The Pennsylvania State University Schreyer Honors College

THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE

DEPARTMENT OF BIOLOGY

GENETIC ANALYSIS IN DROSOPHILA REVEALS A NOVEL ROLE FOR THE ESCRT COMPONENT VPS24 IN SYNAPTIC TRANSMISSION

KERRY CAO SPRING 2013

A thesis submitted in partial fulfillment of the requirements for a baccalaureate degree in Chemistry with honors in Biology

Reviewed and approved* by the following:

Fumiko Kawasaki Assistant Professor of Biology Thesis Supervisor

Bernhard Lüscher Professor of Biology Honors Adviser

* Signatures are on file in the Schreyer Honors College. i ABSTRACT

Understanding the in vivo molecular mechanisms of synaptic transmission remains a major objective in cellular and molecular neuroscience. Our genetic analysis of synaptic transmission has identified a novel synaptic mechanism involving the Endosomal Sorting Complexes Required for

Transport (ESCRT) pathway previously implicated in intracellular membrane trafficking. A forward genetic screen for new mutations affecting synaptic transmission involved chemical mutagenesis with ethyl methanesulfonate (EMS) followed by screening for third chromosome mutations affecting fly behavior. A new mutation was recovered in the vps24 gene, which encodes a conserved protein component of the ESCRT-III complex which functions in targeting proteins to multivesicular bodies

(MVBs) for degradation. The new Drosophila vps24 mutant exhibits defects in neurotransmitter release and provides the first evidence implicating the ESCRT pathway in synaptic transmission. Surprisingly,

VPS24 was found to be localized in proximity to neurotransmitter release sites at active zones (AZs), suggesting a novel ESCRT-mediated mechanism contributing to neurotransmitter release. Furthermore, our preliminary studies of other ESCRT proteins indicate function and localization of this pathway within the presynaptic terminal. Ongoing studies have involved generation of Drosophila transgenic lines and further in vivo analysis of the VPS24 protein properties and functions in synaptic transmission. Given the strong evolutionary conservation of the ESCRT pathway, it is anticipated that these studies will be of general significance to our understanding of the ESCRT pathway and its importance in neural function.

TABLE OF CONTENTS

List of Figures ...... iii

Acknowledgements ...... iv

Contributions ...... v

Introduction ...... 1

Results ...... 10

Discussion ...... 19

Materials and Methods ...... 23

References ...... 26

iii

LIST OF FIGURES

Figure 1. Multivesicular body biogenesis ...... 2

Figure 2. Interactions among the four ESCRT complexes and associated molecules ...... 3

Figure 3. Sequence of intralumenal vesicle formation ...... 6

Figure 4. Purse string model of ESCRT-III disassembly ...... 8

Figure 5. Forward genetic screen for new mutations affecting synaptic transmission ...... 10

Figure 6. Behavioral phenotype of 748 mutant ...... 11

Figure 7. Electrophysiology recordings of 748/Df(3R)Exel6140 flies ...... 12

Figure 8. Genetic mapping of 748 mutation ...... 13

Figure 9. Preservation of presynaptic morphology and organization in vps241 mutant ...... 15

Figure 10. Localization of VPS24 in active zones at neuromuscular synapses ...... 16

Figure 11. An ultrastructural synaptic phenotype in the vps241 mutant ...... 17

Figure 12. Localization of VPS28 at neuromuscular synapses ...... 18

Figure 13. The synaptic vesicle cycle ...... 21

ACKNOWLEDGEMENTS

First and foremost, I would like to thank Dr. Fumiko Kawasaki and Dr. Richard Ordway for their boundless knowledge, guidance, patience, and constant support throughout my time in this research lab. I am fortunate to have two established faculty as open and accessible resources. I would also like to thank

Dr. Bernhard Lüscher for his generosity, patience, and flexibility during this process. I am grateful for Dr.

Janani Iyer, Rie Danjo, and Yunzhen Zheng for making me feel like a welcomed member of the lab and for providing insight into the theory behind the methods of the lab. I am thankful for Sean Stevens for being one of many mentors, being someone to talk to during my early days in the lab, and showing me the protocols of the lab. I am grateful for and appreciate Chris Wahlmark and Giselle Ahnert for partnering with me during our molecular biology and flywork experiments, which made learning new techniques less stressful and also more informative. I would like to thank Alex Strauss, Julie Oliver, and Ximena

Chica for being the role models I strove to emulate. I am thankful for Rolfy Perez and the other undergraduate members of the lab for being my friends during this journey as well as providing a unique and pleasant dynamic in the lab. I would like to thank Dr. Noreen Reist for providing the anti-SYT antibody, Dr. Helmut Kramer for providing the anti-VPS28 antibody, and the Developmental Studies

Hybridoma Bank for supplying the anti-BRP antibody. I would also like to thank the Bloomington

Drosophila Stock Center for supplying the deficiency and P element stocks used in this thesis as well as the Drosophila Genomics Resource Center (Bloomington, IN) for providing us with the vps24 cDNA needed to conduct our transgenic experiments.

CONTRIBUTIONS

Sean Stevens introduced me to the majority of the flywork done in this thesis. He, along with Giselle

Ahnert and Christ Wahlmark, assisted with the execution of the forward genetic screen and behavior test.

Giselle Ahnert and Chris Wahlmark were responsible for the mutagenesis of Iso 3 male flies used in the genetic screen. Giselle Ahnert and Chris Wahlmark were also collaborators for the molecular biology experiments conducted to generate the UAS-EGFP-vps24 construct used to generate the transgenic flies mentioned in this thesis. Chris Wahlmark assisted with the larval dissections used for immunocytochemical analysis and the rescue of the vps241 mutant. Dr. Fumiko Kawasaki conducted the electrophysiological experiments and performed confocal microscopy on the larval and adult immunocytochemical preparations.

INTRODUCTION

Eukaryotic endosomes coordinate the movement of proteins between the plasma membrane, the trans-Golgi network, and the lysosome/vacuole [1]. The endosomal system conducts this intracellular trafficking by internalizing membrane proteins and sorting these to multivesicular bodies (MVBs), which consist of membrane enclosing intralumenal vesicles (ILVs) formed by the invagination of the outer membrane into the lumen [2]. Ubiquitination serves as the major entry signal for proteins into the MVB pathway [3,4]. A set of approximately 20 proteins, the majority of which are vacuolar protein sorting

(Vps) gene products, conserved from yeast to mammals is thought to regulate and drive formation of

ILVs (Figure 1a). These include components of four ESCRT complexes (endosomal sorting complexes required for transport-0, I, II, and III), the AAA ATPase VPS4, and several associated proteins. Mutations in genes encoding ESCRT subunits cause enlarged, “Class E,” endosomal compartments, and result in sorting defects [5,6].

Recently, non-MVB functions for several ESCRT components have been identified [7]. Because the required membrane curvature is opposite to that of clathrin-mediated endocytosis and other coat- mediated vesicle formation, it is likely that novel mechanisms are involved. Importantly, the role of the

ESCRT machinery extends to topologically similar cellular processes including autophagy, cytokinesis, and viral budding (Figure 1b-d). For example, ESCRT-III has been implicated in the autophagy pathway and in the clearance of protein aggregates that characterize neurodegenerative disorders [8-10].

Mammalian ESCRT-III also has a role in retroviral budding, as do ESCRT-0, -I, and the ESCRT- associated protein AIP/ALIX [11,12]. ESCRT-III components have also been shown to concentrate at the mid-body during cell abscission in cytokinesis [13,14], a process that is topologically similar to retroviral budding and ILV formation. Interest in the ESCRT machinery has risen over the past few years mainly since perturbations in this pathway contribute to a large number of defects in various cellular processes leading to human diseases. The present study advances our understanding of the ESCRT pathway by providing the first evidence revealing a novel role for this degradation pathway in synaptic transmission. 2

Modified from Hurley J. H. & Hanson P. I. (2010) Nat. Rev. Mol. Cell Biol.

Figure 1 Multivesicular body (MVB) biogenesis (a) and autophagy (b) require all the ESCRTs. In contrast, cytokinesis (c) and HIV-1 budding (d) require only ESCRT-I and ESCRT-III.

Components of the ESCRT machinery were first identified in yeast Saccharomyces cerevisiae when interferences in the function of these proteins led to a Class E mutant characterized by missorting and aggregation of cargo normally destined for the vacuole, the lysosome-equivalent in yeast [15]. Since the endocytosis of transmembrane proteins is triggered by ubiquitination [16], it is expected that one or more components of the ESCRT machinery respond to this event. Indeed, ESCRT-0 contains five ubiquitin-binding domains [16] and clusters ubiquitinated cargo in vitro [17]. Recruitment of ESCRT-0 to the early endosomal membrane initiates the recruitment of the ESCRT-I, -II, and –III complexes [18,19].

Based on in vitro reconstitution, ESCRT-I and -II drive membrane budding, whereas ESCRT-III cleaves the bud necks to form intraluminal vesicles [17,20,21]. ESCRT-III subunits recruit the ESCRT-associated protein ALIX [22] and the deubiquitinating enzymes UBPY and AMSH to endosomes [23,24, 25].

Although not addressed in the present study, it is still important to understand the composition and the dynamic interactions of ESCRT-0, ESCRT-I, and ESCRT-II (Figure 2). All three are soluble, stable hetero-oligomeric complexes in the cytosol [3,26,27]. ESCRT-0 is an ~ 8 nm long heterodimer of 3

HRS and STAM that consists of two short intertwined helical domains joined by an antiparallel coiled- coil [26]. Both HRS and STAM bind ubiquitin and clathrin. HRS additionally has a FYVE zinc-finger domain that binds PI(3)P and PSAP sequence motifs that mediate interaction with Tsg101 in ESCRT-I.

Vps4

Vps20 Vps25 Vps32 Mvb12 Vps2 Vps37 Vps24 STAM Vps36 Tsg101 Hrs ESCRT-III ESCRT-I Vps22 ESCRT-0 Vps28 ESCRT-II

Modified from Raiborg C. & Stenmark H. (2009) Nature Figure 2 Interactions among the four ESCRT complexes are indicated, as are interactions with ubiquitinated cargo and accessory molecules such as PtdIns(3)P, de-ubiquitylating enzymes (DUBs), Alix and the ATPase, Vps4. Protein domains are labeled in white. CB, clathrin-box motif. Ub, ubiquitin. Dashed arrow indicates an interaction predicted by genetic studies but not yet confirmed biochemically.

ESCRT-I is an 18-25 nm elongated heterotetrameric complex containing one copy each of

Tsg101, VPS28, VPS37, and MVB12 [28,29]. It consists of a headpiece that binds with high affinity to

ESCRT-II, an elongated stalk, and a tailpiece that binds ubiquitin and ESCRT-0. ESCRT-II is another heterotetramer made up of one subunit of VPS36 and VPS22 and two subunits of VPS25 that form a compact Y-shaped complex with a hydrodynamic radius of ~ 5 nm [30]. VPS36 interacts with PI(3)P, ubiquitin, and ESCRT-I at one end of the complex while VPS25 binds VPS20 to connect the other end to

ESCRT-III. If present together on the membrane, these three complexes are expected to cover an area of membrane similar to that needed to form a lumenal vesicle [30].

Importantly, ESCRT-III consists of small, structurally related cytosolic proteins which exist as soluble monomers that assemble into a detergent-insoluble complex on the endosomal membrane [31,32].

Yeast has six similarly-sized ESCRT-III proteins, each a 221–241 residue protein with basic N-terminal 4 and acidic C-terminal halves [31]. The ESCRT-III family is expanded to 11 proteins in humans, where they are referred to either as orthologs of their yeast counterparts or as CHMPs (charged multivesicular body proteins). Hence, the crystal structure of hVps24/CHMP3 (residues 9–183) reveals structural elements that are probably common to all ESCRT-III subunits, which also includes Vps2/CHMP2,

Vps20/CHMP6, and Snf7/Vps32/CHMP4 [33]. Five alpha helices (α1– α5) span two-thirds of the N- terminal of the protein and are connected by a relatively long linker to a sixth short predicted helix (α6) near the C-terminus (α6 and the loop linking α5 to α6 were not seen in the crystal structure, suggesting conformational flexibility). The most prominent structural element observed in the crystal structure is the

N-terminal helical hairpin that is 70 Å long and composed of helices α1 and α2, which are critical for membrane binding and homo- or heterodimerization events [33]. The C-terminal region that includes helix α5, the microtubule-interacting and transport (MIT)-interacting motif (MIM), and helix α6 constitutes a putative auto-inhibitory region that is thought to interact with helix α2 of the core and thereby block homo- or heterodimerization of ESCRT-III components [34]. Such intramolecular interactions may therefore prevent ESCRT-III dimerization and thereby maintain the ESCRT-III proteins as metastable monomers in the cytosol in a “closed” state. ESCRT-III protein assembly may then occur only upon recruitment to endosomes, where the auto-inhibitory intramolecular interaction is lost during a presumed “closed” to “open” state transition that now permits ESCRT-III proteins to associate. The molecular trigger for the proposed release of this inhibition and ESCRT-III assembly on endosomal membranes remains unclear. However, studies on hSnf7-1 (CHMP4A) have shown that overexpressed hSnf7-1/CHMP4A spontaneously forms membrane-attached filaments 5 nm in width that curve and self- associate to create circular arrays. These can promote or stabilize negative membrane curvature and outward budding of plasma membrane tubules [35]. These data indicate that a single ESCRT-III protein can form an ESCRT-III lattice, presumably by Snf7 binding to itself. This possibility is further supported by observations that Snf7 is the most abundant ESCRT-III subunit in yeast [32] and that most Snf7 isoforms self-associate in yeast two-hybrid analyses [18]. 5

Still, the stoichiometry and the size of the ESCRT-III complex on the endosome are not known.

Previous studies have suggested that Vps20 is the first ESCRT-III protein to associate with ESCRT-II on the endosome, followed by the recruitment of the first of two ESCRT-III subcomplexes, Vps20-Snf7 [31].

The Vps20-Snf7 subcomplex binds to the endosomal membrane, in part through the N-terminal myristoylation of Vps20, an ESCRT-III component that binds directly to Vps25 of ESCRT-II [36].

Recruitment of the second ESCRT-III subcomplex Vps2-Vps24 appears to be dependent on the Vps20-

Snf7 subcomplex, suggesting that the two subcomplexes are recruited sequentially [32]. The Vps2-Vps24 subcomplex also recruits the AAA-ATPase, Vps4, to endosomes [31]. In particular, the direct interaction of the MIM of Vps2 with the MIT domain at the N-terminus of Vps4 is required for MVB sorting [37,38].

Yeast two-hybrid and biochemical studies indicate a complex network of homo- and heteromeric interactions among ESCRT-III subunits [12,39]. It has also been shown that when ESCRT-III subunits are present at a high concentration, either in vivo upon overexpression or in vitro, they can oligomerize and even form filaments at the plasma membrane that inhibit viral budding and endosomal function [40].

Yet, how the four core subunits interact to form a functional ESCRT-III complex on endosomes, that contributes the final steps of MVB sorting, is not understood.

VPS4 is an AAA ATPase that releases the ESCRT machinery from the endosomal membrane

[3,27,31,41], and as the only energy consuming enzyme directly implicated in the ESCRT pathway has long been considered to be a key factor controlling the creation of MVB vesicles. This thinking is supported by an elegant electron microscopy (EM) based tomography study demonstrating that yeast lacking VPS4 have no ILVs [42]. VPS4 consists of an N-terminal MIT domain connected to a single

AAA domain [43,44]. It binds to short helical MIT interacting motifs (MIM1 and MIM2) in ESCRT-III proteins [37,38,45]. Like other AAA+ ATPases [46], VPS4 is thought to function as a ring-shaped oligomer that uses alternating movements of central “pore loops” to unfold and thereby disassemble its polymeric ESCRT-III substrates [47,48]. 6

Strikingly, ESCRT-I, -II, and -III all localize to the bud neck [29]. ESCRT-III subunits assemble into tubular structures in vitro and when overexpressed [35,49,50]. The initial budding is driven by the assembly of ESCRT-I and -II with one another and with the endosome membrane as mentioned earlier.

However, the structure of this assembly is unknown and the characteristics of the assembly are currently a pressing question in the field. Composite structures of the ESCRT-I and -II complexes have been developed on the basis of crystal structures of the separate components supplemented with hydrodynamic information of the complete complexes in solution [18,28]. These structures have revealed that multiple membrane and ESCRT-III attachment sites were separated by rigid spacers of up to 18 nm across, suggesting a mechanism that induces or stabilizes formation of a membrane neck of roughly those dimensions. Subsequent recruitment and polymerization of ESCRT-III into spiral domes [51] would then narrow and sever the neck in the current model [20].

ESCRT-0 ESCRT-I Cytosol

ESCRT-II Ubiquitinated cargo Lumen

VPS4

ESCRT-III

ILV

Modified from Bassereau P. (2010) Nat. Cell Biol. Figure 3 (a) ESCRT-0 clusters ubiquitylated cargoes. (b) ESCRT-I and ESCRT-II together form membrane invaginations and are localized inside the bud neck. Cargoes are sequestered in the bud. (c) ESCRT-III assembles at the neck of the bud, colocalizing with ESCRT-I and -II. (d) Vesicle scission by ESCRT-III leading to the formation of ILV. The ATPase VPS4 is recruited to dissociate ESCRT-III oligomers. 7

This observation that the ESCRT complexes localize to the bud neck explains how they bud membranes away from the cytosol without themselves being consumed in the uncoated bud (Figure 3).

Such a mechanism stands in sharp contrast to the familiar budding of coated vesicles toward cytosol. In particular, the regulated disassembly of ESCRT-III is essential for the completion of the sorting of cargos to the intraluminal vesicles of MVBs [22,50,52]. In a yeast vps4 mutant, ESCRT-III components aggregate in so-called class E compartments while cargo sorting is blocked [53]. In fact, the thermodynamic driving force for the pathway is the disassembly and recycling of ESCRT-III via ATP hydrolysis by the dodecameric Vps4 [21,22]. Although the overall thermodynamic driving force is clear, the energetic trajectory of neck-directed bud formation is currently unknown. Theoretical analysis of the membrane mechanics of this process is urgently needed, as is a better understanding of the roles of lipids.

One recently developed hypothesis proposes that Vps24 and Vps2 serve as adaptors for Vps4- mediated disassembly of the Snf7 oligomer (ESCRT-III). Results from studies that have been performed in vivo [32,37,38] are consistent with a model in which Vps24 terminates oligomerization, possibly by capping Snf7 oligomers via recruitment of Vps2 to the Snf7 oligomer in order to initiate Vps4-mediated disassembly. Thus, the Vps2-Vps24 subcomplex can be viewed as a “capping” complex and may itself be in the form of a short mixed oligomer [50], thereby providing multiple binding sites (such as the Vps2

MIM domain) for Vps4 recruitment [38]. It has been speculated that the Vps4 AAA ATPase may drive the sequential disassembly of Snf7 molecules from one end of the Snf7 filament spiral. This would result in closure of the Snf7 spiral and concentration of cargo encircled by the ring. The Vps4 dodecamer could stabilize the shrinking Snf7 spiral via a combination of strong interactions with Vps2 and weak interactions with Snf7 mediated by the N-terminal MIT domain on each Vps4 subunit [38]. The closure of the Snf7 ring may also drive invagination and fission of the sorting domain, thereby directly coupling

ESCRT-III disassembly to cargo sorting and vesicle formation in what has become known as the purse string model (Figure 4).

Figure 4 The purse string model where Vps4 (red) removes one ESCRT-III component at a time (blue), which includes proteins like the Vps2-Vps24 capping complex and the Snf7 polymer (left). This gradual removal of ESCRT-III subunits shrinks the diameter of the ESCRT-III filament and constricts the membrane (center). As ATP hydrolysis continues, Vps4 mediates the disassembly of the entire ESCRT-III complex and completes the membrane scission, reproducing free ESCRT-III monomers and generating an ILV (right).

A major goal in neurobiology in recent years has been to gain insight into the molecular machinery that mediates neurotransmitter release [54]. Synaptic transmission is initiated when an action potential triggers neurotransmitter release from a presynaptic nerve terminal [55]. An action potential induces the opening of Ca2+ channels, and the resulting Ca2+ influx stimulates synaptic vesicle exocytosis via priming and docking of the synaptic vesicles to the presynaptic plasma membrane [54]. After exocytosis, synaptic vesicles undergo endocytosis, recycle, and refill with neurotransmitters for a new round of exocytosis [54]. This last step occurs by three alternative pathways: (a) vesicles are re-acidified and refilled with neurotransmitters without undocking, thus remaining in the readily releasable pool; (b) vesicles undock and recycle locally to re-acidify and refill with neurotransmitters; or (c) vesicles undergo endocytosis via clathrin-coated pits and re-acidify and refill with neurotransmitters either directly or after passing through an endosomal intermediate [54]. Most successive steps occur without much vesicle movement except for docking and recycling [54].

The fact that the ESCRT-III complex is comprised of four protein subunits that undergo a membrane-dependent monomer to hetero-oligomer transition raises a number of mechanistic questions.

Indeed, while the ESCRT pathway has been implicated in several cellular processes, its role in synaptic transmission is not clear. The aim of this study was to investigate the role of Vps24, a crucial component 9 of ESCRT-III, in synaptic transmission. Here, we demonstrate the requirement of VPS24 (ESCRT-III) function in proper neurotransmitter release as well as preliminary observations implicating components of

ESCRT-I and -II in synaptic transmission as well.

RESULTS

A genetic screen identifies a third chromosome mutation 748

Our previous genetic analysis has employed phenotypic screens to identify new mutants affecting synaptic transmission. The 748 mutant (referred to as vps241 in later section: Identification of genetic locus for 748) was recovered in a forward genetic screen for third chromosome mutations affecting synaptic transmission (Figure 5).

Figure 5 Male flies with an isogenized third chromosome (Iso3) were exposed to the mutagen, ethyl methanesulphonate (EMS). Mutagenized males were mated with females carrying the visible third chromosome marker, Lyra (Ly), in trans to a balancer chromosome, TM6c, to prevent recombination. The F1 male progeny carrying a mutagenized third chromosome (3*) in trans to TM6c were crossed to females carrying the third chromosome deficiencies, Df(3L)GN34 and Df(3R)Exel6140. The resulting F2 flies heterozygous for a mutagenized third chromosome in trans to the deficiency chromosome were screened for motor defects at 38 °C.

Briefly, mutagenized third chromosomes in isogenic male flies were tracked through a series of crosses kept at room temperature (~25 oC) to generate F2 progeny with the mutagenized third chromosome in trans to third chromosome deficiencies. Flies of this generation were then tested for defects in climbing behavior. While motor defects can be elicited at 36 oC, the homozygous 748 mutant exhibited rapid paralysis at 38 oC (Figure 6). This phenotype was shown to be recessive when the 748 mutant was made heterozygous by pairing one copy of the gene with a wild-type chromosome.

38 °C

15.0

10.0

5.0

0.0

Figure 6 The 748/Df(3R)Exel6140 flies (748/Df) exhibit rapid paralysis at 38 °C, whereas wild- type flies (WT) do not paralyze at this temperature. Flies heterozygous for the 748 mutation, w;;748/+, behaved like WT showing that the mutation is recessive. The 748 paralytic phenotype was clearly rescued by neural expression of the wild-type VPS24 protein (748/Df rescue). Rescue of 748 was carried out in Appl-GAL4;UAS-EGFP-vps24/+;748/Df(3R)Exel6140 flies. The tests were truncated at 15 min if 50% paralysis had not occurred. Error bars indicate SEM and asterisks mark values significantly different from control values (p ≤ 0.05).

A synaptic phenotype in the 748 mutant

To examine whether the 748 mutant exhibited deficits in synaptic transmission from wild-type flies, electrophysiological recordings were performed at adult dorsal longitudinal muscle (DLM) neuromuscular synapses [56]. As described previously [57] and as shown in Figure 7, synaptic transmission in 748 was similar to that of wild-type flies in the excitatory post-synaptic current (EPSC) amplitude and rise (time-to-peak) time (Figure 7D, E), but exhibited a striking increase in the EPSC decay

(t1/2) time (Figure 7F). The preceding observations indicate that the 748 gene product serves an important function in synaptic transmission.

A B C G WT 748/Df WT and 748/Df WT and 748/Df (scaled) rescued (scaled)

500 nA 5 msec D E F 1.2 2.0 2.0 1.0

1.5 0.8 1.5

0.6 1.0 1.0 0.4 0.5 0.5 0.2

0.0 0.0 0.0 WT 748/Df WT 748/Df WT 748/Df Figure 7 (A, B) Representative EPSC recordings from DLM neuromuscular synapses of wild type (WT, A) and 748/Df(3R)Exel6140 (748/Df, B) at 20 °C. 748 mutant synapses exhibit wild- type EPSC amplitude and slowing of the EPSC decay time. (C) The EPSC in WT (black) was superimposed with a scaled version of the EPSC in 748/Df (orange). (D, E and F) No change in the EPSC amplitude (D) and rise (time-to-peak) time (E) was observed in the 748 mutant. 748 exhibited a significant increase in the EPSC decay (t1/2) time (F). (G) The EPSC synaptic phenotypes in 748 were rescued by neural (presynaptic) expression of the wild-type VPS24 protein. Rescue of 748 was carried out in Appl-GAL4;UAS-EGFP-vps24/+; 748/Df(3R)Exel6140 flies. A scaled version of the EPSC in the rescued fly (green) was superimposed with the EPSC in WT (black).

Identification of genetic locus for 748

It was known from the forward genetic screen that the 748 mutation was located in one of two regions on the third chromosome, a ~600 kb (63E6-64A10) region on the left arm or a ~155 kb (82A-

82A4) region on the right arm. Therefore, deficiency mapping of the recessive 748 phenotype was carried out using deficiency chromosomes covering the two regions in the left and right arms using Df(3L)GN34 and Df(3R)Exel6140, respectively.

Because Df(3L)GN34 complemented 748, whereas Df(3R)Exel6140 did not, the vps241 mutation was placed within the ~155 kb region of the right arm of the third chromosome (Figure 8A). Further complementation tests were then performed using existing mutations or P element insertions disrupting genes within the Df(3R)Exel6140 region (Figure 8B).

A Complementation test I Sequence location 3L:0 3L:24 3R:0 3R:27 (Mbp) Cytogenetic map 61 71 80 81 91 100 3L 3R Df(3L)GN34 Df(3R)Exel6140

B Complementation test II

3R:107-197 (Kbp) existing mutants or : P element insertions

C Sequence analysis

vps24 Gene span Transcript CDS

vps241 11 bp deletion at the beginning of the 1st intron

Figure 8 (A) The deficiency within the right arm, Df(3R)Exel6140, failed to complement 748, whereas Df(3L)GN34 did not. (B) A second round of complementation testing was performed using existing mutants or P element insertions (boxed in red) disrupting genes within the region of Df(3R)Exel6140. A chromosome carrying a P element insertion in the vps24 gene failed to complement 748, indicating that the 748 mutation resides in vps24. (C) Sequence analysis of the vps24 gene over the coding sequence, read right to left, revealed an 11 nucleotide deletion at the beginning of the 1st intron, including its splicing signal. This may result in splicing of the intron such that the open reading frame extends beyond the first exon (15 amino acids) by 29 incorrect amino acids and a stop codon truncating the protein at 44 amino acids.

A chromosome carrying a P element insertion in the vps24 gene (yw; P{EPgy2}vps24EY04708/TM3) failed to complement 748, indicating that the 748 mutation resides in vps24 (Figure 8C). Sequence analysis of the vps24 gene over the coding sequence revealed an 11 nucleotide deletion at the beginning of the first intron, including its splicing signal. This may result in unsuccessful splicing of the intron such that the 14 open reading frame extends beyond the first exon (15 amino acids) by 29 incorrect amino acids and a stop codon truncating the protein at 44 amino acids.

Transformation rescue confirm a role for vps24 in neurotransmitter release

To confirm that the vps241 mutation produces the observed paralytic and synaptic phenotypes, transformation rescue experiments were carried out using the GAL4-UAS (upstream activation sequence) system [58]. UAS transgenic lines were generated to express a form of wild-type VPS24 tagged with

EGFP at the N-terminus, EGFP-VPS24. These UAS-EGFP-vps24 transgenic lines express the wild-type

VPS24 protein and the Appl-GAL4 driver line was used to achieve neural expression in the vps241 mutant background, as described in our previous work [cf. 57,59]. Expression of wild-type VPS24 in the nervous system produced clear rescue of the vps241 paralytic phenotype (Figure 6), demonstrating an important function for VPS24 in neurons. Furthermore, electrophysiological recordings at adult DLM neuromuscular synapses revealed that loss of presynaptic VPS24 function produced the altered EPSC waveform (Figure 7C). This synaptic phenotype was restored to that of wild type with the neural expression of wild-type VPS24, confirming the rescue of the vps241 synaptic phenotype (Figure 7G).

Preservation of presynaptic morphology and organization in the vps241 mutant

As a first step in examining the possible functions of VPS24 in synaptic transmission, basic synaptic morphology was assessed in the vps241 mutant. Although it was considered unlikely that the vps241 mutation was associated with changes in the morphology of presynaptic terminals, immunocytochemical analysis of DLM (Figure 9A-J) and larval (Figure 9K-T) neuromuscular synapses from wild type or the vps241 mutant was performed using markers for the neuronal membrane, the active zone, and synaptic vesicles. These studies indicated no obvious changes in basic synaptic morphology, including the distributions of active zones and synaptic vesicle clusters. With the observed synaptic phenotype deficits in the vps241 mutant, these immunocytochemical observations suggest that a loss of

VPS24 protein produces a functional defect at morphologically normal synapses.

p a n

s u

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Figure 9 Confocal immunofluorescence images of adult (A-J) and larval (K-T) neuromuscular 1 1 synapses in wild type (WT) (A-E, K-O) and vps24 /Df(3L)Exel6140 (vps24 /Df) (F-J, P-T). Anti- HRP labels the neuronal plasma membrane such that the motor axon and its presynaptic boutons (swellings) are visualized. Anti-BRP labels presynaptic active zones. Anti-SYNAPTOTAGMIN (SYT) labels synaptic vesicles. The surrounding postsynaptic muscle membrane is not fluorescent and 1 appears dark. Neuromuscular synapses of vps24 mutants show that overall synaptic morphology and the distribution of active zones and synaptic vesicles resemble that of wild type. All images are maximum projections of two consecutive optical z-sections.

Localization of VPS24 in active zones at DLM neuromuscular synapses

Previous studies indicate that ESCRT-III proteins are localized to the viral plasma membrane

[24,40]. Expression of EGFP-VPS24 in the nervous system of vps241 mutants produced rescue of paralysis (Figure 6), indicating that EGFP-VPS24 retains its function. 16

Figure 10 Confocal immunofluorescence and native EGFP fluorescence images of DLM adult (A-E) and VLM larval (F-J) neuromuscular synapses exhibiting neuronal expression of UAS- EGFP-vps24. At both adult and larval neuromuscular synapses, distribution of EGFP-VPS24 is localized in proximity to neurotransmitter release sites at AZs. AZs are labeled with anti- BRUCHPILOT (BRP). Anti-HRP labels the neuronal plasma membrane.

Following neural expression of EGFP-VPS24, imaging native EGFP fluorescence at DLM and ventral longitudinal muscle (VLM) neuromuscular synapses demonstrated clear neuronal plasma membrane localization. More interestingly, EGFP-VPS24 exhibited presynaptic localization in proximity to neurotransmitter release sites at AZs (Figure 10D, I).

Disruption of presynaptic organization in vps241 mutant

Understanding the mechanisms of VPS24 function as a protein component of ESCRT-III involved in membrane scission will require visualization of the budding event of the early endosome.

Previous studies on the sequence of events of endosome formation established an invagination of the plasma membrane opposite in direction to that of coat-mediated transport vesicle formation followed by cleavage of the bud neck. Adult DLM neuromuscular synapses from wild-type and homozygous vps241 mutant flies were viewed under transmission electron microscopy (TEM) imaging to determine whether any abnormalities in endosome formation existed in the mutants. The vps241 mutant synapses retain a 17 wild-type synaptic vesicle cluster at the active zone (Figure 11A). However, unusual structures were observed within presynaptic terminals of the mutant. These included large and complex endosome-like structures and large plasma membrane invaginations (Figure 11B, C). These preliminary observations suggest a novel and specific role of VPS24 in controlling bud size or budding rate previously thought to be the result of ESCRT-I and -II.

1 1

Figure 11 An ultrastructural synaptic phenotype in the vps241 mutant. TEM images of DLM neuromuscular synapses from WT (A) and vps241/Df(3R)Exel6140 (vps241) (B, C) preparations. The vps241 mutant synapses retain a wild-type synaptic vesicle cluster at the active zone (arrows). However, unusual structures were observed within presynaptic terminals of the mutant. These include large and complex endosome-like structures (arrowhead in B) and large plasma membrane invaginations (arrowhead in C).

Localization of the ESCRT-I Component, VPS28, at neuromuscular synapses

After elucidating the roles and functions of vps24 in synaptic transmission, we were interested in characterizing some of the properties of other ESCRT components. Previous studies indicate that

ESCRT-I proteins are localized to the viral plasma membrane and, in particular, at the bud neck of early endosomes [17,20,21]. Due to the apparent importance of VPS28 in the survival of Drosophila embryos, the anti-VPS28 antibody [60] was used to detect the expression of VPS28 in the fly nervous system.

Imaging native VPS28 fluorescence at DLM and VLM neuromuscular synapses demonstrated clear active zone localization (Figure 12D, I).

Figure 12 Confocal immunofluorescence images of DLM adult (A-E) and VLM larval (F-J) neuromuscular synapses. Endogenous VPS28 localization was detected using an anti-VPS28 antibody. VPS28 is distributed in puncta which surround and include active zones. Anti-HRP and anti-BRUCHPILOT (BRP) label the neuronal plasma membrane and presynaptic AZs, respectively.

DISCUSSION

Our forward genetic screen for synaptic transmission mutants recovered a new mutation in the

Drosophila vps24 gene, which encodes an evolutionarily conserved component of the ESCRT-III complex. Loss of VPS24 function in the new vps241 mutant resulted in marked changes in neurotransmitter release, reflected in a selective slowing of the EPSC decay. Localization of VPS24

(ESCRT-III) and VPS28 (ESCRT-I) was highly enriched in proximity to neurotransmitter release sites at presynaptic active zones. Taken together, these preceding results suggest that ESCRT-I, and -III complexes may cooperate to regulate neurotransmitter release through novel mechanisms of ESCRT function at the active zone during synaptic activity.

Novel Role of ESCRT Pathway in Synaptic Transmission

The ESCRT pathway has been studied extensively and therefore its roles in trafficking membrane proteins to the lysosome and other cellular processes are well established. Thus, it was exciting that an unbiased approach to identify and characterize mechanisms of synaptic transmission, such as our forward genetic screen, recovered the vps241 mutant in the context of synaptic transmission. This raises questions regarding the mechanisms by which the ESCRT machinery plays in neurotransmitter release.

Synaptic defects were evident in electrophysiological recordings of the vps241 mutant at 20oC, where synaptic transmission of the mutant was similar to that of wild-type flies in the EPSC amplitude and rise time while exhibiting a significant increase in the ESPC decay time due to the loss of presynaptic

VPS24 function (Figure 7D-G). The behavior of the vps241 mutant was similar to that of wild-type flies at room temperature (~25 oC), but exhibited paralysis when exposed to 38 oC (Figure 6). Because higher temperatures shorten the time course for neurotransmitter release, a higher degree of precision among the presynaptic protein components of the vesicle cycle is required for proper synaptic transmission. Thus, the vps241 mutant, which exhibits an altered EPSC waveform, lacks this necessary precision and may be a contributing factor towards the observed temperature-sensitive (TS) paralytic phenotype. 20

To confirm that the vps241 mutation produces the observed paralytic and synaptic phenotypes, transformation rescue experiments using UAS-EGFP-vps24 transgenic lines expressing the wild-type

VPS24 protein and the Appl-GAL4 driver line to achieve neural expression were conducted in the vps241 mutant background, as described in our previous work [cf. 57,59]. Expression of wild-type VPS24 in the nervous system produced clear rescue of the vps241 paralytic phenotype (Figure 6). The observed synaptic phenotype was also restored to that of wild type with the neural expression of wild-type VPS24, confirming the rescue of the vps241 synaptic phenotype (Figure 7G). These observations with respect to the effect of VPS24 function on the synaptic phenotype indicate that the vps24 gene product serves an important function in neurotransmitter release.

Relationship to Conventional Synaptic Vesicle Membrane Trafficking

In the synaptic vesicle cycle, filled vesicles dock at the active zone where they are primed for

Ca2+ triggered fusion-pore opening followed by release of neurotransmitter [54]. As shown in Figure 13, synaptic vesicles undergo endocytosis after fusion pre-opening and recycle via several routes: local reuse

(step 6), fast recycling without an endosomal intermediate (step 7), or clathrin-mediated endocytosis (step

8) with recycling via endosomes (step 9). In the vps241 mutant, although endosomal compartment defects were observed (Figure 11B), the synaptic vesicle population appeared normal and the overall synaptic morphology and the distribution of active zones (BRP) and synaptic vesicles (SYT) resemble that of wild type (Figure 9 and Figure 11). Based on these observations, an ongoing project has been the investigation of other protein localization within the neurotransmitter release apparatus, including CAC [61] and t-

SNAREs, to further elucidate the interactions of the ESCRT machinery with other presynaptic players in synaptic transmission. It has been shown that while endosomal compartments exist within the presynaptic terminal, they do not co-localize with the active zone [59]. Yet, we have shown that ESCRT components

VPS24 (ESCRT-III) and VPS28 (ESCRT-I) are found at the active zones of the presynaptic plasma membrane. Based on these preceding studies, considerations of the possible mechanisms by which 21

ESCRT machinery may contribute to neurotransmitter release were made and are discussed in the following section.

Modified from Sudhof, T.C. (2004) Annu. Rev. Neurosci. Figure 13 The synaptic vesicle cycle. Synaptic vesicles are filled with neurotransmitters by active transport (step 1) and form the vesicle cluster (step 2). Filled vesicles dock at the active zone (step 3) and undergo a priming reaction (step 4) that prepares them for Ca2+ triggered fusion-pore opening (step 5). Synaptic vesicles then undergo endocytosis and recycle via several routes: local reuse (step 6), fast recycling without an endosomal intermediate (step 7), or clathrin- mediated endocytosis (step 8) with recycling via endosomes (step 9). Steps in exocytosis are indicated by red arrows and steps in endocytosis and recycling by yellow arrows.

Potential Models for ESCRT Machinery Function in Neurotransmitter Release

Study of the ESCRT machinery has mainly focused on their conventional function in endosomal protein trafficking to the lysosome. However, given our findings, we provide evidence supporting a novel

ESCRT machinery function that is distinct from their conventional function. Immunocytochemical 22 analysis revealed localization of ESCRT-III (VPS24) and ESCRT-I (VPS28) components at presynaptic active zones (Figure 10D, I; Figure 12D, I). Drosophila ESCRT mutants exhibiting major disruptions of cortical actin beneath the plasma membrane of several cell types demonstrated the requirement of

ESCRT-I, -II, and -III in maintaining cell survival [60,62]. Recent findings in Aspergillus nidulans saw that the ESCRT machinery may be present at the plasma membrane and regulate plasma membrane- associated protein interactions [63]. Interestingly, our recent neural-specific RNAi-mediated knockdown of ESCRT-II components, VPS22 and VPS36, produced TS paralytic phenotypes resembling that of the

VPS24 (ESCRT-III) loss-of-function mutant vps241 (data not shown), suggesting that loss of ESCRT-II and ESCRT-III may have similar effects on neural function. Therefore, it is a plausible hypothesis that the

ESCRT-I, -II, and -III complexes participate in neurotransmitter release through a novel mechanism involving interaction with neurotransmitter release sites at the presynaptic plasma membrane.

Our work identified the first vps24 mutant in any metazoan, which will serve as a starting point for broader analysis of ESCRT machinery function in synaptic transmission. Further investigation of the molecular interactions of the ESCRT machinery within the presynaptic terminal expects to define the functional contributions of the ESCRT machinery to neurotransmitter release.

MATERIALS AND METHODS

Drosophila strains

Appl-GAL4 was from our laboratory stock collection. Deficiency lines Df(3L)GN34 and Df(3R)Exel6140 were generously provided by the Bloomington Stock Center. UAS-EGFP-vps24 transgenic line was generated in the current study (see Generation of Transgenic Lines). Wild-type flies were Canton-S (CS).

Stocks and crosses were cultured on a conventional cornmeal-molasses-yeast medium at 20 oC.

Mutagenesis and screening

To isolate new third chromosome mutations that affect synaptic transmission, a forward genetic screen was carried out as summarized in Figure 5. Briefly, CS males with an isogenized third chromosome were exposed for 24 hr to 25 mM ethyl methanesulfonate (EMS) [64]. F2 flies heterozygous for a mutagenized third chromosome in trans to a chromosome carrying two deficiencies, Df(3L)GN34 and Df(3R)Exel6140, were screened for motor defects at room temperature. A 38 oC water bath served as a behavior test to elicit any motor defects.

Molecular characterization of the vps241 mutant

Sequence analysis of candidate genes was carried out essentially as described previously [59]. Briefly, genomic DNA was prepared from the vps241 mutant or flies carrying the parent third chromosome used in the mutagenesis. This was used as a template for PCR, and gel-purified PCR products were sequenced at the Penn State University Nucleic Acids Facility.

Generation of transgenic lines

Transformation construct for UAS-EGFP-vps24 transgenic line was generated by inserting the open reading frame (ORF) for vps24 with EGFP fused to its N-terminus into the P element transformation vector, pUAST [59]. A cDNA clone for vps24 was obtained from the Drosophila Genomics Resource 24

Center (clone ID: GH14561, corresponding cDNA accession number: AY058381). The vps24 ORF, including the flanking BglII and KpnI restriction sites, was amplified with PCR using Pfu DNA polymerase. These restriction sites were used to shuttle the vps24 ORF into the P element cloning vector pBluescriptI SK- (pBlue) and ligated with a pre-existing EGFP sequence in pBlue. The EGFP-vps24 segment was sequentially digested with NotI and KpnI and shuttled into the P element transformation vector, pUAST. Generation of transgenic lines was achieved as described previously [61]. Neural expression of UAS transgenes was achieved using the Appl-GAL4 driver as in the case of experiments for rescue of the vps24 mutant behavioral phenotype.

Immunocytochemistry and confocal microscopy

Immunocytochemistry and confocal microscopy were performed essentially as described previously

[56,59]. These studies employed the following primary antibodies: rabbit anti-SYT Dsyt CL1 (1:5000)

[Noreen Reist (Colorado State University, Fort Collins, CO)]; mAb nc82 anti-BRP (BRUCHPILOT)

(1:50) [Developmental Studies Hybridoma Bank]; rabbit anti-GFP (1:1000) (Invitrogen); Cy5-conjugated rabbit anti-HRP (1:2000) (Jackson Immunoresearch Laboratories, West Grove, PA); anti-VPS28 (1:5000)

[Helmut Kramer (University of Texas Southwestern, Dallas, TX)]. Secondary antibodies included Alexa

Fluor 488-conjugated anti-rabbit IgG (1:200) and Alexa Fluor 568-conjugated anti-rabbit IgG (1:200)

(Invitrogen). Adult dorsal longitudinal flight muscle (DLM) and larval ventral longitudinal flight muscle

(VLM) neuromuscular synapse preparations were imaged using an Olympus FV1000 confocal microscope (Olympus Optical, Tokyo, Japan) with a PlanApo 60x 1.4 numerical aperture oil objective

(Olympus Optical) and a z-step size of 0.2 µm. Images were obtained and processed with Fluoview software (Olympus Optical). All images shown in figures are maximum projections of two consecutive optical z-sections.

Synaptic electrophysiology

Excitatory postsynaptic currents (EPSC) were recorded at DLM neuromuscular synapses of 3- to

o 5-day-old adults reared at 20 C. Saline solution consisted of (in mM): 128 NaCl, 2 KCl, 4.0 MgCl2, 1.8

CaCl2, 5 HEPES, and 36 sucrose. The pH was adjusted to 7.0 using NaOH. These experiments were performed as described previously [56].

REFERENCES

1. Katzmann, D.J., Odorizzi, G., and Emr, S.D. (2002) Receptor downregulation and multivesicular-

body sorting. Nat. Rev. Mol. Cell Biol. 3: 893-905.

2. Haigler, H.T., McKanna, J.A., and Cohen, S. (1979) Direct visualization of the binding and

internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431. J.

Cell Biol. 81: 382-395.

3. Katzmann, D.J., Babst, M., and Emr, S.D. (2001) Ubiquitin-dependent sorting into the multivesicular

body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I.

Cell 106: 145-155.

4. Piper, R.C., and Luzio, J.P. (2007) Ubiquitin-dependent sorting of integral membrane proteins for

degradation in lysosomes. Curr. Opin. Cell Biol. 19: 459-465.

5. Raymond, C.K., Howald-Stevenson, I., Vater, C.A., and Stevens, T.H. (1992) Morphological

classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment

in class E vps mutants. Mol. Biol. Cell 3: 1389-1402.

6. Odorizzi, G., Babst, M., and Emr, S.D. (1998) Fab1p PtdIns(3)P 5-kinase function essential for

protein sorting in the multivesicular body. Cell 95: 847-858.

7. Slagsvold, T., Pattni, K., Malerod, L., and Stenmark, H. (2006) Endosomal and non-endosomal

functions of ESCRT proteins. Trends Cell Biol. 16: 317-326.

8. Forman, M.S., Trojanowski, J.Q., and Lee, V.M. (2004) Neurodegenerative diseases: a decade of

discoveries paves the way for therapeutic breakthroughs. Nat. Med. 10: 1055-1063.

9. Lee, J.A., Beigneux, A., Ahmad, S.T., Young, S.G., and Gao, F.B. (2007) ESCRT-III dysfunction

causes autophagosome accumulation and neurodegeneration. Curr. Biol. 17: 1561-1567.

10. Filimonenko, M., Stuffers, S., Raiborg, C., Yamamoto, A., Malerod, L., Fisher, E.M., Isaacs, A.,

Brech, A., Stenmark, H., and Simonsen, A. (2007) Functional multivesicular bodies are required for 27

autophagic clearance of protein aggregates associated with neurodegenerative disease. J. Cell Biol.

179: 485-500.

11. Martin-Serrano, J., Yarovoy, A., Perez-Caballero, D., and Bieniasz, P.D. (2003) Divergent retroviral

late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins.

Proc. Natl. Acad. Sci. USA 100: 12414-12419.

12. von Schwedler, U.K., Stuchell, M., Muller, B., Ward, D.M., Chung, H.Y., Morita, E., Wang, H.E.,

Davis, T., He, G.P., Cimbora, D.M., et al. (2003) The protein network of HIV budding. Cell 114:

701-713.

13. Morita, E., Sandrin, V., Chung, H.Y., Morham, S.G., Gygi, S.P., Rodesch, C.K., and Sundquist, W.I.

(2007) Human ESCRT and ALIX proteins interact with proteins of the midbody and function in

cytokinesis. EMBO J. 26: 4215-4227.

14. Carlton, J.G., and Martin-Serrano, J. (2007) Parallels between cytokinesis and retroviral budding: a

role for the ESCRT machinery. Science 316: 1908-1912.

15. Hanson, P.I.; Shim, S.; Merrill, S.A. (2009) Cell biology of the ESCRT machinery. Curr. Opin. Cell

Biol. 21: 568-574.

16. Ren, X.F., and Hurley, J.H. (2010) VHS domains of ESCRT-0 cooperate in high-avidity binding to

polyubiquitinated cargo. EMBO J. 29: 1045-1054.

17. Wollert, T., and Hurley, J.H. (2010) Molecular mechanism of multivesicular body biogenesis by

ESCRT complexes. Nature 464: 864-869.

18. Saksena, S., Sun, J., Chu, T., and Emr, S.D. (2007) ESCRTing proteins in the endocytic pathway.

Trends Biochem. Sci. 32: 561-573.

19. Williams, R.L., and Urbé, S. (2007) The emerging shape of the ESCRT machinery. Nat. Rev. Mol.

Cell Biol. 8: 355-368.

20. Hurley, J.H., and Hanson, P.I. (2010) Membrane budding and scission by the ESCRT machinery: it’s

all in the neck. Nat. Rev. Mol. Cell. Biol. 8: 556-566. 28

21. Wollert, T., Wunder, C., Lippincott-Schwartz, J., and Hurley, J.H. (2009) Membrane scission by the

ESCRT-III complex. Nature 458: 172-177.

22. Kim, J., Sitaraman, S., Hierro, A., Beach, B.M., Odorizzi, G., and Hurley, J.H. (2005) Structural basis

for endosomal targeting by the Bro1 domain. Dev. Cell 8: 937-947.

23. Row, P.E., Liu, H., Hayes, S., Welchman, R., Charalabous, P., Hofmann, K., Clague, M.J.,

Sanderson, C.M., and Urbe, S. (2007) The MIT domain of UBPY constitutes a CHMP binding and

endosomal localization signal required for efficient epidermal growth factor receptor degradation. J.

Biol. Chem. 282: 30929-30937.

24. Zamborlini, A., Usami, Y., Radoshitzky, S.R., Popova, E., Palu, G., and Gottlinger, H. (2006)

Release of autoinhibition converts ESCRT-III components into potent inhibitors of HIV-1 budding.

Proc. Natl. Acad. Sci. USA 103: 19140-19145.

25. Agromayor, M., and Martin-Serrano, J. (2006) Interaction of AMSH with ESCRT-III and

deubiquitination of endosomal cargo. J. Biol. Chem. 281: 23083-23091.

26. Ren, X., Kloer, D.P., Kim, Y.C., Ghirlando, R., Saidi, L.F., Hummer, G., and Hurley, J.H. (2009)

Hybrid structural model of the complete human ESCRT-0 complex. Structure 17: 406-416.

27. Babst, M., Katzmann, D.J., Snyder, W.B., Wendland, B., and Emr, S.D. (2002) Endosome-associated

complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body. Dev.

Cell 3: 283-289.

28. Kostelansky, M.S., Schluter, C., Tam, Y.Y., Lee, S., Ghirlando, R., Beach, B., Conibear, E., and

Hurley, J.H. (2007) Molecular architecture and functional model of the complete yeast ESCRT-I

heterotetramer. Cell 129: 485-498.

29. Gill, D.J., Teo, H., Sun, J., Perisic, O., Veprintsev, D.B., Emr, S.D., and Williams, R.L. (2007)

Structural insight into the ESCRT-I/-II link and its role in MVB trafficking. EMBO J. 26: 600-612.

30. Im, Y.J., and Hurley, J.H. (2008) Integrated structural model and membrane targeting mechanism of

the human ESCRT-II complex. Dev. Cell 14: 902-913. 29

31. Babst, M., Katzmann, D.J., Estepa-Sabal, E.J., Meerloo, T., and Emr, S.D. (2002) Escrt-III: an

endosome-associated heterooligomeric protein complex required for mvb sorting. Dev. Cell 3: 271-

282.

32. Teis, D., Saksena, S., and Emr, S.D. (2008) Ordered assembly of the ESCRT-III complex on

endosomes is required to sequester cargo during MVB formation. Dev. Cell 15: 578-589.

33. Muzio1, T., Pineda-Molina, E., Ravelli, R.B., Zamborlini, A., Usami, Y., Göttlinger, H., and

Weissenhorn, W. (2006) Structural basis for budding by the ESCRT-III factor CHMP3. Dev. Cell 10:

821-830.

34. Shim, S., Kimpler, L.A., and Hanson, P.I. (2007) Structure/function analysis of four core ESCRT-III

proteins reveals common regulatory role for extreme C-terminal domain. Traffic 8: 1068-1079.

35. Hanson, P.I., Roth, R., Lin, Y., and Heuser, J.E. (2008) Plasma membrane deformation by circular

arrays of ESCRT-III protein filaments. J. Cell Biol. 180: 389-402.

36. Teo, H., Perisic, O., Gonzalez, B., and Williams, R.L. (2004) ESCRT-II, an endosome-associated

complex required for protein sorting: crystal structure and interactions with ESCRT-III and

membranes. Dev. Cell 7: 559-569.

37. Stuchell-Brereton, M.D., Skalicky, J.J., Kieffer, C., Karren, M.A., Ghaffarian, S., and Sundquist, W.I.

(2007) ESCRT-III recognition by VPS4 ATPases. Nature 449: 740-744.

38. Obita, T., Saksena, S., Ghazi-Tabatabai, S., Gill, D.J., Perisic, O., Emr, S.D., and Williams, R.L.

(2007) Structural basis for selective recognition of ESCRT-III by the AAA ATPase Vps4. Nature

449: 735-739.

39. Bowers, K., Lottridge, J., Helliwell, S.B., Goldthwaite, L.M., Luzio, J.P., and Stevens, T.H. (2004)

Protein-protein interactions of ESCRT complexes in the yeast Saccharomyces cerevisiae. Traffic 5:

194-210. 30

40. Lin, Y., Kimpler, L.A., Naismith, T.V., Lauer, J.M., and Hanson, P.I. (2005) Interaction of the

mammalian endosomal sorting complex required for transport (ESCRT) III protein hSnf7-1 with

itself, membranes, and the AAA+ ATPase SKD1. J. Biol. Chem. 280: 12799-12809.

41. Babst, M., Wendland, B., Estepa, E.J., and Emr, S.D. (1998) The Vps4p AAA ATPase regulates

membrane association of a Vps protein complex required for normal endosome function. EMBO J.

17: 2982-2993.

42. Nickerson, D.P., West, M., and Odorizzi, G. (2006) Did2 coordinates Vps4-mediated dissociation of

ESCRT-III from endosomes. J. Cell Biol. 175: 715-720.

43. Scott, A., Chung, H.Y., Gonciarz-Swiatek, M., Hill, G.C., Whitby, F.G., Gaspar, J., Holton, J.M.,

Viswanathan, R., Ghaffarian, S., and Hill, C.P. (2005) Structural and mechanistic studies of VPS4

proteins. EMBO J. 24: 3658-3669.

44. Scott, A., Gaspar, J., Stuchell-Brereton, M.D., Alam, S.L., Skalicky, J.J., and Sundquist, W.I. (2005)

Structure and ESCRT-III protein interactions of theMIT domain of human VPS4A. Proc. Natl. Acad.

Sci. USA 102: 13813-13818.

45. Kieffer, C., Skalicky, J.J., Morita, E., De Domenico, I., Ward, D.M., Kaplan, J., and Sundquist, W.I.

(2008) Two distinct modes of ESCRT-III recognition are required for VPS4 functions in lysosomal

protein targeting and HIV-1 budding. Dev. Cell 15: 62-73.

46. Hanson, P.I., and Whiteheart, S.W. (2005) AAA+ proteins: have engine, will work. Nat. Rev. Mol.

Cell Biol. 6: 519-529.

47. Landsberg, M.J., Vajjhala, P.R., Rothnagel, R., Munn, A.L., and Hankamer, B. (2009) Three-

dimensional structure of AAA ATPase Vps4: advancing structural insights into the mechanisms of

endosomal sorting and enveloped virus budding. Structure 17: 427-437.

48. Gonciarz, M.D., Whitby, F.G., Eckert, D.M., Kieffer, C., Heroux, A., Sundquist, W.I., and Hill, C.P.

(2008) Biochemical and structural studies of yeast Vps4 oligomerization. J. Mol. Biol. 384: 878-895. 31

49. Bajorek, M., Schubert, H.L., McCullough, J., Langelier, C., Eckert, D.M., Stubblefield, W.M., Uter,

N.T., Myszka, D.G., Hill, C.P., and Sundquist, W.I. (2009) Structural basis for ESCRT-III protein

autoinhibition. Nat. Struct. Mol. Biol. 16: 754-762.

50. Lata, S., Roessle, M., Solomons, J., Jamin, M., Gottlinger, H.G., Svergun, D.I., and Weissenhorn, W.

(2008) Structural basis for autoinhibition of ESCRT-III CHMP3. J. Mol. Biol. 378: 818–827.

51. Fabrikant, G., Lata, S., Riches, J.D., Briggs, J.A., Weissenhorn, W. and Kozlov, M.M. (2009)

Computational model of membrane fission catalyzed by ESCRTIII. PLoS Comput. Biol. 5:

e1000575.

52. Ghazi-Tabatabai, S., Saksena, S., Short, J.M., Pobbati, A.V., Veprintsev, D.B., Crowther, R.A., Emr,

S.D., Egelman, E.H., and Williams, R.L. (2008) Structure and disassembly of filaments formed by the

ESCRT-III subunit Vps24. Structure 16: 1345-1356.

53. Babst, M., Sato, T.K., Banta, L.M., and Emr, S.D. (1997) Endosomal transport function in yeast

requires a novel AAA-type ATPase, Vps4p. EMBO J. 16: 1820-1831.

54. Sudhof, T.C. (2004) The synaptic vesicle cycle. Annu. Rev. Neurosci. 27: 509-547.

55. Katz, B. (1969) The Release of Neural Transmitter Substances, Liverpool University Press.

56. Kawasaki, F., and Ordway, R.W. (2009) Molecular mechanisms determining conserved properties of

short-term synaptic depression revealed in NSF and SNAP-25 conditional mutants. Proc. Natl. Acad.

Sci. USA 106: 14658-14663.

57. Kawasaki, F., Felling, R., and Ordway, R.W. (2000) A temperature-sensitive paralytic mutant defines

a primary synaptic calcium channel in Drosophila. J. Neurosci. 20: 4885-4889.

58. Brand, A.H., and Perrimon, N. (1993) Targeted gene expression as a means of altering cell fates and

generating dominant phenotypes. Development 118: 401-415.

59. Kawasaki, F., Iyer, J., Posey, L.L, Sun, E., Mammen, S.E. et. al. (2011) The DISABLED protein

functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the

active zone. Proc. Natl. Acad. Sci. USA 108: E222-E229. 32

60. Sevrioukov, E.A., Moghrabi, N., Kuhn, M., and Kramer, H. (2005) A mutation in dVps28 reveals a

link between a subunit of the endosomal sorting complex required for transport-I complex and the

actin cytoskeleton in Drosophila. Mol. Biol. Cell 16: 2301-2312.

61. Kawasaki, F., Zou, B., Xu, X., and Ordway, R.W. (2004) Active zone localization of presynaptic

calcium channels encoded by the cacophony. J. Neurosci. 24: 282-285.

62. Vaccari, T., Rusten, T.E., Menut, L., Nezis, I.P., Brech, A., Stenmark, H., and Bilder, D. (2009)

Comparative analysis of ESCRT-I, ESCRT-II and ESCRT-III function in Drosophila by efficient

isolation of ESCRT mutants. J. Cell Sci. 122: 2413-23.

63. Galindo, A., Calcagno-Pizarelli, A.M., Arst, H.N., Jr. and Penalva, M.A. (2012) An ordered pathway

for the assembly of fungal ESCRT-containing ambient pH signalling complexes at the plasma

membrane. J. Cell Sci. 125: 1784-95.

64. Dellinger, B.B, Felling, R., and Ordway, R.W. (2000) Genetic modifiers of the Drosophila NSF

mutant, comatose, include a temperature-sensitive paralytic allele of the calcium channel α1 subunit

gene, cacophony. Genetics 155: 203-211.

ACADEMIC VITA Kerry Cao [email protected]. [email protected] Current Address Permanent Address 478 East Beaver Ave, Apt. 320 5925 Derick Drive State College, PA 16801 Orefield, PA 18069 484-634-6019 610-336-8582

Education The Pennsylvania State University, University Park, PA Schreyer Honors College, B.S. in Chemistry, Anticipated: May 2013

Experience Independent Research, Neurobiology Lab, Department of Biology 12/10- present Thesis Work under Dr. Fumiko Kawasaki  Planned and executed a genetic screen employing classical Drosophila genetics and RNAi to identify mutations that affect synaptic transmission  Participate in molecular analysis of new mutants  Planned and executed the generation of related molecular constructs and transgenic Drosophila lines using a wide range of basic molecular biology methods  Performed live imaging of fluorescently tagged proteins in dissected preparations  Presented at 2012 Undergraduate Research Symposium

Emergency Department Volunteer, St. Luke’s Hospital & Health Network 6/10-9/10  Worked with Patient Representatives to escort and cater to the needs of patients  Observed treatment of medical emergencies in trauma bay and designated rooms

Activities Vice President, Penn State’s Free Wheelchair Mission, Pennsylvania State University 8/10-present  Created first satellite student-run branch, established at Penn State University  Work with headquarters to fundraise for wheelchairs for the disabled in developing countries  3rd Annual Classy Awards - Top 10 National Finalist for Most Influential Student Organization

National Honors Member, Alpha Epsilon Delta, National Pre-Medical Honor Society, Pennsylvania State University 9/09-present  Organized Blood Cup, a blood donation competition between Penn State and Michigan State  Organized Public Health Fair, an annual forum of healthcare discussion

Tutor, Tutor.com 8/11-present  Serve as a tutor in chemistry for middle- and high-school students as well as undergraduate students

SHO-Time Mentor, Pennsylvania State University 8/10-present  Serve as peer advisor for incoming students into the Schreyer Honors College and ongoing mentor  Work with the Dean and administration to execute a 4-day freshmen orientation

Scholarships Braddock Scholarship for Eberly College of Science Scholars, 2009-present