B) Confocal microscope sections showing

localization of amphiphysin (Amph, green) with the T-tubule marker, Discs-large (DLG, red) in

larval body wall muscles of wildtype (WT) and Amph null flies. The T-tubule network in Amph

null flies is largely longitudinal, with expanded tubules and few transverse elements (From

Razzaq et al., 2001). (C) Schematic diagram illustrating reduced transverse elements in Amph

null flies.

33

1.6 Rationale

I hypothesize that dNedd4Lo is a suppressor of dAmph, which regulates stability of dAmph and

hence muscle architecture and function. In order to elucidate the mechanism of dNedd4Lo

induced inhibition of muscle function, I investigated:

1. If dNedd4Lo interacts with dAmph in Drosophila S2 cells and in flies, and if it is a

substrate for dNedd4Lo

2. The levels of dAmph in flies overexpressing dNedd4Lo postsynaptically and in muscles

3. The T-tubule architecture of muscles in flies overexpressing dNedd4Lo

34

Chapter 2: Materials and Methods

2. Methodology

2.1 Fly Stocks

Fly strains and crosses are depicted in Table 2. Overexpression of dNedd4 or dAmph was

achieved using the Gal4/UAS system and the early muscle driver, 24B-Gal4 (Figure 10). Flies

were maintained at room temperature on standard food. All experiments were performed at 25oC.

All flies used in this experiment have a wild type genetic background because dNedd4 null

(dNedd4T121FS

homozygote) flies are homozygous lethal at the embryonic stage and cannot be

rescued with dNedd4Lo overexpression (Zhong et al., 2011).

2.2 Plasmid Constructs

I. Generation of N-terminal and Middle unique sequences of dNedd4Lo: GST tagged

dNedd4Lo N-terminus (Nterm, residues 1-63) and dNedd4Lo Middle (Mid, residues

304-473) region were subcloned into the pQE30 vector for expression in bacteria.

II. Generation of WT, SH3 WA mutant, ΔSH3 and SH3 dAmph: The PCR-amplified

DNA fragments corresponding to WT dAmph, ∆SH3 dAmph (residues 1-522), and

SH3 dAmph (residues 523-602) were subcloned into pDEST17 with an N-terminal

His tag using the Gateway Cloning System (Life technology). PCR-amplified full-

length WA dAmph SH3 mutant (W580A) was synthesized using the Quick

Change Site Directed Mutagenesis Kit (Stratagene).

III. Generation of HA-dAmph WT in pRmHA-3 vector for expression in S2 cells: The

expressed sequence tag (EST) LD19810, representing the cDNA of Amphiphysin

was obtained from Berkeley Drosophila Genome Project. PCR amplified full-length

dAmph containing EcoRI and KpnI sites was subcloned into pRmHA-3 vector for

35

expression in S2 cells. An N-terminal HA tag was inserted using the Quick Change

Site Directed Mutagenesis Kit (Stratagene).

36

Figure 10: The GAL4/UAS system for targeted expression of dNedd4 in the muscles.

The GAL4/UAS system is based on the properties of the yeast GAL4 transcription factor, which

activates transcription of its target genes by binding to the upstream activating system, UAS,

cis-regulator sites. In Drosophila, the two components are carried in separate lines. The driver

lines provide tissue-specific GAL4 expression and the responder lines carry the coding sequence

for the gene of interest under the control of the UAS sites. In all the experiments described, the

muscle-specific driver 24B-GAL4 is used to drive expression of UAS-dNedd4S, UAS-

dNedd4Lo or dNedd4Lo C->A mutant in the muscles (Adapted from Johnston, 2002).

38

2.3 Mass Spectrometry

Bacterially expressed GST tagged dNedd4Lo Nterm and Mid region were lysed in sonication

buffer (1xPBS, 10 g/ml aprotinin, 5 g/ml pepstatin, 1 g/ml leupeptin, 50 g/ml DNase I,

1mg/mL lysozyme, and 1mM PMSF) at 4oC. Cell lysates were incubated with Glutathione

sepharose beads for 1 hr at 4oC. After incubation, beads were washed with low salt HNTG (20

mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol and 0.1% Triton-X100) and 1xPBS. Proteins

were eluted in 1xPBS (pH 8.0) containing reduced glutathione for 1 hr at 4oC.

I. Drosophila embryo collection and preparation: Wildtype (WT) adult flies were placed

on grape fruit agar plates with yeast for embryo collection (0-24 hrs) at 22oC in fly cages.

~300 embryos were collected and dechorionated in 50% bleach for 2 min. Embryos were

lysed in RIPA Buffer (% NP-40, 0.5% Na deoxycholate, 0.1% SDS, 50 mM Tris-HCl,

pH 7.4, 150mM NaCl, 10% Glycerol and 2 mM EDTA) plus protease inhibitor cocktail

(Roche Complete tablet) on ice.

II. Mass Spectrometry Analysis of Drosophila Embryo Lysate using dNedd4Lo N-terminal

and Middle regions: Bacterially expressed and purified GST dNedd4Lo Nterm, Mid or

GST control on Glutathione sepharose beads were incubated with embryo lysates (~100

embryos) at 4oC for 2.5 hours and then washed with 1xPBS. Samples were run on SDS-

PAGE and Coommassie blue -stained prior to analysis by a Thermo LTQ-Orbitrap

Hybrid Mass Spectrometer (Hospital for Sickkids, SPARC BioCentre). X! Tandem

(version: Cyclone, 2010.12.01.2) was set up to search the Drosophila melanogaster

database in order to identify the peptides fragments generated from trypsin digestion. The

mass spectrometry data was analyzed using the Scaffold 4.4 software (Proteome Software

Inc).

39

2.4 In vitro binding assay between dAmph variants and

dNedd4Lo N-terminus

Bacterially expressed and purified His tagged WT dAmph, ΔSH3 dAmph, SH3 dAmph, or W-

>A dAmph SH3 mutant was incubated with bacterially expressed GST-dNedd4Lo Nterm at 4°C

for 2 hrs. Supernatant was removed and the beads were washed with low salt HNTG followed by

1xPBS. Following SDS-PAGE, membranes were blocked and incubated with mouse anti-His to

detect dAmph. Mouse anti-glutathione S transferase (GST) antibody and HRP-conjugated goat

anti-mouse antibody were used to detect bacterially expressed GST-dNedd4Lo Nterm (Table 1).

2.5 Co-immunoprecipitation of dNedd4Lo and dAmph in

Drosophila Schneider 2 tissue culture cells

Two mL of sub-confluent Drosophila S2 tissue culture cells (~1.0x106

cells) were transferred

into 6-well plates and allowed to grow for ~16 hrs to obtain ~90% confluency prior to

transfection. Effectene transfection kit (Qiagen) was used to transfect FLAG-dNedd4S or FLAG-

dNedd4Lo and HA-dAmph in pRmHa3 vectors into S2 cells using standard protocol. 0.4µg of

DNA each for dNedd4 and dAmph were transfected per well. 24 hours post-transfection, 500µM

CuSO4 was added in each well to induce expression of the transfected dNedd4 and dAmph

under the metallothionein promoter. 48 hours post-transfection, cells were lysed in lysis buffer

(150 mM NaCl, 50 mM HEPES, 1% Triton-X, 10% glycerol, 1.5 mM MgCl2 and 1.0 mM

EGTA) plus

40

Table 1: Antibody Staining

Primary Antibody Blocking 1

o antibody

(dilution)

Source of 1o

antibodies

2o antibody (dilution)

3rd

instar Larval Muscle Immunohistochemistry

Mouse anti-Dlg 2%BSA+2% NGS 4F3 anti-discs

large (1:200) Iowa University

Goat anti mouse- Alexa

Fluor488

(1:600)

(Invitrogen)

Mouse anti-Flag 2%BSA+2%NGS Anti-Flag (1:250) Sigma

Goat anti mouse-Cy3

(1:600) (Jackson

ImmunoResearch

Laboratories, Inc)

HRP 2%BSA+2%NGS Anti-HRP Alexa

Fluor488

(1:400)

Jackson

ImmunoResearch

Laboratories, Inc

Rabbit anti-V5 2%BSA+2%DS Anti-V5 (1:200) Millipore

Donkey anti rabbit-cy3

(1:600) (Jackson

ImmunoResearch

Laboratories, Inc)

Rabbit anti-Myc 2%BSA+2%DS Anti-Myc (1:250) Millipore


(1:600) (Jackson

ImmunoResearch

Laboratories, Inc)

Rabbit anti-Amph 2%BSA+2%DS Anti-Amph

(1:100) G. Boulianne


(1:600) (Jackson

ImmunoResearch

Laboratories, Inc)

Western Blotting

Mouse anti-actin 3% skim milk Anti-actin, JLA20

(1:100) Iowa University

Anti-mouse HRP

(1:10,000)

Mouse anti-HA 3% skim milk Anti-HA Covance Anti-mouse HRP

(1:2000) (1:10,000)

Mouse anti-Flag 3% skim milk Anti-Flag

(1:5000) Sigma

Anti-mouse HRP

(1:10,000)

Mouse anti-GST 5% dry milk Anti-GST

(1:5000) Covance

Anti-mouse HRP

(1:10,000)

Mouse anti-His 3% skim milk Anti-His (1:1000) Qiagen Anti-mouse HRP

(1:10,000)

Rabbit anti-Amph 3% skim milk Anti-Amph

(1:500) G. Boulianne

Anti-rabbit HRP

(1:10,000)

41

protease inhibitors (1 mM PMSF, 1 µg/ml each of aprotinin, leupeptin and pepstatin A).

dNedd4S or dNedd4Lo was immunoprecipitated (IP) with M2 anti-Flag agarose beads and the

membrane was immunoblotted with anti HA antibody. Flag-dNedd4S and dNedd4Lo were

detected using M2 anti-Flag antibody and HRP-conjugated goat anti-mouse antibody (Table 1).

2.6 Third instar larval fillet preps and immunofluorescent staining

of body wall muscles

Wandering third instar larvae were dissected using a standard fillet preparation technique (Brent

et al., 2009), fixed in 4% paraformaldehyde (PFA) (20 min), washed in PBT (0.1% Tween-20 in

PBS), blocked (2% BSA, 2% Normal Goat Serum (NGS) and/or 2% Donkey Serum (DS) in

PBT) for 1 hr at RT prior to overnight incubation with primary antibodies at 4oC. See Table 1 for

antibodies and dilutions used, and Table S1 for fly line crosses used in this study. Muscle

segments and postsynaptic regions from muscle 6/7 were visualized using a Zeiss AxioVert

200M confocal microscope with a Zeiss objective lens set to 100x (oil imaging medium),

numerical aperture 1.4 or 63x (water imaging medium), numerical aperture 1.3. Confocal data

was acquired at RT with a Hamamatsu C9100-13 EM-CCD camera and images were analyzed

and deconvolved to reduce background signal using Volocity 6.3.0 (Perkin Elmer).

I. Analysis of postsynaptic dAmph and T-tubule architecture in Drosophila larva:

Larvae were incubated overnight with rabbit anti-Amph antibody and mouse anti-

Dlg antibody at 4oC. The samples were incubated for 2 hrs with Cy3-conjugated

donkey anti-rabbit and Alexa488

conjugated goat anti-mouse antibody to visualize

dAmph and Dlg, respectively (Table 1).

42

Table 2: Fly line crosses

Overexpression of dNedd4 in the muscle for immunohistochemistry and western blot analysis

dAmph5E3

(Amph null,

Leventis et al., 2001)

Control for anti amph antibody

W1118

(Wild type ) [source:

G. Boulianne]

X 24B-GAL4 (driver control) [source: Bloomington]

24B-Gal4 X 24B-Gal4 (driver control)

UAS-dNedd4S-Flag

(Zhong et al., 2011)

X 24B-GAL4

UAS-dNedd4Lo WT-Flag


X 24B-GAL4

UAS-dNedd4Lo CA-Flag


X 24B-GAL4

Overexpression of dAmph and dNedd4 in the muscle for immunohistochemistry

UAS-dAmph WT-V5

[source: G. Boulianne]

X 24B-GAL4

UAS-dAmph WT-V5 X UAS-dNedd4S-Flag; 24B-GAL4

UAS-dNedd4Lo WT-Flag;

UAS-dAmph WT-V5

X 24B-GAL4

UAS-dAmph WT-V5 X UAS-dNedd4Lo CA-Flag; 24B-GAL4

UAS-dAmph ΔSH3-Myc

[source: G. Boulianne]

X 24B-GAL4

43

II. Co-localization of V5-dAmph and Flag-dNedd4Lo: Larvae were immunostained with

M2 anti-Flag antibody and rabbit anti-V5 primary antibodies overnight at 4oC. The

samples were incubated for 2 hrs with Cy3-conjugated goat anti-mouse antibody,

Alexa Fluor488

conjugated goat anti-rabbit antibody, and Alexa Fluor647

HRP to label

the presynaptic region, and visualized by confocal microscopy (See Table 1).

III. Postsynaptic localization of V5-dAmph and MYC-ΔSH3 dAmph: Larvae were

immunostained with mouse anti-Dlg and rabbit anti-V5 or rabbit anti-MYC primary

antibodies overnight at 4oC. The samples were incubated for 2 hrs with Cy3-

conjugated donkey anti-rabbit antibody, Alexa Fluor488

conjugated goat anti-mouse

antibody, and Alexa Fluor647

HRP (Table 1). Co-localization of Dlg and dAmph (V5-

WT or MYC-ΔSH3) was assessed by Volocity 6.3.0 (Perkin Elmer) and expressed in

terms of the Pearson's correlation coefficients.

2.7 Western Blot analysis of muscle protein levels of dAmph in

dNedd4 transgenic larvae

Third instar larvae were collected (35 larvae per line, or 10 larvae for the overexpressed V5-

dAmph), washed in 1x PBS and manually homogenized in lysis buffer plus protease inhibitors (1

mM PMSF, 1 µg/ml each of aprotinin, leupeptin, and pepstatin A) and 40µM MG132, and 0.2

mM chloroquine on ice. Samples were examined by Western blotting, using rabbit anti-Amph or

mouse anti-V5 antibody and M2 anti-Flag antibody to detect dAmph and dNedd4, respectively

(Table 1). The amount of dAmph was determined relative to Actin using Image Studio software

after subtracting the background signal from Amph null flies for the endogenous dAmph.

2.8 Statistical Analysis

I. Quantification of postsynaptic levels of dAmph in dNedd4 transgenic fly: The

postsynaptic region was identified as the area demarcated by anti-Dlg immunostaining,

44

and the intensity of endogenous or overexpressed (using V5 antibodies) dAmph in this

region was measured using Volocity 6.3.0 software (Perkin Elmer). The intensity of

dAmph and Dlg in this region was standardized to the levels of dAmph in WT or 24B-

Gal4 flies for overexpressed V5-dAmph. For endogenous dAmph levels, the intensity of

dAmph measured in Amph null was subtracted from the intensity of dAmph measured

for each sample to eliminate background signal from the dAmph antibody. Student’s t-

test was performed to analyze levels of dAmph in the presence of dNedd4 isoforms and

the dNedd4Lo(CA) mutant. A p-value of ≤ 0.05 was considered significant.

II. Quantification of defective muscle T-tubules in dNedd4Lo transgenic fly: The indicated

numbers of muscle 6/7 hemi-segments for Amph null (28), wildtype (80), dNedd4S (30),

dNedd4Lo (39) and dNedd4Lo(CA) (94) were analyzed and compared to wildtype to

determine whether segments appeared normal or abnormal. Muscle segments that showed

reduced transverse tubules numbers were defined as abnormal. GraphPad Prism 5.0

software (GraphPad Software, San Diego, CA) and Fisher's exact test in a 2×2

contingency table were used to analyze the data for T-tubule defects of body wall muscle

segments. The number of transverse (T) tubules that appeared to branch from a

longitudinal tubule segment were also counted blindly. For each muscle segment

analyzed, the mean T-tubules along three different longitudinal segments of a hemi-

segment were counted. GraphPad Prism 5.0 software and student’s t-test was performed

to quantify the number of T-tubules branching along segments of longitudinal tubules.

45

Chapter 3: Results

3. Results

3.1 DAmph interacts with dNedd4Lo

Previously, we found that dNedd4S promotes neuromuscular (NM) synaptogenesis (Ing

et al 2007), but that dNedd4Lo negatively regulates this process and inhibits muscle function

(larval locomotion) (Zhong et al., 2011). The unique N-terminal and Middle region, as well as

the HECT domain of dNedd4Lo, are involved in regulating its adverse function in

synaptogenesis (Zhong et al., 2011). Since this adverse effect was not caused by inhibition (by

dNedd4Lo) of the HECT domain of dNedd4S, nor by AKT phosphorylation of dNedd4Lo

(Zhong et al 2011), we suspected that the unique regions of dNedd4Lo might negatively regulate

NM synaptogenesis and muscle function by interacting with other proteins. To identify such

putative interacting partners, embryo lysates were incubated with purified (immobilized) unique

regions of dNedd4Lo (N-terminus and Middle regions), and several high confidence (>95%)

interacting proteins were identified by mass spectrometry (Table 3). Drosophila Amphiphysin

(dAmph), which contains BAR and SH3 domains, was identified as a binding partner of the

unique N-terminal region of dNedd4Lo (Table 3 and Fig. 1A). To validate the MS results, we

tested whether dAmph can bind dNedd4Lo by co-immunoprecipitation (co-IP). We transiently

expressed HA-tagged dAmph and Flag-tagged dNedd4Lo or dNedd4S in Drosophila S2 cells,

and found binding (co-IP) of dAmph with dNedd4Lo, but not dNedd4S (Fig. 1B).

46

Table 3: Mass spectrometry identification of binding partners to Nedd4Lo unique N-

terminus and Middle regions. (A) Bacterially expressed GST tagged dNedd4Lo N-terminus

(Nterm), residues 1-63, or dNedd4Lo Middle (Mid), residues 304-473, were incubated with

embryo lysates (~100 embryos) and analyzed by a Thermo LTQ-Orbitrap Hybrid Mass

Spectrometer. The BAR-SH3 domain containing proteins: dAmph, syndapin as well as sorting

nexin 9 were identified as interacting proteins of the N-terminus and middle region of

dNedd4Lo, respectively, using X! Tandem to search the Drosophila melanogaster database. (B)

dAmph peptides identified as binding partners to dNedd4Lo N-terminus in the MS analysis.

47

Total Spectral Counts

Identified Proteins (80)

Accession

Number

MW,

kDa GST GST

GST

dNedd4-

Lo,

Middle

GST

dNedd4-

Lo,

Middle

GST

dNedd4-

Lo, N-

Term

GST

dNedd4-

Lo, N-

Term

Nedd4, isoform H [Drosophila melanogaster] gi|221512757 113 0 0 378 410 123 173

heat shock protein cognate 3, isoform A

[Drosophila melanogaster]

gi|24641402

(+3) 72 2 5 38 41 46 49

SH3PX1 [Drosophila melanogaster] gi|21355219 63 0 0 53 67 0 0

Rm62, isoform A [Drosophila melanogaster]

gi|24644479

(+6) 79 0 0 39 39 0 0

glutathione S transferase D1, isoform A


gi|17737923

(+1) 24 4 3 11 12 11 11

CG6459 [Drosophila melanogaster] gi|20130085 29 0 3 20 18 0 2

Prp19 [Drosophila melanogaster] gi|17647459 55 0 0 31 29 0 0

heat shock protein cognate 4, isoform A


gi|17737967

(+5) 71 0 4 29 26 18 20

glutathione S transferase E6 [Drosophila

melanogaster] gi|19922532 25 0 0 21 24 0 0

CG31012, isoform C [Drosophila

melanogaster] gi|28571934 92 0 0 0 0 16 21

ribosomal protein S4, isoform A [Drosophila

melanogaster]

gi|24663668

(+1) 29 0 0 17 20 0 0

beta-Tubulin at 56D, isoform B [Drosophila

melanogaster] gi|24655737 50 4 9 0 3 0 0

heat shock protein cognate 5 [Drosophila

melanogaster] gi|24653595 74 0 0 5 7 6 6

ribosomal protein S17 [Drosophila

melanogaster] gi|17647889 15 0 2 9 9 2 3

Roe1 [Drosophila melanogaster] gi|24653432 24 0 0 0 0 8 6

CG6905, isoform A [Drosophila melanogaster]

gi|19922992

(+1) 93 0 0 13 12 0 0

ribosomal protein S3A, isoform A [Drosophila

melanogaster] gi|17864162 30 0 0 10 9 0 2

belle [Drosophila melanogaster] gi|17985987 85 0 0 12 16 0 0


melanogaster]

gi|24661707

(+1) 23 0 0 11 5 0 0

ribosomal protein S15Aa, isoform D


gi|17975567

(+4) 15 0 0 7 6 0 0

receptor of activated protein kinase C 1

[Drosophila melanogaster] gi|17137396 36 0 0 7 11 0 0

ribosomal protein L5, isoform A [Drosophila

melanogaster]

gi|116007382

(+1) 34 0 0 7 10 0 0


melanogaster] gi|24651181 24 0 0 7 7 0 0

cabeza [Drosophila melanogaster] gi|24642436 39 0 0 7 7 0 0

string of pearls [Drosophila melanogaster] gi|17136734 29 0 0 5 8 0 0

actin 5C, isoform B [Drosophila melanogaster]

gi|17530805

(+4) 42 0 7 0 0 2 3

growl, isoform A [Drosophila melanogaster] gi|21355167 60 0 0 6 6 0 0

Table 3: Mass spectrometry identification of binding partners to Nedd4Lo

unique N-terminus and Middle regions

A. Performed by Yunan Zhong

48

ribosomal protein L23A [Drosophila

melanogaster] gi|24655502 29 0 0 8 4 0 2

ribosomal protein LP0 [Drosophila

melanogaster] gi|17737731 34 0 0 3 7 0 0

ribosomal protein L26 [Drosophila

melanogaster] gi|21357853 17 0 0 0 4 0 0

alpha-Tubulin at 84B [Drosophila

melanogaster]

gi|17136564

(+2) 50 3 4 0 0 0 0

argonaute 2, isoform B [Drosophila

melanogaster]

gi|24664664

(+1) 137 0 0 4 3 0 0


melanogaster] gi|19922390 17 0 0 5 4 0 0

syndapin [Drosophila melanogaster] gi|28571785 56 0 0 0 0 4 5


melanogaster]

gi|17136322

(+1) 24 0 0 5 5 0 0

heterogeneous nuclear ribonucleoprotein at

87F, isoform A [Drosophila melanogaster]

gi|17136624

(+1) 39 0 0 3 9 0 0

downstream of receptor kinase, isoform A


gi|17136708

(+5) 24 0 0 0 0 4 6

amphiphysin [Drosophila melanogaster] gi|17647155 66 0 0 0 0 4 4


melanogaster] gi|17137152 31 0 0 5 4 0 0


melanogaster]

gi|24645865

(+1) 13 0 0 3 2 0 0

Fmr1, isoform A [Drosophila melanogaster]

gi|19922726

(+4) 76 0 0 4 5 0 0

vasa intronic gene, isoform A [Drosophila

melanogaster]

gi|17737419

(+3) 53 0 0 5 4 0 0

glutathione S transferase D5 [Drosophila

melanogaster] gi|45549270 25 0 0 2 4 0 2

capping protein alpha [Drosophila

melanogaster] gi|19922662 33 0 0 0 0 4 6

capping protein beta [Drosophila

melanogaster] gi|17136938 31 0 0 0 0 4 4

elongation factor 1alpha48D, isoform A


gi|17137572

(+1) 50 0 0 0 3 0 0


melanogaster] gi|17647883 15 0 0 5 5 0 0


melanogaster] gi|17137026 21 0 0 3 4 0 0

glyceraldehyde 3 phosphate dehydrogenase 2,

isoform B [Drosophila melanogaster]

gi|17933600

(+1) 35 2 3 0 2 0 0


melanogaster]

gi|21358459

(+3) 22 0 0 0 4 0 0


melanogaster] gi|22024141 16 0 0 0 2 0 0

histone H1 [Drosophila melanogaster]

gi|24585669

(+21) 26 0 0 2 4 0 0

beta'-coatomer protein [Drosophila

melanogaster] gi|24584107 103 0 2 0 0 0 0

ypsilon schachtel [Drosophila melanogaster] gi|24663131 38 0 0 3 2 0 0

SF2 [Drosophila melanogaster] gi|21358097 28 0 0 2 5 0 0

Qm, isoform C [Drosophila melanogaster]

gi|221513692

(+1) 26 0 0 0 3 0 0

49

ribosomal protein S11, isoform C [Drosophila

melanogaster]

gi|24652922

(+1) 18 0 0 0 2 0 0


melanogaster] gi|19921254 18 0 0 0 4 0 0

mitochondrial ribosomal protein L11

[Drosophila melanogaster] gi|17737961 22 0 0 3 2 0 0

Aly [Drosophila melanogaster] gi|21356157 28 0 0 0 2 0 0


melanogaster]

gi|17864452

(+2) 18 0 0 3 2 0 0


melanogaster]

gi|17136832

(+1) 17 0 0 3 2 0 0

trailer hitch [Drosophila melanogaster] gi|45550607 69 0 0 4 2 0 0

ribosomal protein S6, isoform B [Drosophila

melanogaster] gi|17737290 28 0 0 2 3 0 0


melanogaster]

gi|17137274

(+2) 9 0 0 4 2 0 0


melanogaster]

gi|17647879

(+1) 25 0 0 2 2 0 0



CG6995, isoform A [Drosophila melanogaster]

gi|45550823

(+1) 73 0 0 2 3 0 0


melanogaster]

gi|17864264

(+1) 12 0 0 3 3 0 0

ribosomal protein L31, isoform B [Drosophila

melanogaster]

gi|19921922

(+2) 15 0 0 0 3 0 0

ribosomal protein S27A [Drosophila

melanogaster] gi|17136574 18 0 0 2 0 0 0

transport and golgi organization 4 [Drosophila

melanogaster] gi|18859793 53 0 0 0 2 0 0

twinstar [Drosophila melanogaster] gi|17136986 17 3 0 0 0 0 0

glutathione S transferase E3 [Drosophila

melanogaster] gi|24654975 25 0 0 2 0 0 0

ribosomal protein LP1 [Drosophila

melanogaster] gi|17136320 12 0 0 2 0 0 0

alpha-coatomer protein, isoform A [Drosophila

melanogaster]

gi|17137608

(+1) 139 0 2 0 0 0 0

vig2, isoform D [Drosophila melanogaster]

gi|161078610

(+3) 45 0 0 0 2 0 0

fibrillarin [Drosophila melanogaster] gi|17647425 35 0 0 0 2 0 0

protein on ecdysone puffs, isoform B


gi|17864514

(+1) 78 0 0 0 2 0 0

50

Unique peptides of Amphiphysin identified as interactors of dNedd4Lo N-terminus

Sequence Prob X! Tandem

(R)HSFQNLQANANK(R) 100% 4.958607

(K)GREQLEEAR(R) 98% 3.3372421

(K)ATTTTQTSPTEDK(A) 95% 2.7447276

(R)VIEYDDPEDQEEGWLMGQK(E) 100% 10.207608

(K)GLFPANFTRPI(-) 100% 4.2676063

B.

51

Figure 11: dNedd4Lo co-immunoprecipitates with dAmph

(A) Schematic representation of dNedd4S and dNedd4Lo showing the C2, WW(x3) and HECT

domains. dNedd4Lo contains unique N-terminal (Nterm) and Middle (Mid) regions that are

absent in dNedd4S. The Drosophila Amphiphysin (dAmph) protein was identified as an

interacting partner of the unique N-terminal region of dNedd4Lo by mass spectrometry. (B)

DAmph binds to dNedd4Lo in Drosophila S2 cells: Drosophila S2 cells were transfected (Tfxn)

with Flag-tagged dNedd4S or dNedd4Lo and HA-tagged dAmph. Transfected cells were lysed

and the lysates were immunoprecipitated (IP) with anti-Flag (dNedd4) antibodies and

immunoblotted with anti-HA to detect co-immunoprecipitated dAmph (upper panel).

Precipitated Flag-dNedd4 proteins are shown in the second panel. DAmph expression in the

lysates was verified using anti-HA antibodies; actin was used as a loading control (lower panels).

53

3.2 dNedd4Lo N-terminus directly binds to the SH3 domain of

dAmph

DAmph contains a C-terminal SH3 domain, a domain that usually binds proline-rich

motifs (Grabs et al., 1997). dNedd4Lo N-terminus contains a Pro-rich sequence with a perfect

consensus motif for SH3 binding (PRPPPR). We therefore tested in vitro binding of a purified

SH3 domain of dAmph to the purified N-terminus of dNedd4Lo. Purified HIS tagged full-length

(WT) dAmph, SH3 domain deletion mutant (dAmph-𝛥𝑆𝐻3), SH3 mutant (dAmph SH3(W->A),

containing a mutation in the conserved Trp required for binding the Pro-rich motif), or the SH3

domain of dAmph alone (SH3 dAmph) (Fig. 12A), were incubated with purified GST-tagged

dNedd4Lo N-terminus (residues 1-63). Our results show that WT dAmph and the dAmph-SH3

domain bound to GST-dNedd4Lo N-terminus, while the dAmph(𝛥𝑆𝐻3) or dAmph-SH3(W->A)

mutants did not (Fig 12B). This demonstrates that the SH3 domain of dAmph directly binds the

N-terminus of dNedd4Lo.

54

Figure 12: Drosophila Amphiphysin directly binds the N-terminus of dNedd4Lo via its SH3

domain. (A) Schematic representation of dNedd4Lo, the unique N-terminal region of dNedd4Lo

(Nterm, residues 1-63), wildtype dAmph (WT dAmph), the SH3 mutant containing a tryptophan

to alanine substitution in the SH3 domain (dAmph SH3(W->A)), SH3 deletion mutant (ΔSH3

dAmph) and the SH3 domain alone (SH3 dAmph). (B) The SH3 domain of dAmph mediates

direct binding to the N-terminal region of dNedd4Lo: HIS-tagged purified dAmph (WT, ΔSH3,

SH3(W->A) or SH3 domain alone) were incubated with GST beads or GST fused to dNedd4Lo

N-terminal region (GST dNedd4Lo(Nterm)). DAmph was detected using HIS antibodies and

dNedd4Lo(Nterm) using GST antibodies.

56

3.3 dAmph is enriched postsynaptically and co-localizes with

dNedd4Lo

Drosophila dAmph is enriched postsynaptically at neuromuscular junctions (NMJs) and

in muscle T-tubules (Leventis et al., 2001), and our earlier work detected dNedd4 expression in

larval muscles (Ing et al., 2007). To investigate the biological importance of the

dAmph:dNedd4Lo interaction in vivo, we first analyzed co-localization of dAmph and

dNedd4Lo in the postsynaptic region and the muscle T-tubule network. It has been previously

shown that endogenous dAmph co-localizes with the postsynaptic marker Dlg at NMJs (Leventis

et al., 2001). Here, we overexpressed Flag-tagged dNedd4Lo and V5-tagged dAmph in flies

using the muscle-specific driver 24B-Gal4, and examined larval NMJs located on muscles 6/7 by

immunohistochemistry with antibodies against Flag (dNedd4), V5 (dAmph) and HRP

(presynaptic marker). dNedd4Lo co-localized with dAmph in muscles but not with HRP

labeling, which is specific for (presynaptic) neuronal membranes (Fig. 13A). To verify that V5-

dAmph is localized postsynaptically and in muscle T-tubules, we examined NMJs using

antibodies against V5, HRP, and a postsynaptic/T-tubule marker Disc large (Dlg) (Fig. 13B).

Similar to what was previously observed with endogenous dAmph (Leventis et al., 2001), V5-

dAmph co-localized with Dlg labeling (Fig.13B), indicating that dNedd4Lo co-localizes with

dAmph in the postsynaptic region and muscle T-tubules.

57

Figure 13: Drosophila Amph and dNedd4 are enriched postsynaptically at the

neuromuscular junctions and in muscle T-tubules.

(A) dNedd4Lo is expressed in the muscles and the postsynaptic region of the neuromuscular

junction (NMJ) where it co-localizes with Drosophila Amphiphysin (dAmph). (B) DAmph is

enriched postsynaptically in NMJ and in muscle T-tubules where it co-localizes with

postsynaptic and T-tubule marker Dlg. Expression was driven by 24B-Gal4 in larval muscles,

dNedd4Lo (UAS-dNedd4Lo) was detected with an anti-Flag antibody (green) and dAmph (UAS-

V5-dAmph) was detected with V5 antibody (red). Synaptic boutons from muscles 6/7 were

analyzed. Presynaptic marker: HRP (blue). Scale bar: 3 m.

59

3.4 dAmph lacking its SH3 domain is mislocalized in muscles

Since we found that the interaction between dNedd4Lo and dAmph is mediated by the

SH3 domain of dAmph, we expect this interaction to be lost in vivo in dAmph(ΔSH3) mutant

flies. To determine the localization of the dAmph(ΔSH3) mutant in Drosophila, we analyzed

UAS transgenic fly lines that overexpress MYC-tagged dAmph(ΔSH3) in muscles. In contrast to

wildtype dAmph, dAmph(ΔSH3) did not co-localize in muscle T-tubules with Dlg. Deletion of

the SH3 domain of dAmph shifts its localization from the muscle T-tubules and postsynaptic

region to a region near the plasma membrane of the muscles (Fig. 14A and B). Quantification of

the Pearson’s correlation coefficient further shows that dAmph WT co-localizes with the

postsynaptic marker Dlg (r= 0.83) (Fig. 14C), but that the SH3 domain deletion mutant

dAmph(ΔSH3) shows a significantly reduced co-localization (r=0.45). These results indicate that

the SH3 domain of dAmph is important for its localization in the postsynaptic region and muscle

T-tubules.

60

Figure 14: dAmph(ΔSH3) mislocalizes in Drosophila muscles.

(A) ΔSH3 dAmph (red) localization with Dlg (green) is reduced in the postsynaptic region and

muscle T-tubules. (B) Wild-type (WT) dAmph (red) is enriched postsynaptically and in muscle

T-tubules, where it co-localizes with Dlg (green). Muscle overexpression of dAmph(ΔSH3) and

WT dAmph was achieved using 24B-Gal4 muscle driver. dAmph(ΔSH3) and WT dAmph were

visualized in third instar larva postsynaptic regions of muscle 6/7 using anti-MYC and anti-V5

antibodies, respectively. Presynaptic marker: HRP (blue). Scale bar: 3 m. (C) dAmph ΔSH3 or

WT colocalization with the postsynaptic marker Dlg is expressed in terms of the Pearson’s

correlation coefficient. Graph illustrates changes in the mean Pearson's coefficients of dAmph

V5-WT or MYC-ΔSH3 with Dlg (WT dAmph, n=24;ΔSH3 dAmph, n=30). Error bars indicate

SEM. *** denotes p

62

3.5 dNedd4Lo regulates the levels of dAmph in the postsynaptic

region

As dNedd4Lo is an E3 ubiquitin ligase, we investigated the possibility that it regulates

levels of dAmph in vivo in the postsynaptic region. Thus, we analyzed the levels of endogenous

dAmph in our UAS transgenic fly lines that overexpress dNedd4S, dNedd4Lo and

dNedd4Lo(CA) (a catalytically inactive mutant generated by mutating the catalytic cysteine in

the HECT domain). The flies used in this experiment have a wild type genetic background

because dNedd4 null (dNedd4T121FS

) flies are homozygous lethal during embryogenesis and

cannot be rescued with dNedd4Lo overexpression (Zhong et al., 2011).

If dNedd4Lo regulates dAmph, then we expect dAmph abundance in the postsynaptic

region to be reduced in flies overexpressing dNedd4Lo but not the dNedd4Lo(CA) mutant or

dNedd4S. We examined larval NMJs located on muscles 6/7 by immunohistochemistry with

antibodies against dAmph and Dlg. w1118

(wildtype) flies and damph5E3

(Amph null) transgenic

flies that have their entire amph gene deleted were used as controls (Leventis et al., 2001). The

mean intensity of dAmph that overlapped with Dlg labeling in the postsynaptic region was used

to quantify the levels of dAmp

drosophila nedd4-long reduces amphiphysin levels in ...€¦ · chapter 3: results ... c2 –...

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