transmitter release at the neuromuscular junction · mitter release indirectly, that is, mutations...

TRANSMITTER RELEASE AT THE NEUROMUSCULAR JUNCTION

Thomas L. Schwarz

Program in Neurobiology, Children’s Hospital and Department of NeurobiologyHarvard Medical School, Boston, Massachusetts 02115, USA

I.

INTE

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DOI:

I

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1

ntroduction

NATIONAL REVIEW OF 105OBIOLOGY, VOL. 75

Copyright 2006, Elsev

All rights re

0.1016/S0074-7742(06)75006-1 0074-7742/06

II.
P hysiological Properties of Transmitter Release
III.
E xperimental Advantages and Limitations of the Fly NMJ
A. C
a2þ Measurements
B. D
ye Loading
C. A
lternative Measures of Exocytosis
IV.
H ow Do Vesicles Fuse: Full Fusion or Kiss-and-Run?
V.
C ore Machinery of Exocytosis: Syntaxin, VAMP/Synaptobrevin, and SNAP-25
A. S
ynaptobrevins in the Fly
B. T
etanus Toxin as a Synapse-Silencing Reagent
C. S
yntaxin1
D. S
NAP-25 and SNAP-24
E. S
NARE Proteins Act Late in the Vesicle Cycle
F. S
ummary of SNARE Proteins in Drosophila
VI.
V esicular ATPase and Membrane Fusion
VII.
N SF and the Resetting of the SNARE Machinery
VIII.
S ynaptotagmin and the Regulation of Transmitter Release
A. S
ynaptotagmin and Its Binding Properties
B. S
ynaptotagmin Phenotypes at the NMJ
C. O
ther Functions of Synaptotagmin
D. M
ultiple Synaptotagmins in the Fly
E. S
ummary of Synaptotagmin Function at the Fly NMJ
IX.
E xocyst at the NMJ
X.
O ther Mutations of Proteins on the Target Membrane
A. R
op/Unc-18/n-Sec1
B. U
nc-13 and Vesicle Priming
C. C
AST/ERK at the Active Zone
XI.
M utations in Peripheral Synaptic Vesicle Proteins
A. S
ynapsin
B. C
ysteine String Protein and Hsc70
XII.
S ummary
R
eferences
The mechanism of transmitter release at the Drosophila neuromuscular junc-

tion (NMJ) continues to be an area of active investigation in many laboratories.

The undertaking has enhanced our knowledge of synaptic transmission and

exocytosis by permitting in vivo tests of the significance of synaptic proteins.

To explore the proteins that mediate exocytosis and synaptic transmission,

ier Inc.

served.

$35.00

106 THOMAS L. SCHWARZ

mutations in nearly two-dozen genes are available. Others that influence trans-

mitter release indirectly, that is, mutations in endocytosis, development, or Kþ

channels, are largely outside the scope of this chapter. From these studies a

detailed picture emerges in which some proteins form a core apparatus for fusion

while others contribute regulation and eYciency to the system. Yet other proteins

may act in docking vesicles at active zones and in regulating their availability for

fusion. Several interesting questions have been addressed. Is exocytosis primarily

by the full fusion of vesicles with the plasma membrane or by ‘‘kiss-and-run’’? Do

the proteins that mediate fusion also account for the specificity of fusion at active

zones? To what extent are the proteins of the synapse specialized and distinct

from those that mediate exocytosis in nonneuronal cells? Are the soluble

N-ethylmaleimide-sensitive factor attachment receptors (SNAREs) suYcient for

synaptic vesicle fusion? How is the release of transmitter coupled to the action

potential and its consequent influx of Ca2þ? In addition, the dissection of the

fusion apparatus has given rise to the development of at least one reagent, tetanus

toxin (TNT) light chain that is used as a transgene in Drosophila to block synapses

and thereby to silence selected circuits. Thus, the investigation of synaptic

mechanisms in Drosophila is helping us to understand synaptic cell biology in

general and also provides tools with which to probe behavioral and developmen-

tal questions. This chapter will begin with a general discussion of the physiologi-

cal properties of transmission at the NMJ and its cell biological underpinnings,

and will then consider the individual proteins and their genetic analysis.

I. Introduction

The Drosophila neuromuscular junction (NMJ) has played a significant role in

our understanding of neurotransmitter release. Genetic strategies have been used

to test hypotheses concerning the function of proteins that had been implicated in

this process by biochemistry or by yeast genetics. These studies have also led to

physiological insights into the nature of the exocytotic event, distinctions among

exocytotic pathways in neurons, and the mechanisms of modulating synaptic

strength. The knowledge of the mechanism of transmitter release is also impor-

tant as a basis for understanding how synaptic function develops and how

synaptic activity can in turn shape development. Moreover, as investigators

increasingly turn their attention to the functions of the central nervous system

(CNS), including the circuits that govern behavior, synaptic proteins have gained

further importance as a means of manipulating neurons within a circuit. What we

know about these proteins in Drosophila is largely derived from studies of the NMJ.

It is in this manner, rather than by the isolation of novel proteins in genetic

screens, that Drosophila has influenced the field.

TRANSMITTER RELEASE AT THE NMJ 107

This chapter will first review our knowledge on transmitter release from a

functional, physiological perspective, and will examine the nature of the exocy-

totic event. Thereafter, the proteins that mediate transmitter release will be

reviewed with regard to their mutant phenotypes, and how the study of these

phenotypes in Drosophila relates to the larger field of membrane traYc in neurons.

II. Physiological Properties of Transmitter Release

The release of neurotransmitter at the Drosophila NMJ is fundamentally akin

to chemical transmission at vertebrate synapses. Neurotransmitter is packaged

into synaptic vesicles and released into the synaptic cleft by the exocytosis of those

vesicles. Single vesicles may fuse spontaneously, giving rise to unitary ‘‘quantal’’

events—miniature excitatory junctional potentials (mEJPs), also called miniature

excitatory postsynaptic potentials (mEPSPs), or most colloquially just called

‘‘minis.’’ The chief distinction between Drosophila and vertebrate NMJs is that

the fly motoneurons use glutamate as their transmitter rather than acetylcholine.

In addition, whereas vertebrate NMJs are specialized to produce large responses,

with each presynaptic action potential triggering an action potential in the

muscle, the Drosophila NMJ gives rise to a subthreshold, graded response. In this

regard, and in the use of glutamate as a transmitter, the fly NMJ is more akin to a

central excitatory synapse in a vertebrate.

In response to an action potential, the opening of voltage-dependent Ca2þ

channels at the presynaptic terminal causes a large increase in the probability of

exocytosis. The consequent release of the contents of numerous vesicles gives rise

to postsynaptic responses that represent the summed responses of individual

quanta. If one is monitoring the voltage of the membrane (i.e., in ‘‘current

clamp’’ mode), the altered voltage at the postsynaptic cell is called an EJP or

sometimes EPSP. If instead, one is recording the current flowing through the

channels (i.e., in voltage-clamp mode), the response is called an excitatory

junctional current (EJC) or excitatory junctional postsynaptic current (EPSC).

The latter mode of recording is generally preferable because the voltage change is

not simply proportional to the number of channels opened or vesicles released,

while the current flux under constant voltage is.

The delay between the arrival of the action potential to the presynaptic

terminal and the fusion of the vesicles at the active zone is too brief to permit

any large-scale movements of vesicles. Therefore, the vesicles are not released

from the general pool at large in the nerve terminal, but only from a subset of

vesicles that are immediately adjacent to the plasma membrane. These vesicles

are often referred to as ‘‘docked’’ and sometimes are said to comprise the ‘‘readily

releasable pool.’’ The concept of a docked vesicle is itself somewhat loose.


In some papers it refers only to an anatomical criterion of juxtaposition with the

plasma membrane, while in others it implies a biochemical state in which vesicle

proteins are interacting with proteins of the plasma membrane.

Although the details of Ca2þ influx and its spatial properties have not been

studied in Drosophila, it is likely that, as in mammalian neurons, the release of

neurotransmitter is triggered by microdomains of high Ca2þ that are formed

near the mouth of voltage-dependent Ca2þ channels. From vertebrate studies

it is known that resting Ca2þ in the cytosol is approximately 100 nM and that

Ca2þ concentration ([Ca2þ]) may rise to several hundred micromoles within

approximately 100 nm of the Ca2þ channel, while the channel is open. These

microdomains—flashes of elevated Ca2þ that, as soon as the channel is closed,

dissipate by diVusion into the larger volume of the terminal and by binding to

cytosolic buVers—are formed in the immediate vicinity of docked, fusion-

competent vesicles. Vesicles further away from the open Ca2þ channels will see

much smaller increases in [Ca2þ] and will have low probabilities of fusion.

Which Ca2þ channels mediate the critical influx of Ca2þ in Drosophila

presynaptic terminals? Although several genes coding for Ca2þ channels are

present in the genome, the cacophony (cac) locus appears to be responsible for most,

and perhaps all, of the Ca2þ influx that triggers transmitter release (Kawasaki

et al., 2000, 2004; Rieckhof et al., 2003). These channels are localized at active

zones and hypomorphic cac alleles cause reductions in transmitter release at the

NMJ of third instar larvae. cac null animals die as embryos, presumably due to

severe reductions in transmission. However, it might be unwise to presume that

this gene mediates all Ca2þ influx at this or any synapse in Drosophila, as multiple

Ca2þ channels can be present at mammalian synapses (Reid et al., 2003).

Once inside the nerve terminal, multiple Ca2þ ions act cooperatively to

trigger the fusion of a synaptic vesicle. This has been determined explicitly at

the third instar NMJ by varying extracellular [Ca2þ] and observing the changes

in the magnitude of transmitter release (Jan and Jan, 1976). The relationship

between [Ca2þ] and vesicles released is nonlinear. The slope of this relationship on

a log–log plot is equivalent to the Hill coeYcient used by enzymologists as

a measure of cooperativity in a biochemical reaction. Similarly, the slope of ln

[Ca2þ] versus ln[vesicles released] is an indication of the number of Ca2þ ions

needed to trigger vesicle fusion, or, more accurately, the cooperativity of Ca2þ in

triggering fusion (Dodge and RahamimoV, 1967). Because this method has been

applied frequently, and not always accurately, some technical issues should be

noted here. (1) The slope is related to the cooperativity of Ca2þ only at very low

[Ca2þ], typically under 300 mM. At higher [Ca2þ] two factors can distort the

measurement. Influx through Ca2þ channels may no longer be proportional to

the extracellular [Ca2þ] and therefore the eVective intracellular [Ca2þ] cannotbe inferred from the external [Ca2þ]. Furthermore, the release apparatus itself

may become saturated or partially limiting at high [Ca2þ]. In the extreme, the


quantal content of the response cannot be further increased by raising Ca2þ and

the relationship has a slope of zero. (2) At the necessary low [Ca2þ], the number

of quanta released per impulse can be very low, particularly when examining a

mutation that decreases release. These measurements frequently involve deter-

mining probabilities of release that are much less than one vesicle per action

potential. In this range, it is important to distinguish spontaneous minis that arise

coincidentally after the stimulus, from release evoked by the stimulus itself.

Determining the frequency of spontaneous minis and subtracting their contribu-

tion from the observed responses is therefore a necessary correction. Both

potential errors (saturation in higher [Ca2þ] or the inclusion of spontaneous

events) tend to flatten the measured dependency on Ca2þ and decrease the

measured cooperativity. The best estimates of the cooperativity at wild-type third

instar NMJs falls in the range of 3.5–4.5 ( Jan and Jan, 1976; Okamoto et al.,

2005; Robinson et al., 2002), a value consistent with similarly derived estimates of

the number of Ca2þ ions needed for exocytosis at mammalian synapses. One

further technical note: Ca2þ influx is not only a function of the extracellular [Ca2þ]but it also depends on [Mg2þ] ions, which can compete for access to Ca2þ

channels. Thus, for a given [Ca2þ], influx, and hence transmitter release, will be

lower in a high Mg2þ saline, such as HL3 (Stewart et al., 1994), than in salines

with less Mg2þ such as that of Jan and Jan (1976).

Most of the recordings made at the Drosophila NMJ, either in third instar

larvae or earlier, have been made from muscles 6 and 7 in the abdominal

segments. These muscles are among the largest and most easily accessed if

the larva is opened along the dorsal midline. However, there is at least one

feature of this preparation that is not ideal and that must be kept in mind in

experimental design and analysis. These muscles are innervated by two gluta-

matergic neurons and each contributes to the EJP (Hoang and Chiba, 2001;

Lnenicka and Keshishian, 2000). Damage to a preparation, improper adjustment

of the stimulating voltage, high-frequency stimulation, or a mutant phenotype,

may cause only one of the two axons to be stimulated and this will decrease the

postsynaptic response. When working in high Ca2þ salines with large EJPs, it

may be quite obvious when the amplitude of the response abruptly drops to a

lower value, signaling that stimulation of one of the axons has failed. If one of the

two axons is intermittently activated, the response will fluctuate between two

amplitudes. In other conditions, where the quantal content is low and stimulus to

stimulus variation is therefore large relative to the mean response amplitude,

failure to conduct an action potential can be much more diYcult to spot and may

be confused with a true phenotype or with use-dependent fatigue of the synapse.

Even more problematic is the possibility of branch-point failure—the failure of

an action potential to invade all the terminal branches of the axons onto the

muscle. Should this occur, the amplitude of the resulting change in EJP is

unpredictable. The preference for recording from muscles 6 and 7 is largely


historical. In the future, it may be preferable to record from muscle 5, which

receives input from only a single axon (Hoang and Chiba, 2001).

III. Experimental Advantages and Limitations of the Fly NMJ

The Drosophila NMJ has been invaluable for the analysis of mutations in

synaptic proteins. The third instar larval preparation is among the easiest dissec-

tions in any organism. Themuscle fibers are large and easy to impale and the nerve

is easy to pick up and stimulate with a suction electrode.With an appropriate saline

(Stewart et al., 1994) it is possible to record for extended periods. This synapse,

above all, gives an outstanding opportunity to record the detailed physiological

properties of a great many mutations. The embryonic NMJ and first instar larval

NMJ are considerably harder preparations to master. In particular, the dissections

require patience and practice, but only this preparation permits recordings from

strains with lethal mutations that will not develop beyond these stages. In both

third instar and younger preparations, however, the individual quantal events are

easily resolved from the noise and evoked responses can be studied in response to

nerve stimulation. Individualmuscles and their innervation are easy to identify and

their synapses are stereotyped, showing comparatively little variation from animal

to animal. Compared to neurons in the vertebrate CNS, these identified muscles

provide admirable electrical properties and ease of access.

Nevertheless, some shortcomings of the preparation must be confessed, even

while hoping that future work will overcome some of these obstacles.

A. CA2þ MEASUREMENTS

In Drosophila, it is not presently possible to measure directly the [Ca2þ] thatarises in the immediate vicinity of synaptic release sites. Loading terminals with

Ca2þ indicators, however, has permitted some measurements of the bulk rise in

Ca2þ that occurs in the cytosol of the terminal after trains of action potentials

(Dawson-Scully et al., 2000). Methods for photolytic uncaging of Ca2þ in the

cytosol have not been applied for the study of these synapses as a means of

gaining more direct control of instantaneous changes in cytosolic Ca2þ.

B. DYE LOADING

The study of synaptic vesicle release has been restricted to two methods.

The principle method is recordings from the postsynaptic cell whereby the


channels in the postsynaptic membrane serve as sensors for the transmitter

released. The second method is the loading of synaptic vesicles with fluorescent

dyes such as FM1-43 (Cochilla et al., 1999). The uptake of these dyes into

reforming vesicles by endocytosis and the subsequent release of the dye can

monitor vesicle fusion and endocytosis independently of the presence of neuro-

transmitter in those vesicles or the sensitivity of the postsynaptic cell (Chapter 7

by Kidokoro). However, the size of embryonic terminals and the vesicle pool

within them has made it diYcult to apply this method to study mutations with

early lethal periods. Moreover, because the endings of larval motor neuorons are

typically embedded within the muscle cell, there are diVusion barriers for access

to the synapse and the dye cannot be applied and removed as rapidly as it can, for

example, in cultures of mammalian neurons. Therefore, while very precise rates

of dye loading and unloading can be calculated with this method in mammalian

systems, and dyes of diVerent hydrophobicities can be compared (Richards et al.,

2000), at the Drosophila NMJ this method cannot determine rates of endocytosis

or exocytosis. Dye experiments, instead, are limited to the demonstration of the

existence of cycling vesicles and measurements of the size of the cycling pool

(Chapter 7 by Kidokoro).

C. ALTERNATIVE MEASURES OF EXOCYTOSIS

The Drosophila physiologist can also look with envy on some other prepara-

tions that are amenable to additional sophisticated methods for analyzing exocy-

tosis. These include amperometry, capacitance measurements, and total internal reflection

fluorescence (TIRF) microscopy.

Amperometry uses a carbon-fiber electrode to sense certain biogenic amines,

chiefly dopamine, norepinephrine, and serotonin, and thereby measure the

amount of biogenic amine that is released by the fusion of a vesicle independently

of the sensitivity of the postsynaptic membrane (Zhou and Misler, 1995). At its

best, amperometry can resolve partial release of vesicular contents. However, this

method is not appropriate for glutamate and requires direct access to the synaptic

cleft with the sensing electrode.

By patching directly onto endocrine cells or cells with large nerve terminals,

and measuring the capacitance of the cell membrane, physiologists have been able

to detect exocytosis, sometimes of single vesicles, in a manner that is altogether

independent of the transmitter content of the vesicle. This means that the fusion

of empty or partially loaded vesicles can be determined. In addition, capacitance

measurements have been used to determine the resistance of the fusion pore and

the time course of its opening at the onset of vesicle fusion (Beutner et al., 2001).

However, because the Drosophila NMJ is typically very small and also embedded


in the muscle, it is not possible to patch directly onto the boutons for capacitance

measurements.

TIRF microscopy employs fluorescent tags on or in synaptic vesicles and, by

imaging vesicles within approximately 100 nM of the coverslip surface, can

resolve the movements and fusions of single vesicles (Steyer et al., 1997). Again

the geometry of the Drosophila NMJ prevents having release sites directly facing a

coverslip, and therefore this technique has not been used in the fly. If it were to be

adapted for Drosophila, it would probably require using cultured neurons and

inducing them to synapse directly onto a coverslip.

IV. How Do Vesicles Fuse: Full Fusion or Kiss-and-Run?

Before discussing the individual proteins that are involved in transmitter

exocytosis, it is necessary to examine the larger question of precisely how the

transmitter leaves the synaptic vesicle and enters the synaptic cleft. This has been

a long-running controversy in cell biology and neuroscience, dating back at least

30 years (Ceccarelli and Hurlbut, 1980; Heuser and Reese, 1973). The issue can

be summarized as follows. Classical exocytosis, as observed in many cell types,

involves the complete fusion of a vesicle with the plasma membrane. The vesicle

flattens out and is incorporated into the plasma membrane. Its contents are

completely disgorged into the extracellular space. At a nerve terminal, where

vesicles must be recycled rapidly for reuse, this exocytosis would be followed by

endocytosis, probably via clathrin, to recover the membrane and its proteins into

a new synaptic vesicle (Chapter 7 by Kidokoro). An alternative model, often

called kiss-and-run, invokes a mechanism in which the vesicular membrane does

not completely merge with and flatten out onto the plasma membrane. Instead, a

brief, partial fusion is envisioned, in which a transient fusion pore is opened

between the vesicle lumen and the extracellular space. The vesicle is reformed by

the reversal of this process and the closing of the fusion pore. If the pore is open

very briefly, it is possible that transmitter will only partially be unloaded.

A hallmark of the kiss-and-run process is that fusion is not complete and that

classical endocytosis is not needed to remake a synaptic vesicle. The literature

from mammalian systems, for and against the existence of a kiss-and-run mecha-

nism, is too extensive to be reviewed here (An and Zenisek, 2004; Jarousse and

Kelly, 2001). On several points the field has not yet reached a consensus, but

some points are generally accepted: (1) classical exocytosis and endocytosis are

likely to exist at most chemical synapses; (2) partial release of transmitter due to

incomplete unloading appears to occur, at least some of the time, in the case of

large dense-cored vesicles released from nonsynaptic sites; and (3) if kiss-and-run


does occur from small clear vesicles at synapses, it may occur only under certain

circumstances or alongside classical exocytotic events.

At the Drosophila NMJ, the issue of kiss-and-run has surfaced and has

provoked some debate (Dickman et al., 2005; Verstreken et al., 2002). There is

no doubt that full fusion and classical endocytosis occur and account for many of

the fusion events at this synapse. The proteins of the classical endocytotic

pathway are present at presynaptic endings and mutations in a clathrin adaptor

protein, Lap-180, can alter the size of synaptic vesicles (Bao et al., 2005; Zhang

et al., 1998a). Mutations in shibire (shi) have been extremely informative. This gene

encodes the protein dynamin, a component of the classical endocytotic pathway

that severs nascent vesicles from the plasma membrane. As discussed elsewhere in

this volume (Chapter 7 by Kidokoro), shi temperature-sensitive mutations, at a

nonpermissive temperature, can cause the run down of a synapse on stimulation.

Vesicles that fuse to the plasma membrane are trapped there until eventually the

releasable pool of vesicles is completely transferred to the plasma membrane

(Koenig and Ikeda, 1989; van de Goor et al., 1995). These observations are

consistent with classical exocytosis serving as the dominant form of transmitter

release at the Drosophila NMJ. The coexistence of kiss-and-run, however, was

reported from an examination of mutations in other proteins in the classical

endocytotic pathway, Endophilin (Endo) and Synaptojanin (Synj) (Verstreken

et al., 2002, 2003). The response of endo and synj mutants to repeated stimulation

at 10 Hz is quite diVerent from that of a shimutant. Whereas the amplitude of the

EJP declines in shi until a negligible response remains, amplitudes in endo and synj

larvae decline only partially and then reach a plateau level that can be sustained,

seemingly indefinitely. During this plateau many more vesicles are released than

were originally present in the terminal, and thus, there must be a means to

recycle and reuse synaptic vesicles even in the absence of these proteins. Arguing

that these mutations block the classical recycling pathway, the authors concluded

that the plateau phase must be maintained by another pathway and in particular

by kiss-and-run. Further analysis of these mutations, however, has demonstrated

that they do not completely block classical endocytosis, but rather diminish the

maximum rate of endocytosis that can be achieved at the NMJ (Dickman et al.,

2005). The residual slowed pathway has the properties of classical endocytosis,

including the ability for vesicles to be loaded with the dye FM1–43 (Chapter 7

by Kidokoro) and a dependency on Dynamin/Shi. The residual slow pathway

is suYcient to account for the ability of synj and endo terminals to sustain a

reduced level of transmitter release even under high-frequency stimulation. Thus,

although it is not possible to completely exclude the existence of kiss-and-run at

this synapse, there is presently no evidence for its occurrence and the full-fusion

pathway is suYcient to explain the physiology of the system to date, including

the absolute requirement for Dynamin/Shi for sustained vesicle cycling.


V. Core Machinery of Exocytosis: Syntaxin, VAMP/Synaptobrevin, and SNAP-25

A wealth of data from yeast to mammals has demonstrated the existence of a

core machinery for intracellular membrane fusion. Versions of the proteins that

form this machinery are present at synapses and can also be found on vesicles

that shuttle between the endoplasmic reticulum (ER) and Golgi in yeast, and in

all membrane traYcking compartments of every eukaryotic cell. In the eVort tounderstand the workings of these proteins, the Drosophila NMJ has proven

valuable. It has the advantages, of course, of having the ability to examine

mutations in the genes encoding for these proteins. However, it also provides

the capacity to resolve single vesicle fusions with rapid kinetics by electrophysio-

logical means and to examine the ability of vesicles to find and dock at their

target membranes by electron microscopy. Additionally, the manipulation of one

of these proteins by the expression of tetanus toxin (TNT) is now used extensively

to alter the activity of circuits within the fly brain (Heimbeck et al., 1999; Kaneko

et al., 2000; Martin et al., 2002; Suster et al., 2003; Sweeney et al., 1995). For this

reason, the role of the fusion complex will be examined here in detail.

The proteins of the core complex are often referred to as soluble

N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins (Sollner

et al., 1993b) and the prevailing model, compiled from experiments in yeast,

in synaptic preparations, and in vitro fusion assays, is that these proteins, by

binding to one another, can form a bridge between a vesicle and its target

membrane. Moreover, the energy released by the formation of this complex

may provide the driving force for the actual fusion of the two membranes (Chen

and Scheller, 2001). Fusion has been shown to require complementary proteins

on either side of the reaction, that is, on both the vesicle and target membranes.

These are sometimes referred to as v- and t-SNARES, although the distinction as

to who is the vesicle and who the target is sometimes not so clear. At the synapse,

the t-SNARES (found principally on the plasma membrane) are called Syntaxin

and SNAP-25. The v-SNARE (that resides chiefly on the synaptic vesicles) is

called either Synaptobrevin or VAMP. The complex that forms between these

proteins consists of a four-stranded coiled coil. Two of the component strands are

contributed by SNAP-25 and one each by the other proteins. The complex they

form is exceptionally stable and it has been hypothesized that vesicles docked at

the plasma membrane can form a loose complex first, which, at the time of

vesicle fusion, is allowed to ‘‘zipper closed.’’ This zippering drives the two

membranes together (Chen and Scheller, 2001; Lin and Scheller, 2000).

In addition, it was noted that diVerent traYcking compartments within the cell

can have diVerent isoforms of these proteins and that not all isoforms will

function in conjunction with one another (McNew et al., 2000). Thus, it has been

hypothesized that the cognate pairing of v- and t-SNAREs may provide the


specificity that allows a vesicle to fuse selectively with its appropriate target

membrane.

There have been several key questions regarding the function of this core

complex that have been investigated at the fly NMJ. (1) Are these proteins

essential for all types of synaptic vesicle fusion? (2) Is an individual SNARE used

only for synaptic vesicle fusion or is it used for other types of exocytosis? (3) Are

SNAREs necessary for an early step in transmitter release, particularly for the

docking of vesicles at the active zone, or only for the final membrane fusion step?

(4) Do the SNAREs show specificity in their ability to support synaptic vesicle

fusion? Some or all of these questions have been addressed with mutations in the

genes coding for each of the v- and t-SNAREs.

A. SYNAPTOBREVINS IN THE FLY

There are at least two isoforms of Synaptobrevin/VAMP in Drosophila. One,

called Synaptobrevin (Syb), is ubiquitously expressed in the organism (Chin et al.,

1993; DiAntonio et al., 1993a; Sudhof et al., 1989). Mutations in the syb gene are

embryonic lethals, causing arrest of development at what is probably the time

when the maternally contributed syb runs out. Clones of syb null mutants are cell

lethal in the eye (Bhattacharya et al., 2002). Thus, although there have been no

direct assays of membrane traYc in these cells, it seems likely, based on its

phenotype and homology to Synaptobrevins in other species, that Syb mediates

the constitutive traYcking of proteins to the cell surface. Without this traYc, cells

cannot grow or divide.

The second Synaptobrevin isoform is called neuronal-Synaptobrevin (n-Syb)

and it is detected exclusively in the nervous system, where it appears to be highly

concentrated at all synapses (Deitcher et al., 1998; DiAntonio et al., 1993a).

The first analysis of n-syb function in Drosophila was not via mutations in the gene,

but by the expression of a transgene encoding the light chain of TNT (Broadie

et al., 1995; Sweeney et al., 1995). TNT is a product of a Clostridial bacterium,

and the light chain of TNT is a protease that selectively cleaves certain isoforms

of Synaptobrevin (Pellizzari et al., 1999). In Drosophila, it cleaves n-Syb, but not

Syb. Neuronal expression of TNT did not impair the diVerentiation of moto-

neurons or their ability to extend axons and form synapses, but embryos were

paralyzed and could not hatch (Sweeney et al., 1995). Physiological recordings

from embryonic NMJs uncovered a surprising result: EJPs could not be evoked in

the TNT-expressing flies, but spontaneous minis were still observed. The former

finding was, of course, consistent with the hypothesis that n-Syb is the essential

v-SNARE for the fusion of synaptic vesicles, but the persistence of minis was

confounding. This phenomenon has been studied more intensively in null alleles

of n-syb, which circumvent some potential problems of TNT expression—that


some protein may persist intact or that the cleaved products may retain some

activity. The phenotype of the n-syb nulls, however, was identical to that observed

on TNT expression. Null mutations lacking n-syb develop normally but are

paralyzed and do not hatch. Even under conditions where evoked EJPs should

be very large (high Ca2þ or the presence of Kþ channel blockers) no EJP was

seen. Minis, however, were normal in size and present at approximately 25% the

frequency of wild type (Deitcher et al., 1998). These findings are novel because

they distinguish the mechanistic requirements for spontaneous and evoked vesicle

fusions. If evoked release were merely the same process as a mini, but with

increased probability and synchronized, the two types of fusion would be equally

dependent on the same SNARE proteins.

Do the remaining minis fuse via a novel mechanism, completely independent

of SNARE proteins? This is unlikely because, as discussed later, minis are

reported to be completely absent in flies lacking the t-SNARE Syntaxin. However,

SNARE-dependent fusion requires a SNARE on the vesicle membrane as

well (Nichols et al., 1997). What then is the mechanism for the release of these

minis? One possibility is that they employ the other v-SNARE, Syb. Another is

that a t-SNARE, perhaps Syntaxin, is partially localized on synaptic vesicles and

can substitute for n-Syb in the formation of the core complex. There is precedent

for fusion proceeding, albeit poorly, by means of t-SNARE–t-SNARE complexes

(Nichols et al., 1997). Whatever this fusion pathway might be, it is not capable of

the fast, synchronous fusions that form an EJP. In a detailed study of these

residual events (Yoshihara et al., 1999), it was determined that their frequency

can be modulated by cytosolic Ca2þ, but the elevated Ca2þ must be sustained.

Thus, in an n-syb null mutant, the frequency of minis could be increased up to

17-fold by a steady state depolarization of the terminal, a tetanic stimulation of

the nerve, or direct Ca2þ influx via an ionophore. Thus, n-syb mutant terminals

retain a Ca2þ-sensor that can couple to the remaining release apparatus.

The inability of Ca2þ influx during a single action potential to have a similar

eVect could indicate that the triggering is too slow to be activated by a transient

change in local Ca2þ, or that the vesicles are no longer in the immediate vicinity

of the open Ca2þ channels.

Another fascinating aspect of these mutant terminals is that the release

apparatus is no longer modulated by cAMP (Yoshihara et al., 1999). In wild-

type larvae, cAMP, via Protein Kinase A, has two actions at the synapse. One

depends on extracellular Ca2þ and likely involves the modulation of Ca2þ

channels. The other is independent of extracellular Ca2þ and likely involves a

modulation of the release apparatus that increases mini frequency. This Ca2þ-independent action was absent from n-syb larvae (Yoshihara et al., 2000) and thus

the pathway downstream of cAMP must pass through this particular v-SNARE.

Another interesting feature of the n-syb phenotype was noted in viable hypo-

morphic alleles (Stewart et al., 2000): a shift in the apparent cooperativity of Ca2þ


ions in triggering release, as measured by systematically altering the extracellular

[Ca2þ]. This finding may reflect the fact that Ca2þ, having bound to its sensor,

must act via the SNARE complex.

Are v-SNAREs specific for particular types of membrane traYc? The speci-

ficity of SNARE pairing is a key element of the hypothesis that SNAREs direct

vesicles to the correct target membrane. Through the availability of null muta-

tions in both syb and n-syb, and of transgenes to drive expression of each, it has

been possible to ask whether they are either interchangeable or selective in their

particular roles, that is, in cell survival and growth versus synaptic transmission

(Bhattacharya et al., 2002). The expression of n-syb in the developing syb null eye

could in fact rescue its development and the expression of syb could partially

restore the EJP at an n-syb null NMJ. Moreover, these two v-SNARES have

behaved identically in biochemical assays of complex formation (Niemeyer and

Schwarz, 2000). Thus, Synaptobrevins may or may not have selectivity for fusion

with the plasma membrane instead of with intracellular organelles. However,

they cannot account for the selective fusion of synaptic vesicles at active zones, as

opposed to the fusion of other vesicles that deliver proteins to other sites on the

cell surface.

B. TETANUS TOXIN AS A SYNAPSE-SILENCING REAGENT

As mentioned earlier, the light chain of TNT can cleave n-Syb and it does

so with great specificity as well as great eYciency: little or no intact n-Syb remains

when TNT expression is driven throughout the nervous system (Sweeney et al.,

1995). There has therefore been great interest in selectively expressing TNT

in subsets of neurons to silence their output (Martin et al., 2002). This method has

been used to establish the functional importance of neurons for such behaviors

as feeding and locomotion (Heimbeck et al., 1999; Kaneko et al., 2000; Suster

et al., 2003; Sweeney et al., 1995). However, some important caveats need to be

considered, because it is not possible in the CNS to determine directly whether

or not TNT expression in the selected neurons is eVective in preventing trans-

mitter release. Instead, there is a presumption, not necessarily accurate, that

those cells will be aVected in the same manner as the well-studied motoneurons.

We do not know, however, that every neuron contains its target, n-Syb, although

it is certainly widespread (DiAntonio et al., 1993a). Moreover, since overexpres-

sion of Syb, which is not cleaved by TNT, can substitute, at least in part,

for n-Syb (Bhattacharya et al., 2002), it is possible that high levels of Syb or a

related SNARE might sustain transmission in subpopulations of CNS synapses.

Similarly, even at the NMJ, TNT does not abolish transmitter release. It only

abolishes the eYcient, synchronous release of transmitter in response to individual

action potentials. Spontaneous minis can therefore be expected to remain


in the CNS and any sustained electrical activity, such as high-frequency trains

of action potentials, or sustained depolarizations in nonspiking neurons, such

as photoreceptors, would be expected to increase the release of transmitter

despite the removal of n-Syb. Finally, there are many forms of transmitter release

that may diVer mechanistically from what we have studied in detail at the

glutamatergic motoneuron synapses. The eYcacy of TNT in preventing the

release of aminergic vesicles or dense-cored peptidergic vesicles is only now being

examined. Release of transmitter from nonvesicular pools, for example, from

plasma membrane transporters running ‘‘backward’’ is presumed to be TNT-

insensitive (Yang and Kunes, 2004). Finally, although loss of n-Syb does not cause

gross abnormalities in the development of neurons, the possibility remains that

the eVects of TNT will not be exclusively on the fusion of synaptic vesicles. TNT

may alter the release of signaling molecules, or the surface expression of some

receptors. In summary, although TNT expression is a highly valuable tool for

neurogenetics, it may not silence all forms of synaptic transmission or be devoid

of developmental consequences.

C. SYNTAXIN1

Of the SNAREs that are localized to the plasma membrane, the most

attention has been given to Syntaxin. Although Syntaxin isoforms are present

on many compartments within the cell, a particular Syntaxin, called Syntaxin1

(Syx1) in Drosophila, appears to be the homolog of the Syntaxins found on the

plasma membrane in mammalian cells (Schulze et al., 1995). Genetic analysis of

this protein at the synapse is complicated by the fact that Syx1 is not exclusively

synaptic. Mutations in syx1 are cell lethal, suggesting that it is important in cell

growth and division, like the v-SNARE syb (Schulze et al., 1995; Stowers and

Schwarz, 1999). Maternal germ-line mosaics of null syx1 alleles are infertile due

to arrest of oocyte development. Similarly, mosaics of weaker alleles give rise to

embryos with defects in cellularization, a process that involves a large increase in

membrane surface area (Burgess et al., 1997). These phenotypes attest to the

essential role that Syx1 plays in membrane traYc to the cell surface.

However, due to maternally contributed protein and mRNA, homozygous

null mutant embryos from heterozygous parents can develop until late stages of

embryogenesis before dying (Broadie et al., 1995; Burgess et al., 1997; Schulze

et al., 1995). These embryos have abnormalities indicative of secretory defects in

nonneuronal cells and also mild anatomical abnormalities in their central and

peripheral nervous system. Nevertheless, the maternal contribution appears to be

just barely enough that, at least in most segments, motoneurons are born and can

extend axons to the appropriate muscle targets, but not enough for synapses to

function properly (Schulze et al., 1995). Several papers have appeared in which


the electrophysiological and developmental properties of these synapses are

described (Broadie et al., 1995; Featherstone and Broadie, 2002; Saitoe et al.,

2001, 2002; Schulze et al., 1995), and their findings are not always in agreement.

The diVerences are likely, at least in part, to be due to the fact that these embryonic

synapses are poised at a threshold where the maternally contributed Syx1 is

disappearing. Therefore, the age of the embryo, the position of the muscle, and

other unknown variables, may cause some recordings to be made from synapses

that still contain low levels of Syx1, while others have so little Syx1 that the synapse

never formed or formed and then began to retract. In no case has an EJP been

evoked from a homozygous null syx1 embryo, consistent with a requisite role

for Syx1 in transmitter release (Broadie et al., 1995; Saitoe et al., 2001; Schulze

et al., 1995). It has also been reported that, contrary to initial reports (Broadie et al.,

1995), quanta are not released from the syx terminals by the application of

hypertonic sucrose solutions (Aravamudan et al., 1999). Hypertonic sucrose has

been used in mammalian systems to trigger the release of vesicles from nerve

terminals by an unknown mechanism that bypasses the normal requirement for

Ca2þ influx. This finding again emphasizes the importance of Syntaxin for the

fusion of synaptic vesicles, and further suggests that sucrose triggers release in a

SNARE-dependent manner. However, one group has observed spontaneous

minis at early stages of synapse formation in syx1 mutants (perhaps while some

Syx1 still remains). Subsequently this group observed a large decrease in the

sensitivity of the muscle membrane to glutamate and an absence of glutamate

receptors clusters. These findings were interpreted as showing that minis are

needed to form or preserve glutamate receptors clusters opposite to active zones

(Saitoe et al., 2001, 2002). Others have observed normal levels of glutamate

sensitivity and the absence of minis (Featherstone and Broadie, 2002). These

findings underscore the diYculty of analyzing the function of a protein in trans-

mitter release when that protein is also required for constitutive membrane traYc

and hence for the viability of the cell and the formation of the synapse.

Biochemical studies of the H3 domain of mammalian Syntaxin1 indicate that

it can bind to several proteins other than Synaptobrevin/VAMP and SNAP-25,

the proteins of the core complex. In particular, Syntaxin1 can bind to the protein

known as n-Sec1 or Munc-18 in mammalian systems and encoded by ras opposite

(rop) in Drosophila. This is a very high aYnity interaction, and its function is not yet

clear. However, n-Sec1 is not present in the core complex, and therefore, one

hypothesis is that it holds Syntaxin in a closed state that prevents it from

associating with the other SNAREs (Dulubova et al., 1999). Mutations in rop

are embryonic lethal and, like syb or syx1 mutations, have defects in nonneuronal

structures, suggesting a requirement in constitutive membrane traYcking

(Harrison et al., 1994). This phenotype (and the phenotype of sec1 mutations in

yeast; Novick et al., 1980) is not compatible with n-Sec1 functioning solely as a

negative regulator of release: its phenotype indicates a decrease in secretion,


not an increase. Syntaxin also binds to the vesicle protein Synaptotagmin (see in

a later section) and may bind to a region of mammalian Ca2þ channels

(Bezprozvanny et al., 2000; Leveque et al., 1994). Attempts have been made to

understand the physiological significance of these interactions in Drosophila by

using point mutations that selectively alter one or another of the protein–protein

interactions (Fergestad et al., 2001; Wu et al., 1999). Portions of these studies,

however, have been challenged on the basis that the mutations used do not

selectively or suYciently alter the interactions in question (Matos et al., 2000).

D. SNAP-25 AND SNAP-24

The other plasma membrane t-SNARE, SNAP-25, has been particularly

diYcult to study in Drosophila, in part because the gene is located within hetero-

chromatin and is composed of multiple exons that are small and widely spaced

(Risinger et al., 1997). In addition, it is partially redundant with a homolog

protein called SNAP-24 (Niemeyer and Schwarz, 2000; Vilinsky et al., 2002).

The first allele of Snap25 to be isolated was a temperature-sensitive paralytic

mutation that resulted in a single amino acid change in the first amphipathic coil

of SNAP-25 (Rao et al., 2001). Biochemical analysis indicated that this protein

remained capable of forming SNARE complexes. The only temperature-sensitive

defect that was detected in this mutant was in the little-understood phenomenon

of SNARE complex multimerization—the presence of higher molecular weight

complexes of SNARE proteins on SDS gels. These presumed multimers of the

SNARE complex were less stable if SNAP-25 contained the point mutation.

In vivo, the temperature-sensitive allele had the unexpected property of increasing

EJP amplitude at the permissive temperature, perhaps reflecting an enhanced

ability of the mutant protein to proceed toward fusion (Rao et al., 2001).

At elevated temperatures, however, transmission was reduced relative to wild

type, which is the likely cause of the temperature-sensitive paralysis and suggests

that the multimerization of SNAREs may be important to their function in vivo.

The isolation of null alleles of Snap25 oVered an additional surprise:

homozygotes could survive through pupal stages and synaptic transmission at

the larval NMJ was largely unaVected by the absence of the protein (Vilinsky

et al., 2002). This phenomenon is likely to be explained by the presence of the

closely related t-SNARE SNAP-24. SNAP-24 is less abundant than SNAP-25

within the nervous system and is more abundant in nonneuronal tissues, suggest-

ing that it is primarily concerned with constitutive membrane traYc. Instead,

SNAP-25 predominates at nerve terminals (Niemeyer and Schwarz, 2000).

However, in the complete absence of SNAP-25 protein (which is not the case

in the temperature-sensitive Snap25 allele) SNAP-24 can replace SNAP-25

(Vilinsky et al., 2002). In biochemical studies, SNAP-24 and SNAP-25 were


indistinguishable in their shared ability to form SNARE complexes with Syntaxin

and n-Syb, consistent with their interchangeability during transmitter release

(Niemeyer and Schwarz, 2000). In addition, both isoforms could form SNARE

complexes with Syntaxin and Syb, the likely complex involved in constitutive

membrane traYc. Thus, as discussed earlier for n-Syb and Syb, the t-SNAREs

SNAP-24 and SNAP-25 are interchangeable. Specificity of cognate recognition

by SNARE proteins cannot explain the distinctions in membrane targeting

between synaptic vesicles that fuse at active zones and constitutive vesicles that

fuse elsewhere on the surface.

E. SNARE PROTEINS ACT LATE IN THE VESICLE CYCLE

The study of n-Syb (as well as Syntaxin, see in a later section) has been useful

in determining the step at which SNAREs act, that is, whether in the docking of

vesicles at their target membranes, in the priming of them for fusion, or in the

fusion step itself. For these questions of general cell biological importance, the

Drosophila NMJ has several advantages. Not only are there unambiguous null

alleles for SNARE proteins, but there is also a well-defined target membrane that

normally contains a population of docked vesicles that are visible by electron

microscopy. It is not clear just how this pool, defined as ‘‘docked’’ by anatomical

criteria, correlates with the biochemist’s model of ‘‘docked’’ vesicles, which

presumes an association of the vesicle and target membranes via protein–protein

interactions. Nonetheless, in the case of syx1 null alleles or in nerve endings

expressing TNT, there is no detectable loss of synaptic vesicles in close juxtaposi-

tion to the active zone membrane. On the contrary, the number of docked

vesicles appears to be increased (Broadie et al., 1995). These findings indicate

that synaptic vesicles can find nerve terminals, cluster around active zones, and

dock at the active zone membrane in the absence of the SNARE proteins. The

absence of evoked release, in contrast, argues strongly for a block at a late stage in

the process, most likely in fusion itself. The intermediate steps that are hypothe-

sized to occur in order to make a vesicle ready for rapid fusion after it contacts the

plasma membrane (collectively referred to as priming), are more diYcult to

judge. The presence of minis in n-syb null alleles, and their ability to fuse in

response to sustained elevation of Ca2þ, may indicate that some vesicles are

primed for fusion, but their slower rate of fusion indicates that they are not as

competent for fusion as vesicles in wild type.

F. SUMMARY OF SNARE PROTEINS IN DROSOPHILA

In summary, null alleles are available for each of the v- and t-SNAREs

that function at the synapse, syx1, n-syb, and Snap25, as well as for the close


homolog syb that principally functions in nonsynaptic traYc to the plasma

membrane. In addition, TNT can selectively cleave n-Syb and thereby prevent

the evoked release of transmitter. By selective expression in particular cell types,

TNT may be a powerful means to silence the output of a cell, although some

caution is needed in extrapolating from the NMJ to the CNS. From studies of the

fly NMJ we have learned that mutations in SNAREs can diVerentially aVectevoked release and minis, suggesting mechanistic diVerences in the nature of

vesicle fusion in these two processes. Because morphologically docked vesicles

persist in these mutants, the SNAREs are likely to act specifically in a late step of

the vesicle cycle, most probably in fusion itself. In addition, although there are

distinctions between the Syb isoforms that normally predominate in constitutive

versus synaptic membrane traYc, those isoforms can substitute for one another in

functional assays. In addition, they show no biochemical specificity in their ability

to complex with one another. Moreover, a single Syx isoform is used in both

pathways. Therefore, SNARE pairing does not provide specificity for targeting

these classes of vesicles to their particular target membrane at the active zone.

VI. Vesicular ATPase and Membrane Fusion

The demonstration that SNARE proteins function in a late stage of exocytosis

raises the question as to whether they are the sole requirement for membrane

fusion. There have been in vitro studies to demonstrate that SNAREs by them-

selves can promote the fusion of lipid vesicles in vitro (Weber et al., 1998), but this

does not necessarily mean that this minimal reconstituted system represents the

normal in vivomechanism. In this context, one of the most intriguing studies using

the Drosophila NMJ (Hiesinger et al., 2005) proposes that a subunit of the vesicular

ATPase, an abundant and required protein in the synaptic vesicle membrane,

also functions at a late step of exocytosis, perhaps by forming a fusion pore

bridging the membranes. The vesicular ATPase is the proton pump responsible

for the acidification of the vesicle lumen at the expense of ATP. As such it

provides, in the form of a proton gradient, the energy that drives the uptake of

transmitter into vesicles. A hypothesis is that at least one of the proteins in this

large complex (the v100–1 subunit of the V0 complex) functions directly in

membrane fusion.

This model has origins in studies of vacuolar fusion in yeast (Bayer et al., 2003;

Peters et al., 2001) wherein it was found that a particular subunit of the vacuolar

ATPase, Vph1p, was necessary in assays of vacuole fusion with one another

in vitro. This ATPase comprises two large complexes, one that hydrolyzes ATP

and another that forms a proteolipid pore through which protons can flux.

The Vph1p subunit is in the latter, the V0 complex. However, the particular


mutations studied did not block proton transport, the canonical function of the

V0 complex. Rather, the defect in the yeast assay was specifically in fusion, at a

step after the requirement for the SNARE proteins. It was hypothesized that the

large Vph1p protein formed a bridge between two vesicles that were linked by

SNAREs and that this bridge could progress into a proteinaceous pore that

subsequently would resolve into full fusion of the membranes.

In this context, it was striking that mutations in the Drosophila homolog of the

same large subunit were isolated in a phototaxis screen designed to identify

mutations of synaptic transmission (Hiesinger et al., 2005). The subunit, called

v100-1, is concentrated at synapses, and null mutations in the v100-1 gene

showed a sevenfold reduction in EJP amplitude at the embryonic NMJ. Minis

were present and of normal amplitude, although reduced in frequency. The

presence of some minis indicates that some transmitter loading and some fusion

can occur in these embryos, perhaps because of a partially redundant second

isoform. But why is the EJP so small? Is it because of a block in fusion or because

a lack of proton transport has generated empty vesicles? The evidence favors the

former because FM1-43 dye loading was below normal levels (Hiesinger et al.,

2005). Moreover, both in yeast and in the fly, the v100-1 subunit can bind to

t-SNAREs (Hiesinger et al., 2005; Peters et al., 2001). Thus, while the precise role

of this ATPase subunit remains uncertain, the hypothesis that it interacts with

t-SNAREs to form a fusion pore, or to initiate membrane fusion is an attractive

possibility.

VII. NSF and the Resetting of the SNARE Machinery

NSF, a factor that support in vitro vesicle traYcking in cell extracts (Block et al.,

1988), is the key element of a multimeric protein that includes six NSF subunits

and six subunits of an associated protein called alpha-SNAP. NSF can bind to

SNARE complexes and, through the catalysis of ATP, can cause them to

dissociate (Sollner et al., 1993a). Originally, it was thought that this ATP-depen-

dent step might represent the fusion reaction itself, but this model has been

rejected because fusion in many systems is ATP-independent, and because

SNAREs in vitro can stimulate membrane fusion without NSF (Weber et al.,

1998). Models (Lin and Scheller, 2000; Schwarz, 1999) place NSF action after

fusion as a means of undoing SNARE complexes so that v- and t-SNAREs can be

separated from one another and recycled to their proper compartments.

The formation of the SNARE complex is so energetically favorable that energy

(provided by the ATPase activity of NSF) is required to pull the SNAREs apart

and free them for another round of vesicle fusion. Separated, the SNAREs are

in a state with high potential energy and that energy can be harnessed by the


cell when SNARE complexes subsequently reform and thereby drive membrane

fusion.

The comatose (comt) gene in Drosophila encodes an NSF homolog (Pallanck

et al., 1995). The study of comt permitted a crucial genetic test of NSF function

in vivo. The classic comt alleles are temperature-sensitive paralytics (Siddiqi and

Benzer, 1976). These alleles are hypomorphic mutations of Nsf1, one of two NSF

homologs in the fly (Pallanck et al., 1995). Consistent with the model described

above, when shifted to the nonpermissive temperature, comt flies accumulate

SNARE complexes that reside primarily on the plasma membrane (Tolar and

Pallanck, 1998). Using electron microscopy it is observed that these terminals

accumulate vesicles, including a population that is docked at the active zone

by morphological criteria, suggesting a defect in the priming of vesicles for

transmitter release (Kawasaki et al., 1998). A likely explanation (Littleton et al.,

2001) is that each round of vesicle fusion in a comt mutant creates SNARE

complexes on the plasma membrane that cannot be dissociated and that, over

time, the cycling vesicle pool is depleted of usable, free, SNAREs. At this point,

the vesicles can no longer fuse with the membrane and are therefore trapped in a

docked but fusion-incompetent state.

At adult NMJs on the dorsolateral flight muscles, the electrophysiological

phenotype of comt mutants is wholly consonant with this model. At the permissive

temperature the EJP is normal but, at the nonpermissive temperature, there is

a progressive decline in EJP amplitude. This likely reflects the loss of free

SNAREs and hence the decline in releasable vesicles (Kawasaki et al., 1998).

At the third instar larval NMJ, however, comt does not have a detectable pheno-

type (Golby et al., 2001; Mohtashami et al., 2001). This can be explained by the

fact that the Nsf1 gene that is mutant in comt appears to be predominantly

expressed in the adult nervous system, while NSF2 is likely to play an equivalent

role at the larval NMJ (Stewart et al., 2002). Mutations in Nsf2 are early lethals,

perhaps reflecting the importance of this isoform for membrane traYc in non-

neuronal cells (Golby et al., 2001). To study the physiological consequences of

disrupting NSF2 at the larval NMJ, a dominant negative approach has therefore

been taken (Stewart et al., 2002, 2005). Expression of a dominant negative NSF2

in larval neurons has the expected consequence of causing use-dependent run-

down of the amplitude of the EJP. However, it also causes a substantial over-

growth of the synapse, even at early developmental stages, and some axonal

misrouting. The traYcking defect behind these developmental phenotypes is not

yet known.

The relationship of the two Nsf genes in Drosophila is not yet completely

understood. However, the apparent importance of NSF2 in nonneuronal embry-

onic tissues as well as at larval synapses provides another example of membrane

traYcking proteins that are shared between synaptic and nonsynaptic pathways.


There are no known diVerences in the SNARE proteins at adult versus larval

synapses, and so it seems unlikely that the two NSF isoforms will diVer in their

substrate specificities.

VIII. Synaptotagmin and the Regulation of Transmitter Release

A. SYNAPTOTAGMIN AND ITS BINDING PROPERTIES

Ever since the identification of Synaptotagmin as a major synaptic vesicle

protein and the determination of its sequence (Perin et al., 1990), Synaptotagmin

has been the focus of intense study. The biochemical properties of mammalian

Synaptotagmin have been studied in great detail (Chapman, 2002; Li et al., 1995;

Rizo and Sudhof, 1998; Sudhof and Rizo, 1996), and some of these properties

have been confirmed for Drosophila. The hallmark of the Synaptotagmin protein

family is an N-terminal transmembrane domain, followed by a large cytoplasmic

domain containing two C2 domains in tandem. The C2 domain is a much-

studied motif also present in isoforms of Protein Kinase C and Phospholipase A2.

In these proteins the C2 domain binds Ca2þ and causes a conformational change

in the protein that triggers its translocation to the membrane (Nalefski et al.,

2001). The C2 domains of Synaptotagmin also bind Ca2þ via a set of conserved

negative charges that are clustered at one end of the structure (Shao et al., 1996,

1998). In addition, C2 domains of Synaptotagmin also bind phospholipids and

phosphatidyl inositol (Li et al., 1995; Zhang et al., 1998b). These binding proper-

ties strongly suggest an important role for Synaptotagmin in the regulation of

transmitter release. Synaptotagmin is the best candidate for being the sensor for

cytosolic Ca2þ that triggers the release of vesicles in response to an action

potential.

In addition to these binding properties for small ligands, Synaptotagmin

interacts with SNARE proteins, SNARE complexes, the clathrin adaptor protein

AP-2, as well as with lipid membranes (Li et al., 1995; Zhang et al., 1994). Many of

these interactions have been shown to be modulated by Ca2þ. In considering the

function of Synaptotagmin, it is clear that these interactions could be the means

by which Synaptotagmin regulates the fusion step during transmitter release, the

ability of vesicles to translocate to and dock at the membrane, or the rate of

endocytosis. To resolve the significance of these myriad interactions in vivo genetic

analysis of synaptotagmin (syt) mutants was undertaken, first in the fly (Broadie et al.,

1994; DiAntonio and Schwarz, 1994; DiAntonio et al., 1993b; Littleton et al., 1993,

1994) and Caenorhabditis elegans (Jorgensen et al., 1995; Nonet et al., 1993) and

subsequently in the mouse (Geppert et al., 1994).


B. SYNAPTOTAGMIN PHENOTYPES AT THE NMJ

Null mutations of syt, also called syt1 (see in a later section), are poorly viable

and first were believed to be incapable of surviving past the first instar larval stage

(DiAntonio et al., 1993b). However, by segregating homozygotes away from their

more robust heterozygous siblings and by raising them under appropriate

conditions, it is possible for them to survive to the third instar and even to early

adulthood (Loewen et al., 2001). Therefore, data are now available for the

phenotypes of syt null alleles for embryonic, first instar and third instar NMJs,

as well as from weaker alleles. The electrophysiological phenotype of null alleles is

severe—approximately a 10- to 40-fold reduction in the amplitude of the evoked

response—at each of these stages (Broadie et al., 1994; Loewen et al., 2001;

Okamoto et al., 2005; Robinson et al., 2002; Yoshihara and Littleton, 2002).

Yet, as expected from the persistent viability of the flies, transmission is never

abolished. The remaining evoked responses and other physiological parameters

have therefore been examined to determine precisely what is lacking in these

mutants.

One hypothesis has been that Syt serves as a fusion clamp that prevents the

SNARE proteins from interacting in resting Ca2þ, but which releases the

SNAREs and thereby permits vesicle fusion when Ca2þ is bound (Littleton

et al., 1994). Some suggestion of this mechanism might be taken from the

observation that mini frequency is modestly increased at both embryonic and

third instar NMJs that lack Syt (Broadie et al., 1994; DiAntonio and Schwarz,

1994; Littleton et al., 1994; Loewen et al., 2001). In this model, the removal of Syt

would cause the constitutive fusion of synaptic vesicles as soon as they dock at the

active zone, and the evoked response would be reduced because of the inability to

accumulate readily releasable vesicles. This model, however, was rejected

because the number of minis released was not adequate to explain the drastic

reduction in evoked release (DiAntonio and Schwarz, 1994). This finding is also

supported by studies in which Synaptotagmin was acutely inactivated (Marek and

Davis, 2002). For these experiments, the wild-type Syt was replaced with a syt

transgene that included a C-terminus binding site for a fluorescent ligand. After

application of the ligand, laser illumination produces reactive oxygen species

that rapidly destroy the tagged protein (FlAsH-FALI). In this protocol, evoked

transmission could be suppressed within seconds without a concomitant increase

in spontaneous release. Thus, Syt plays a positive role by promoting fusion rather

than by acting as a fusion clamp to prevent spontaneous release.

The dominant hypothesis is that Ca2þ binding to Synaptotagmin is the

necessary and suYcient event to trigger transmitter release in response to an

action potential. However, strong evidence for this hypothesis was not easy to

achieve. The first obstacle has been the persistence of Ca2þ-dependent transmitter

release even in null alleles, a phenomenon also observed in C. elegans and


mice (Geppert et al., 1994; Nonet et al., 1993). Thus, Synaptotagmin can enhance

transmitter release, but is not absolutely necessary. In the absence of a complete

blockade of transmission, two interpretations were available. Synaptotagmin

might not be the Ca2þ-trigger but might instead be a facilitator of release that

increases the availability of vesicles for fusion or the probability of their fusing.

Alternatively, Synaptotagmin might be a trigger for the EJP, but coexists with a

second Ca2þ-sensor that mediates the residual release. The sequencing of the

Drosophila genome revealed additional synaptotagmin genes that might represent

this residual Ca2þ sensor, although none has yet been demonstrated to play this

role (see in a later section). Therefore, attempts to clarify the importance of

Synaptotagmin have centered on examining the properties of exocytosis in

wild-type and syt mutants, in the hope of finding an indication that the residual

release is using a Ca2þ-sensor that is distinct from that in wild-type synapses. The

nonsynaptotagmin Ca2þ-sensor might have a diVerent aYnity for Ca2þ or

involve the binding of a diVerent number of Ca2þ ions. The latter would be

reflected in a diVerent slope for the plot of log[Ca2þ] versus log response

amplitude (see in an earlier section). Particularly, if there were two sensors, both

of which needed to be activated by Ca2þ, the removal of one of them should

decrease the slope of this relationship. Recordings from null embryos, however,

failed to see such a shift (Broadie et al., 1994). Although some alleles were

reported to have a shift in the slope or aYnity (Littleton et al., 1994), most

recordings indicated a predominant eVect on the number of vesicles secreted

(Vmax) rather than on the slope or aYnity. A change in the number of vesicles

secreted could be caused by changes in many aspects of transmission other than a

change in the Ca2þ sensor. These studies are exceptionally diYcult because the

evoked responses are so small in the mutants and because it is necessary to work

in very low Ca2þ to determine the cooperativity of the relationship. The most

thorough study of evoked release in null syt embryos (Okamoto et al., 2005) found

a substantial decrease in the slope, consistent with the hypothesis that Syt is the

major Ca2þ sensor for exocytosis and that the residual release is driven by a

sensor with distinct properties.

Reports of recordings from syt alleles sometimes noted that the time course of

release was altered, with more release occurring slower or asynchronously than in

wild type (Loewen et al., 2001; Yoshihara and Littleton, 2002). This has given rise

to speculation that the terminals contain a fast sensor (Syt) and a slow sensor

(unknown) governing the residual release. However, a loss of synchrony in

transmitter release can be explained by changes in docking, priming, and fusion,

in addition to changes in the Ca2þ sensor, and others have observed low levels of

fast synchronous release to persist in these mutants (Okamoto et al., 2005).

Some of the most compelling evidence that Synaptotagmin is the major

Ca2þ-sensor for triggering fusion has come from studies of the Ca2þ-binding sitesin the C2 domain (Mackler et al., 2002; Okamoto et al., 2005). If Synaptotagmin


is the crucial Ca2þ sensor, these studies reasoned, mutation of the aspartate

residues that form the Ca2þ-binding site should cause drastic and measurable

changes in the Ca2þ-dependent properties of transmission. The Ca2þ-bindingsite in the C2 domain had been described in detail from structural studies, and

the significance of each aspartate for the Ca2þ-dependent properties of Synapto-tagmin had been defined for mammalian Synaptotagmin I (Li et al., 1995; Rizo

and Sudhof, 1998; Shao et al., 1996, 1998; Zhang et al., 1998b). These mamma-

lian studies implicated the first of the two C2 domains (C2A) as the more

important. However, when syt null Drosophila mutants were supplied with syt

transgenes carrying mutations that completely disrupted this site (and therewith

the ability of the C2A domain to undergo Ca2þ-dependent binding to Syntaxin

or phospholipids) synaptic transmission was restored in a manner completely

comparable to rescue by a wild-type transgene. Even the slope of the Ca2þ/response relationship was comparable to that of wild type, indicating that Ca2þ

binding by the C2A domain cannot be required for or have a significant influence

on the release of transmitter (Robinson et al., 2002). Mutations in the C2B

domain resulted in dramatically diVerent consequences (Mackler et al., 2002).

Neutralizing the equivalent aspartates in this second C2 domain were suYcient to

render the protein inactive in rescue experiments, and expression of this trans-

gene had a dominant-negative eVect by suppressing synaptic transmission. Simi-

lar dramatic eVect was observed in syt alleles that either remove the C2B domain

or carry a point mutation near the C2B Ca2þ-binding domain (DiAntonio and

Schwarz, 1994; Okamoto et al., 2005; Yoshihara and Littleton, 2002).

Together, the evidence from studies of Ca2þ-dependency in the syt null

and the eYcacy of alterations in the Ca2þ-coordinating aspartates of the C2B

domain, build a strong case for Synaptotagmin being the crucial sensor for

triggering exocytosis. It remains to be determined why some release persists

and also why the neutralization of charges in the C2A domain (highly conserved

through evolution and partaking in many Ca2þ-dependent interactions) appearsto be of so little consequence to synaptic physiology.

C. OTHER FUNCTIONS OF SYNAPTOTAGMIN

The significance of Synaptotagmin for the Ca2þ-dependence of transmitter

release does not preclude additional functions of the protein. Electron micrographic

studies of central synapses as well as the NMJ (Loewen et al., 2006; Reist

et al., 1998) indicate that syt mutant terminals have fewer vesicles, although those

vesicles present continue to cluster in the vicinity of the active zone. This

phenotype suggests a defect in either the biogenesis of synaptic vesicles or in

their retrieval from the membrane after fusion. A similar paucity of vesicles at

the terminals of C. elegans mutants led to the hypothesis that Synaptotagmin


regulates endocytosis (Jorgensen et al., 1995). Such a function is likely to be

mediated by the ability of Synaptotagmin to bind the clathrin adaptor protein

AP-2 and thereby recruit clathrin to vesicles (Zhang et al., 1994). Quantitative

measurements of endocytotic rates have been undertaken in sytmutants by means

of a GFP-based reporter of vesicle fusion (Poskanzer et al., 2003). Because endocy-

tosis is coupled to exocytosis, it had not previously been possible to distinguish any

direct eVect of Synaptotagmin on the former from its clear eVects on the latter.

Particularly, by using FlAsH-FALI (see in an earlier section) to inactivate Synap-

totagmin after a round of exocytosis, it was possible to measure these rates

independently. Loss of Syt slows the rate of endocytosis and particularly limits

the ability of the synapse to increase its rate of endocytosis in response to increased

exocytosis (Poskanzer et al., 2003).

The interactions of Synaptotagmin with phosopholipid membranes and with

the proteins of the plasma membrane also led to the hypothesis that Synapto-

tagmin may promote the docking of vesicles at active zones, akin perhaps to the

function of C2 domains in various enzymes. Electron microscopy of syt mutants

revealed a selective decrease in the pool of vesicles immediately adjacent to the

active zone (Reist et al., 1998). Loss of the pool that presumably correspond to

vesicles primed and ready for fusion may be a major contributor to the syt

phenotype. Mutations of the C2B domain that retain the rest of the protein do

not cause these ultrastructural changes (Loewen et al., 2006). This may hold a

significant clue for understanding the function of the rest of the molecule.

D. MULTIPLE SYNAPTOTAGMINS IN THE FLY

The Drosophila genome predicts six additional isoforms of Synaptotagmin,

but there is little functional data at present concerning any of them (Adolfsen

and Littleton, 2001). Syt4 is most homologous to mammalian Synaptotagmins

4 and 11 and, like Syt1, is abundant in the nervous system where it can be

found enriched at synapses. At the NMJ it is present in muscle cells, appearing

in puncta near the synapse (Adolfsen et al., 2004), but it can also be found

in neurosecretory endings, including peptidergic terminals at the NMJ (Bohm

and Schwarz, unpublished data). Within the CNS it is not known if Syt4 is

predominantly pre- or postsynaptic. Syt� and Syt� (with no immediate relative

in mammals but closest to Synaptotagmin 12) are also reported to be concen-

trated in some neurosecretory endings (Adolfsen et al., 2004), whereas Syt7,

homolog to mammalian Synaptotagmin 7, is found in many tissues including

nerve and muscle, but has not been observed to be concentrated at synapses.

The remaining genes, syt12 and syt14 are the least understood, although low

levels of Syt14 have been observed by in situ hybridization in the embryonic CNS

(Adolfsen et al., 2004).


Of these additional Synaptotagmins, only Syt4 has been analyzed genetically.

Null alleles [from the excision of a P-element (Adolfsen et al., 2004), or made

by homologous recombination (Bohm and Schwarz, unpublished data)] are

homozygous viable, fertile, and overtly normal. In NMJ morphology and EJP

amplitude, the third instar syt4 mutants are similarly normal. The analysis of null

alleles does not support earlier suggestions that syt4 could account for residual

transmitter release in syt1 null larvae. My laboratory had reported that syt4

transgenes could rescue synaptic transmission in syt1 mutants, but this was in

error (Robinson et al., 2002), as was an earlier report on overexpression of syt4

that concluded that syt4 regulates transmitter release by interfering with syt1

function (Littleton et al., 1999). However, at the NMJ of third instar larvae,

postsynaptic Syt4 translocates to the plasma membrane in response to activity

and this may represent the release of an unidentified retrograde signal (Yoshihara

et al., 2005). Also, in embryonic synapses at hatching stages, an electrophysio-

logical phenotype has been reported: whereas in wild-type embryos high-

frequency trains of stimuli result in an increase in minifrequency, this was not

observed in the syt4 nulls. The eVect on minis could be restored by the rescue of

syt4 expression in the muscle, consistent with a model in which muscle activity

releases a retrograde messenger that modulates the release of minis from the

presynaptic terminal (Yoshihara et al., 2005).

E. SUMMARY OF SYNAPTOTAGMIN FUNCTION AT THE FLY NMJ

The analysis of syt1 mutations indicates an important role for Synaptotagmin

in promoting the fusion of vesicles in response to an action potential. Because syx1

and n-syb mutants are also required for the EJP, Syt and the SNAREs are part of

the same release pathway. The function of Syt is likely to be closely tied to its

ability to bind Ca2þ ions. The Ca2þ-binding site of the C2A domain, however,

appears rather insignificant, whereas the Ca2þ-binding site in the C2B domain

has an essential role. The residual release of vesicles in response to an action

potential or elevated cytosolic Ca2þ remains a puzzle. If it is due to the presence

of a redundant synaptotagmin gene, the gene has not yet been identified. In

addition to a role in promoting fusion, Syt is likely to promote vesicle recycling

and vesicle docking.

IX. Exocyst at the NMJ

The SNARE complex is not the only protein complex that is implicated

in membrane traYc. The genetic screens for secretory defects in yeast that

uncovered mutations in SNARE proteins also uncovered a complex that is


frequently referred to as the exocyst (EauClaire and Guo, 2003; Lipschutz and

Mostov, 2002). The eight components of this complex are Sec3, Sec5, Sec6, Sec8,

Sec10, Sec15, Exo70, and Exo84 and their phenotypes in yeast include blocking

a late stage in vesicle transport to the plasma membrane. In budding yeast,

vesicles are transported into the growing bud but fail to fuse at their target sites

(Novick et al., 1980). One of the most intriguing aspects of the exocyst is that it

localizes to the site of vesicle fusion, that is, at the bud tip when daughter cells are

growing and at the bud neck when membrane is being added for cytokinesis

(Finger and Novick, 1997; Finger et al., 1998). In contrast, the SNARE proteins

are uniformly distributed within the plasma membranes of yeast. The exocyst

might therefore be responsible for docking vesicles at target membranes.

With so many other mechanistic parallels between membrane traYc in yeast

and at the synapse (Bennett and Scheller, 1993), the question naturally arose as to

whether the exocyst plays a similar role in the nervous system, particularly with

regard to targeting synaptic vesicles to active zones. The exocyst complex was

purified from mammalian brain (Hsu et al., 1996) and antibody studies suggested

an important function in membrane traYc in epithelial cells (Hazuka et al., 1999).

A murine mutation of sec8 was discovered (Friedrich et al., 1997), but it caused

lethality at a very early stage of development and therefore was unsuitable for

more detailed studies of membrane traYc.

The analysis of exocyst mutations and neuronal function therefore

commenced with the identification of sec5 mutations in Drosophila (Murthy

et al., 2003). As previously discussed regarding syx mutations, the analysis is

complicated by the fact that members of this complex have vital functions and

cell-lethal phenotypes. sec5 null alleles are cell lethal when clones are made in the

eye or female germ line (Murthy and Schwarz, 2004; Murthy et al., 2003).

However, as in the case of syx, maternally contributed sec5 mRNA is suYcient

to permit the embryo to develop and, in the case of sec5, larvae hatch out and can

survive for up to 3 days. These larvae, however, do not achieve their expected

size but rather remain at the border of first and second instars. This can be

attributed to the need for sec5 in constitutive membrane addition. Although there

is suYcient maternal Sec5 in homozygous null embryos to support normal

development, by hatching it has fallen to 29% of wild-type levels and by 48 h after

egg laying (AEL) to 11%, at which point no further growth is observed. By 72 h

AEL, Sec5 levels are barely detectable, no more than 3% of wild type. At the

NMJ this is manifest in the arrest of muscle growth and a similar arrest in the

development of the synapse. At 96 h, motoneurons on muscles 6/7 have bouton

counts appropriate for 48 h larvae (Murthy et al., 2003). Therefore, by studying

neurons from larvae that were 72 h AEL or older, the significance of Sec5 could be

assessed in the almost complete absence of the protein. It was found that these

neurons had severe defects in membrane traYc. If neurons were dissociated from

larval brains they were incapable of re-extending an axon. If a reporter gene was


turned on at this stage, it could be synthesized but was not eYciently traYcked to

the cell surface (Murthy et al., 2003). These defects not withstanding, the synapse

at the NMJ continued to function and the EJP was slightly larger at 96 h than it

had been at 48 h, perhaps reflecting the limited formation of new release sites.

Although the amplitude of the EJP was well below that of a normal 96 h larva,

presumably due to the small size of the NMJ, release per bouton was quite normal.

Thus, Sec5 is essential in many membrane traYcking steps, including those that

are responsible for the growth of the NMJ and formation of new boutons.

However, it is dispensable for the actual targeting and fusion of synaptic vesicles

once a bouton has formed.

Mutations have been reported in the exocyst components sec6 (Beronja et al.,

2005; Murthy et al., 2005) and sec15 (Mehta et al., 2005). Although these studies

have not closely examined synaptic transmission or the NMJ per se, the mutant

phenotypes reported are consistent with the conclusion that the exocyst is not

needed for the release of neurotransmitter. Synaptic phenotypes at the NMJ have

been examined for mutations in sec8 (Liebl et al., 2005) and through the use of RNAi

to reduce levels of sec10 (Andrews et al., 2002). In each case the release of

transmitter was unaVected. The significance of the exocyst or of individual compo-

nents of it for synaptic development, however, remains an area of great interest

because it has the potential not only to permit but perhaps also to direct the addition

of new boutons presynaptically and the expression of receptors postsynaptically.

To a large extent, synaptic transmission can be thought of as a variation on

exocytosis in all cells, including yeast, but with additional regulatory components,

such as Synaptotagmin, added on. Sec5 and the exocyst as a whole, however, are

a counter example. The exocyst is a protein complex that is essential in yeast and

in the constitutive traYcking pathway for many, perhaps all, membrane proteins,

and yet it is dispensable for transmitter release. One possible explanation is that

the specialized architecture of the active zone provides a unique mechanism for

the docking of synaptic vesicles at release sites. If so, this mechanism may

substitute for the normal role of the exocyst (Murthy et al., 2003).

X. Other Mutations of Proteins on the Target Membrane

A. ROP/UNC-18/N-SEC1

As discussed earlier, neither mutations in SNARE-coding genes nor in genes

encoding components of the exocyst have phenotypes that are adequate to

explain how synaptic vesicles are preferentially clustered near and docked at

the active zone. From studies of mammalian synapses, there are several major

proteins that may contribute to these events, but which presently have received


less attention in Drosophila. One of these is a Syntaxin-binding protein called Sec1

in yeast and n-Sec1 or Unc-13 in mammals and C. elegans, respectively. Mutations

in the Drosophila homolog gene were recovered fortuitously in a screen for ras

mutants and were given the name of rop (Harrison et al., 1994). The phenotype of

rop mutants, including developmental abnormalities in embryos, strongly impli-

cated rop as necessary for both constitutive and synaptic exocytosis, which was

subsequently confirmed by the analysis of point mutations (Wu et al., 1998).

Overexpression of Rop, however, inhibited transmitter release (Schulze et al.,

1994). These findings have led to conflicting interpretations, according to which

Rop is either an inhibitor of transmission or a necessary promoter of vesicle

release. The enhancement of synaptic transmission by mutations in the Rop-

binding region of Syt appeared to favor a model in which this protein restricts the

availability of Syt for SNARE complex formation (Wu et al., 1999). This has

proven controversial due to the diYculty of determining in vivo the extent to

which protein interactions have actually been prevented (Matos et al., 2000; Wu

et al., 2001). From mammalian studies, it is clear that the n-Sec1/Unc-18 protein

is not bound to Syntaxin when Syntaxin is complexed with the other SNARE

proteins. It is therefore most attractive, at present, to envision Rop as a protein

that cycles on and oV Syntaxin, priming it for activity in transmission, but then

releasing it so that Syntaxin could bind to the other SNAREs. Such a cyclical role

could explain both its requirement for secretion and the ability of overexpressed

Rop to interfere with transmitter release.

B. UNC-13 AND VESICLE PRIMING

Unc-13, a protein that has been extensively studied in both C. elegans and the

mouse (Rhee et al., 2002; Richmond et al., 1999), may also regulate the release

apparatus. The prevailing model, from studies in those organisms, is that Unc-13

can regulate the state of Syntaxin and thereby influence its ability to form

SNARE complexes (Richmond et al., 2001). Unc-13 appears to be a crucial focus

for modulating the strength of synaptic transmission in response to changes

in second messengers, particularly diacylglycerol (Rhee et al., 2002). Drosophila

lacking unc-13 have a severe phenotype: a complete loss of both the embryonic

EJP and minis (Aravamudan et al., 1999) and an accumulation of docked vesicles.

In addition, the levels of Unc-13 may be modulated by second messenger systems

(Aravamudan and Broadie, 2003).

C. CAST/ERK AT THE ACTIVE ZONE

The monoclonal antibody nc82 has been a favorite for the analysis of

synaptic structures due to its remarkable specificity for active zones. The gene


encoding the nc82 epitope has been cloned and shown to encode the fly homolog

of ELKS/CAST (Wagh et al., 2006), a protein whose localization to active zones

has also been established in vertebrates (Ohtsuka et al., 2002; Wang et al., 2002).

In Drosophila, the locus was given the name bruchpilot (brp). Although the function

of ELKS/CAST is not known, it binds to a protein called RIM-1 that in turn

binds to Unc-13 (see in an earlier section). Although no mutation in the fly

homolog has yet been reported, protein levels were knocked down by means of

RNAi expression (Wagh et al., 2006). Reducing nc82 immunoreactivity produced

a reduction in the amplitude of the EJP, although minis persisted normally.

A striking ultrastructural phenotype confirmed the importance of the protein in

organizing the active zone. In Drosophila, many although not all active zones

possess an electron dense structure, sometimes called a T-bar. This structure may

be akin to the ribbons and similar structures seen at some mammalian synapses.

RNAi to Drosophila CAST/ELKS prevented the formation of these T-bars.

XI. Mutations in Peripheral Synaptic Vesicle Proteins

A. SYNAPSIN

The Synapsin family was one of the first major synaptic proteins to

be identified and it associates with the cytoplasmic surface of synaptic vesicles.

It was recognized early to be the subject of phosphorylation by both cAMP- and

Ca2þ-dependent protein kinases (Greengard et al., 1993). Consequently, there has

been much hope and speculation that it might be a crucial control point for such

synaptic phenomena as modulation by amine transmitters and paired-pulse

facilitation. By and large, these expectations have not been met by the pheno-

types of knockout mice (Rosahl et al., 1993, 1995), although the presence of three

partially redundant genes in the mouse makes this analysis more complicated.

Murine data suggest a role for the Synapsins in regulating the availability of

reserve pool vesicles during prolonged neuronal activity (Chi et al., 2001; Sun

et al., 2006). In Drosophila, the presence of a single synapsin (syn) gene, albeit

an alternatively spliced gene, has assisted its genetic analysis (Godenschwege

et al., 2004). The syn mutant phenotype, however, remains astonishingly mild—

undetectable at the level of cellular analysis at the NMJ. No alteration was

detected in the ultrastructure of these synapses, including the density of synaptic

vesicles in the vicinity of active zones. Thus, no indication was found that

Synapsins were essential for coupling vesicles to the local cytoskeleton. In addi-

tion, the properties of the EJP and minis were unchanged from control.

Even with 2 s of 5 Hz stimulation, no diVerences were observed, with a modest


degree of synaptic depression in both mutant and control. However, the flies have

distinct behavioral abnormalities, including learning defects, in both adult

and larval stages (Godenschwege et al., 2004; Michels et al., 2005). Thus, the

importance of Synapsin to the functioning of the nervous system was confirmed,

but the specific cell biological function of this vesicle protein has not been

identified at fly synapses. More detailed physiological tests may eventually reveal

a subtle electrophysiological phenotype.

B. CYSTEINE STRING PROTEIN AND HSC70

The cysteine string protein (CSP) is another protein associated with the

cytoplasmic surface of synaptic vesicles (Chamberlain and Burgoyne, 2000).

It has homology to DNAJ/Hsc40 proteins and through its J domain can interact

with the chaperone Hsc70. Therefore, studies have focused on a likely role as a

chaperone protein that can assist in protein folding and refolding (Braun and

Scheller, 1995; Braun et al., 1996). Consistent with such a model, Hsc70 mutants

have also been shown to have a phenotype at the fly NMJ. The significance of

such a protein on synaptic vesicles remains unclear. It may involve interactions

with Syntaxin and Synaptotagmin (Bronk et al., 2001, 2005).

The phenotype of Csp mutants in Drosophila has been crucial to determining

its physiological significance. CSP cannot be central to the process of transmitter

release and exocytosis—transmitter release persists in null mutants (Zinsmaier

et al., 1994). Nevertheless, synaptic function is not normal. Particularly at elevated

temperatures synaptic function is impaired causing smaller evoked responses

(Dawson-Scully et al., 2000; Ranjan et al., 1998; Umbach et al., 1994).

This reduction does not appear to be due to a decrease in Ca2þ-channel activ-ity. The synaptic defects are accompanied by a progressive degeneration of

neurons and eventual paralysis and death. The importance of the interaction

with Hsc70 was confirmed by the isolation of mutations in that gene and the

finding that they give rise to phenotypes very similar to those of Csp (Bronk et al.,

2001), although some eVects of Csp may be independent of Hsc70 (Arnold et al.,

2004; Bronk et al., 2005). The prevailing model, at present, is that repeated

cycling of synaptic vesicles, particularly at elevated temperatures, results in the

production of denatured proteins. It is not yet known who the critically vulnera-

ble proteins are. CSP on the vesicles, working with Hsc70 and perhaps, an

additional associated protein (Tobaben et al., 2001), can restore proper

folding of these proteins and prevent the ensuing degeneration and loss of

synaptic strength (Fernandez-Chacon et al., 2004). In this regard, the neurode-

generative phenotype of CSP may become an interesting model for the study of

degenerative disorders.


XII. Summary

Nearly two decades of work on the release of transmitter at the Drosophila

NMJ has produced a sizeable collection of mutations and a great deal of

phenotypic analysis. Mutations have been generated in all the central compo-

nents of the release machinery and in many additional regulatory and peripheral

components of the nerve terminal. It is interesting that mutant screens in

Drosophila have not yet identified essential elements of the exocytosis apparatus

that were not previously identified by biochemical means. However, by bringing

electrophysiological and ultrastructural analysis in vivo to the study of synaptic

components, the system has made substantial contributions to our understanding

of synaptic function in all organisms.

The task of elucidating synaptic mechanisms is not over. One of the most

significant unanswered questions centers on the active zone. The prominent

electron density of this structure suggests numerous highly specialized compo-

nents, but few have as yet been identified or subject to genetic analysis. We still

do not understand why synaptic vesicles accumulate in terminals and cluster near

active zones. Nor do we understand how synaptic vesicles are selectively targeted

to these sites rather than elsewhere on the cell surface, while other cargo-carrying

vesicles do not fuse at the active zone. Similarly, the mechanistic relationship of

minis to EJPs remains unclear. Perhaps the most important question for synaptic

function entails understanding the diVerences between synapses. Why does one

synapse has a high probability of release while another has a low probability?

How is the release of peptidergic granules for modulatory and neuroendocrine

function distinct from the release of small clear vesicles? How are synapses

specialized for tonic or phasic release of transmitter? It is likely that the Drosophila

NMJ will continue to figure prominently in the exploration of these issues.

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transmitter release at the neuromuscular junction · mitter release indirectly, that is, mutations...

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