[International Review of Neurobiology] The Fly Neuromuscular Junction: Structure and Function Second Edition Volume 75 || Scaffolding Proteins at the Drosophila Neuromuscular Junction

SCAFFOLDING PROTEINS AT THE DROSOPHILANEUROMUSCULAR JUNCTION

Bulent Ataman,* Vivian Budnik,* and Ulrich Thomasy

*Department of Neurobiology, University of MassachusettsMedical School, Worcester, Massachusetts 01605, USA

yDepartment of Neurochemistry, Leibniz Institute for Neurobiology39118 Magdeburg, Germany

INTE

NEUR

DOI:

. I

R

1

ntroduction

NATIONAL REVIEW OF 181OBIOLOGY, VOL. 75

Copyright 2006, Elsev

All rights re

0.1016/S0074-7742(06)75009-7 0074-7742/06

I

II.
S tructure of the Drosophila Larval Neuromuscular Junction
III.
M ultidomain Organization of ScaVolding Proteins
IV.
D lg-Based ScaVold at the NMJ
A. D
iscs-Large
B. S
ynaptic Localization and Targeting of Dlg
C. S
tructure and Physiology of Dlg-Mutant NMJs
D. D
lg-Interacting Partners at the NMJ
V.
D ystrophin
VI.
d GRIP
VII.
d X11/dMint/dLin-10
VIII.
D liprin-�
A. D
liprin Regulates Active Zone Morphology and Synaptic Physiology
B. D
liprin-� Interacts with Drosophila Leukocyte Antigen-Related Receptor Tyrosine
Phosphatases (DLAR) to Control Synaptic Development

C. D
liprin-� as a Target for Regulated Degradation by the APC/C Complex
D. N
ot Just Cargo: Dliprin-� Promotes Proper Trafficking of Synaptic Vesicles
IX.
B ruchpilot: A Crash Pilot Targets the Active Zone
X.
B azooka (Par-3)/Par-6/aPKC
XI.
M issing Prominents: Homer/Vesl, Shank/ProSAP, and GKAP/SAPAP
XII.
P erspectives
R
eferences
The eYcacy of synaptic transmission and its regulation during plasticity rely on a

complex set of proteins that give rise to crucial structures required for synaptic

function, such as the active zone and the postsynaptic density. This organization is

in part due to the existence of a synaptic scaVold that anchors synaptic membrane

proteins, physically links functionally related proteinswithinmultiprotein complexes,

transports protein complexes to appropriate synaptic sites, and insulates individual

synapses. In general terms, scaVolding proteins are composed of multiple protein-

binding domains, of which postsynaptic density-95/discs large/zone occludens-1

(PDZ) domains are a common occurrence. This chapter will focus on the genetic

ier Inc.

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182 ATAMAN et al.

approaches that have been used at theDrosophila neuromuscular junction to elucidate

the in vivo significance of scaVolding proteins for synaptic structure and function.

I. Introduction

The eYcacy of synaptic signaling and its plasticity-related tuning rely on a

complex and compact arrangement of proteins, which is reflected by the distinct

appearance of presynaptic active zones (AZs) and postsynaptic densities (PSDs), two

electron-dense specializations that are linked across the synaptic cleft (Dresbach et al.,

2001; ZiV, 1997). The benefits of such molecular compactness can easily be illu-

strated. AZs, for instance, constitute specialized sites for neurotransmitter release

where synaptic vesicles become docked and primed prior to Ca2þ-dependent fusionwith the presynaptic membrane. The colocalization of voltage-gated calcium chan-

nels within AZs therefore allows an almost instantaneous release of neurotransmitter

on arrival of an action potential. Postsynaptically, neurotransmitter receptors are

associated into high-density clusters precisely apposed to AZs. This organization

allows a rapid response by the postsynaptic cell to neurotransmitter release by the

presynaptic cell. The principal constituents of the neurotransmission machinery are

complemented by a multitude of proteins, which modulate synaptic structure and

function in a short- or long-term fashion (Li et al., 2004; Peng et al., 2004), suggesting

that a sophisticated, yet well-defined mode of assembly underlies the formation of

synaptic junctions. This concept has been supported by the identification and

characterization of multidomain proteins that form scaVolds to which other proteinscan bind concurrently in a highly ordered manner. At synapses, scaVolding mole-

cules are believed to exert the following functions: (1) anchorage of various synaptic

membrane proteins by providing an interface in between synaptic membranes

and their underlying cytoskeleton, (2) optimization and spatial restriction of signaling

events by physical linkage of functionally related proteins within multiprotein

complexes, (3) transport of cytosolic or vesicle-associated protein complexes to

appropriate synaptic sites through coupling to molecular motors, and (4) insulation

of individual synapses by shaping specialized perisynaptic zones which limit the

expansion of AZs and PSDs.

The distinct ultrastructure of synaptic junctions contrasts with their well-

documented plasticity, that is, their ability to undergo considerable modifications

within short periods of time. ScaVolding molecules may be assumed to be first-

order targets of plasticity-related signaling events, which lead to rearrangements

in the molecular composition of synapses. In fact, prominent scaVolding mole-

cules are among proteins that exhibit the highest turnover rates on long-term

potentiation (LTP) in mammalian neurons (Ehlers, 2003). Hence, a comprehen-

sive characterization of synaptic and perisynaptic scaVolds will be indispensable

SCAFFOLDING PROTEINS AT THE DROSOPHILA NMJ 183

to understand the mechanisms and regulatory events associated with dynamic

changes in synaptic structure and function.

In vitro studies and cell culture assays on synaptic scaVolding proteins in mam-

mals have contributed enormously to our concept on the role of these molecules. To

date, however, corresponding genetic analyses in mice are still rare. In this chapter,

wewill focus on the genetic approaches that have been used at the fly neuromuscular

junction (NMJ) to unravel the role of synaptic scaVolds. These studies have also led tothe identification of some intriguing regulatory mechanisms of synapse plasticity.

II. Structure of the Drosophila Larval Neuromuscular Junction

As described in Chapter 4 by Prokop, the Drosophila larval NMJ is character-

ized by branched synaptic terminals containing varicosities, also known as syn-

aptic boutons. Each muscle of the body wall is innervated by a characteristic

complement of motoneurons, and each class of motoneurons gives rise to term-

inals with diVerent structural properties. All muscle cells are innervated by type-I

boutons, which can be subdivided into type-Ib (b, big) and type-Is (s, small)

boutons (Gramates and Budnik, 1999). These boutons release glutamate, the

primary excitatory transmitter at the larval NMJ. Additionally, subsets of muscles

are innervated by peptidergic and/or octopaminergic motoneurons, which are

thought to modulate the glutamatergic response. Although each muscle of the

body wall can be innervated by multiple motoneurons, each motorneuron-

specific synapse is apposed by a characteristic postsynaptic structure (Atwood

et al., 1993; Jia et al., 1993). Therefore, muscle cells can respond in a spatially

segregated manner to the interaction with a specific presynaptic cell. In this

chapter, we will primarily focus on scaVolding proteins functioning at type-I

terminals, the best-studied terminals at the NMJ.

A unique feature of type-I boutons is the presence of a complex postsynaptic

membrane compartment, the so-called subsynaptic reticulum (SSR). The com-

plex organization of the SSR develops gradually during larval development,

beginning as a single postsynaptic membrane and increasing exponentially in

surface area by forming convoluted invaginations of the muscle plasma mem-

brane that surround the presynaptic boutons and that form many membrane

layers (Guan et al., 1996). This increase in SSR surface is correlated to the

increase in muscle volume. Fields of glutamate receptors (GluRs) are embedded

in the membrane layer directly apposed to the presynaptic membrane, but other

molecules, including Shaker-type Kþ channels (Sh), the cell adhesion molecule

FasciclinII (FasII), the actin-binding protein Spectrin, and the scaVolding pro-

teins Discs-large (Dlg), Scribble (Scrib), Bazooka (Baz), and Lin-7 are localized

throughout most if not all layers of the SSR (Chapter 4 by Prokop). The exact

184 ATAMAN et al.

physiological significance of the SSR is poorly understood, but several functions

have been proposed, including glutamate uptake (Faeder and Salpeter, 1970),

maintaining a high concentration of molecules important for synaptic develop-

ment and function, and rapid transport of molecules which are required to

maintain synaptic eYcacy as the muscles and synaptic boutons grow in size

during a short period of time. There is also evidence that the SSR may be the

site for local translation of GluRs (Sigrist et al., 2000) (Chapter 14 by Schuster).

III. Multidomain Organization of Scaffolding Proteins

ScaVolding molecules are commonly found in association with the cytoplas-

mic face of membrane specializations such as epithelial or synaptic junctions.

They can be assorted to a number of evolutionary distinct protein families.

Principally, however, they are all composed of two or more protein–protein

interaction modules. Certain types of domains contribute to the basic modular

organization of most scaVolding proteins. Most notably, PDZ domains have

emerged as a trade mark for scaVolding proteins. In a socket-like manner they

usually bind to defined C-terminal sequence motifs of their ligand proteins.

Subclasses of PDZ domains are distinguished according to diVerences in the

consensus-binding sequence of their ligands (Harris and Lim, 2001). A crucial

feature of most scaVolding proteins is their ability to form homo- or heteromulti-

meric complexes. One type of complex is formed by homotypic interactions

between PDZ domains (Brenman et al., 1996; Im et al., 2003a,b). However, most

PDZ domains contribute to the formation of multimeric complexes via the

oligomerization of their ligands. Other modules, such as coiled-coil regions or

the L27-type domains, also promote multimerization directly through homotypic

interactions (Marfatia et al., 2000; Nakagawa et al., 2004).

A diagram of the scaVolding proteins known to be localized at larval NMJs

is shown in Fig. 1A. Certainly, additional components of the synaptic and

perisynaptic scaVolds are yet to be identified.

IV. Dlg-Based Scaffold at the NMJ

A. DISCS-LARGE

Much of our knowledge about synaptic and perisynaptic scaVolds at the

Drosophila NMJ has been derived from studies centered on the various isoforms

of the protein Dlg (Fig. 1A). Initially described as a tumor suppressor which

localizes at septate junctions of epithelial tissues, Dlg has also been identified as a

FIG. 1. (A) Diagram showing the domain organization of scaVolding proteins located at the NMJ. (B) Model of L27-mediated multimerization

at the synapses. (C) Protein interactions in the Par3/Par6/Baz complex.

185

186 ATAMAN et al.

major scaVolding component at larval NMJs, especially abundant at the post-

synapse (Lahey et al., 1994). Strong Dlg expression is also evident in the neuropil

regions of the central nervous system (CNS) throughout embryonic and larval

development and in the adult fly, suggesting a role for Dlg at synapses in the

brain. Deciphering the role of Dlg at glutamatergic NMJs is of general interest as

the Dlg-like proteins PSD-95, SAP97, PSD-93, and SAP102 have emerged as

core components of glutamatergic synapses in the mammalian brain (Kim and

Sheng, 2004). Dlg and its homologues represent a subfamily of membrane-

associated guanylate kinases (MAGUKs), defined by a common modular struc-

ture consisting of three type-I PDZ domains followed by an SH3-like and a

C-terminal guanylate kinase-like domain (Funke et al., 2005). Aside from these

canonical domains two other conserved segments of Dlg should be noted: the

so-called HOOK region, in between the SH3 and GUK domains, and an

amino terminal region harboring an L27 domain, which is present in Dlg-S97

and absent in Dlg-A isoforms (Mendoza et al., 2003). The presence of diVerentDlg isoforms, such as Dlg-S97 and Dlg-A, is based on the diVerential usage of

alternative transcriptional start sites. Additional diversification of both S97 and

A-type Dlg isoforms due to alternative splicing is evident from Western blot and

cDNA analyses, but their relevance is still unknown (Mendoza et al., 2003). Dlg-

S97 appears to be the predominant form in the CNS, whereas most if not

all epithelia express Dlg-A exclusively. At NMJs, both Dlg-A and Dlg-S97 are

present (Mendoza et al., 2003; Urra, F., Barria, R., Thomas, U., Kobler, O.,

Budnik, V., Delgado, R., and Sierralta, J., in preparation) (Fig. 2), a situation

reminiscent to the simultaneous synaptic expression of �- and �-isoforms of the

mammalian MAGUKs PSD-95 and SAP97 (Chetkovich et al., 2002). The L27

domain of Dlg-S97 mediates specific interactions (see later), implying that Dlg-

S97- and Dlg-A-based complexes are diVerent and may be regulated separately

to at least some extent.

B. SYNAPTIC LOCALIZATION AND TARGETING OF DLG

At larval NMJs, Dlg is localized to glutamatergic type-I synaptic terminals,

being more prominent at type-Ib than type-Is boutons (Lahey et al., 1994). Strong

immunofluorescence surrounding the presynaptic terminals indicates that Dlg is

associated with the postsynaptic membranes formed by the SSR, which is more

extended in type-Ib boutons than in type-Is (Chapter 4 by Prokop). Immuno-

electron microscopy, however, revealed that Dlg is also present at the presyn-

aptic membrane (Lahey et al., 1994). In fact, Dlg is detectable at immature

presynaptic terminals of developing late embryonic NMJs before postsynaptic

recruitment becomes apparent (Guan et al., 1996; Thomas et al., 2000). While

the onset and maintenance of Dlg expression in muscles are independent from

FIG. 2. Coexistence of Dlg-S97 and DlgA isoforms at the NMJ. Panels show third instar NMJs

from (A, B) wild type and (C, D) larvae expressing Dlg-S97-RNAi postsynaptically via the C57-Gal4

(A, C) anti-S97 (green) and (B, D) anti-PDZ1–2 (red). (Images from Urra, F., Barria, R., Thomas, U.,

Kobler, O., Budnik, V., Delgado, R., and Sierralta, J., in preparation.)


innervation, its local clustering at synapses is clearly directed by the physical

contact with the motor nerve terminals (Chen and Featherstone, 2005). Notably,

the initial clustering of Dlg can occur in the absence of functional GluRs (Chen

and Featherstone, 2005). The subsequent enrichment of postsynaptic Dlg immu-

noreactivity parallels the growth of the SSR throughout larval development

(Guan et al., 1996). During this process Dlg remains largely excluded from the

glutamate receptor fields (Sone et al., 2000).

Early on in postembryonic development, considerable Dlg-immunoreactivity

is observed at extrasynaptic sites, including a subcortical membrane compart-

ment and evenly distributed microdomains right underneath the muscle surface

(Thomas et al., 2000). The more Dlg becomes concentrated at the maturing

NMJs, the more it appears to decrease extrasynaptically. These observations

suggest that postsynaptic targeting of Dlg involves at least two steps, that is,

the temporal association with the muscle membrane and/or subcortical com-

partment and the subsequent recruitment to the SSR (Thomas et al., 2000).

188 ATAMAN et al.

This hypothesis is supported by the finding that deletions of certain domains of

Dlg-A interfere with individual targeting steps. Deletion of the HOOK region

prevents association with the subcortical membrane compartment and results in

ineYcient synaptic targeting. Deleting the first two PDZ domains results in

striking extrasynaptic membrane accumulations again accompanied by poor

synaptic localization. Thus, at least one of these PDZ domains has to be present

to ensure recruitment of Dlg from extrasynaptic plasma membrane regions to the

NMJ. Proper traYcking of Dlg-A lacking the GUK domain was found to depend

on the presence of endogenous Dlg, suggesting that the truncated protein may

travel as a ‘‘hitchhiker’’ together with intact Dlg molecules. A role for the GUK

domain in traYcking Dlg is consistent with the identification of various

microtubule-associated proteins that bind the GUK domain of mammalian Dlg-

like MAGUKs (Asaba et al., 2003; Brenman et al., 1998).

Proper anchorage of Dlg at NMJs appears to depend on an intact Actin–

Spectrin network. Both �- and �-Spectrin are present at NMJs (Featherstone

et al., 2001), and as described in Chapter 11 by GriYth and Budnik, the SSR is

particularly enriched in Spectrin (Ruiz-Canada et al., 2004). Loss of �- or

�-Spectrin, which causes late embryonic to early first instar larval lethality, aVectspostsynaptic recruitment of Dlg to the developing NMJ (Featherstone et al.,

2001). However, a direct interaction between Dlg and Spectrin has not been

demonstrated. A pivotal role of the actin cytoskeleton in organizing the SSR has

also been deduced from mutational analysis of dPix, a synaptic Rho-type GDP/

GTP exchange factor (Parnas et al., 2001). Dlg expression is reduced at dpix-

mutant NMJs. However, it is not clear whether this eVect simply reflects the

severe reduction of the SSR or whether conversely loss of dPix primarily causes a

reduction in postsynaptic Dlg leading to defects in SSR formation.

C. STRUCTURE AND PHYSIOLOGY OF DLG-MUTANT NMJS

Maternally supplied Dlg allows dlg mutants to survive embryogenesis and to

develop into third instar larvae. Strong hypomorphic alleles, such as dlgXI-2 or

dlgm52, are associated with tumorous overgrowth of imaginal disks and optic lobes

and with a failure to enter metamorphosis resulting in a giant larvae phenotype

(Perrimon, 1988; Woods and Bryant, 1989, 1991; Woods et al., 1996). These

mutants also exhibit striking alterations in NMJ structure and function, which are

independent of both the tumor phenotype and the extended larval life span. At

the light microscopical level, type-I boutons often appear to be enlarged and

irregularly shaped (Lahey et al., 1994; Thomas et al., 1997a). Ultrastructural

analyses not only confirmed an increase in the cross-sectional area of boutons

but also revealed a poorly developed SSR with a 40% reduction in membrane

complexity (Lahey et al., 1994; Thomas et al., 1997b), a phenotype that arises as a


result of an abnormally slow expansion of the SSR membrane system during

development (Guan et al., 1996). Moreover, the normalized number of T-bar

bearing presynaptic densities is increased in type-I boutons of dlgXI-2 mutants

(Thomas et al., 1997b), whereas dlgm52mutant boutons exhibit strikingly expanded

zones of synaptic contact (Karunanithi et al., 2002). Both aberrations may be

interpreted as allele-specific expressions of a common phenotype: the expansion

of the electron-dense synaptic core area at the expense of the flanking peri-

synaptic zones. Thus, Dlg appears to be crucial for the proper spacing of synaptic

zones, a hypothesis that is supported by the local distribution of the protein (see

earlier).

As revealed by careful quantitative analysis, dlgm52 mutant type-Ib boutons

contain enlarged synaptic vesicles with normal glutamate concentration

(Karunanithi et al., 2002). Despite conflicting results concerning the frequency

and amplitude of miniature excitatory junctional currents (mEJCs), this observa-

tion is probably related to earlier studies, which showed that dlg mutants display

larger amplitudes of evoked EJCs (Budnik et al., 1996). The EJC phenotype can

be rescued by expressing Dlg-A in the presynaptic but not in the postsynaptic

cells (Budnik et al., 1996), supporting the idea that increased levels of neurotrans-

mitter release rather than alterations in postsynaptic receptor fields account for

the higher EJC amplitudes in dlg mutants. The mechanism by which Dlg

regulates vesicle size remains elusive. The presence of Dlg in presynaptic term-

inals is consistent with a more direct role of Dlg in this process. It should be

stressed, however, that enlarged synaptic vesicles are often observed in neurons

which are relatively inactive (Karunanithi et al., 2002). Therefore, Dlg might

exert its eVect on vesicle size in a rather indirect manner, for example, by

controlling synaptic strength of motoneuron inputs.

D. DLG-INTERACTING PARTNERS AT THE NMJ

1. Dlg, FasciclinII, and CaMKII: A Joint Venture in Synaptic Plasticity

Although the pleiotropism of synaptic Dlg function certainly relies on its

ability to bind numerous proteins, relatively few synaptic interaction partners of

Dlg have been identified to date. Among these, FasII, a homophilic cell adhesion

molecule of the immunoglobulin superfamily, has been studied in greatest detail

with regard to synaptic function. At NMJs, FasII is localized at pre- and post-

synaptic membranes and a minimal amount of the protein is required for the

maintenance of synaptic boutons (Schuster et al., 1996b). The carboxyl terminus

of the transmembrane forms of FasII can bind to the first and second PDZ

domains of Dlg with moderate aYnity (Thomas et al., 1997b). The in vivo signifi-

cance of this interaction is indicated by the striking reduction of FasII at

190 ATAMAN et al.

dlg-mutant boutons (Thomas et al., 1997b; Zito et al., 1997). Moreover, boutons of

severe hypomorphic fasII and dlg mutants share specific structural abnormalities,

that is, increased size and a disproportionately high number of AZs (Stewart et al.,

1996; Thomas et al., 1997b) further supporting the idea that FasII acts as a

downstream eVector of Dlg.

Above a certain threshold level, regulated variations in the amount of synap-

tic FasII can modulate the growth of NMJs (Ashley et al., 2005; Davis et al., 1997;

Schuster et al., 1996a). Studies on larvae with genetically altered excitability

revealed that enhanced or reduced neural activity influences the size of NMJs

(Budnik et al., 1990; Mosca et al., 2005). In fact, increased presynaptic activity

results in both a reduction of FasII expression levels and an increment in the

number of boutons (Schuster et al., 1996a). Further analyses involving various

fasII mutant alleles and targeted overexpression revealed that an asymmetric

increase in FasII levels in either the pre- or the postsynaptic cell impairs the

formation of new synaptic boutons, whereas both a symmetric decrease or

increase of FasII promotes the growth of NMJs (Ashley et al., 2005; Schuster

et al., 1996b). Regulating the interaction of FasII with Dlg could be part of a

mechanism that determines the surface expression of FasII (Koh et al., 2002;

Mathew et al., 2003), thus contributing to plasticity-related growth control of

NMJs. In line with this notion, dynamic changes in the local distribution of Dlg,

which occur in the course of bouton formation during NMJ expansion, are

closely followed by changes in FasII distribution. In particular, the temporal

decrease of Dlg observed at budlike protrusions, some of which will eventually

develop into mature boutons, may account for a corresponding reduction in

FasII at these sites (Mathew et al., 2002; Zito et al., 1999). It has been proposed

that this reduction of the FasII levels at buds is necessary for a reduction of the

pre- and postsynaptic membrane adhesion at growing sites (Zito et al., 1999).

One possible mechanism by which synaptic activity may be translated into a

reduction of Dlg and FasII, thus resulting into NMJ expansion, involves Ca2þ/Calmodulin-dependent protein kinase II (CaMKII) (Koh et al., 1999), a primary

eVector of calcium signaling at synaptic junctions. Due to its ability to transform

transient changes in Ca2þ-concentration into persistent states of kinase activity,

CaMKII has been implicated as a major player in models of synaptic plasticity

(Lisman et al., 2002). Accordingly, manipulations of CaMKII activity were found

to interfere with synaptic and behavioral plasticity in both mammals and flies

(Bach et al., 1995; Barria and Malinow, 2005; Jin et al., 1998; Margrie et al., 1998;

Stanton and Gage, 1996; Wang et al., 1994). Dlg and CaMKII were shown to be

present in a protein complex at NMJs and a functional link between both

proteins was suggested by two additional observations: (1) CaMKII phosphor-

ylates Dlg in vitro within a conserved motif in the first PDZ domain. (2) Simulta-

neous expression of a constitutively active form of CaMKII (CaMKII-T287D) in

motoneurons and muscles strongly impairs the localization of Dlg at NMJs, thus,


mimicking dlg-mutant phenotypes including the poor localization of FasII (Koh

et al., 1999). Notably, elimination of the phosphorylation site in PDZ1 was shown

to render the localization of Dlg-A within the SSR largely insensitive to CaMKII

hyperactivity. This suggests that phosphorylation by postsynaptic CaMKII pro-

motes delocalization of Dlg-A. It should be mentioned, however, that expression

of CaMKII-T287D in muscles alone has relatively little eVect on both the locali-

zation of Dlg and the ultrastructure of NMJs (Haghighi et al., 2003). Thus CaMKII

activity in the presynaptic cell substantially contributes in a yet unknown way to

the observed eVect on postsynaptic Dlg.

2. Integrins: Another Link to Cell Adhesion and CaMKII

While the results mentioned earlier imply that CaMKII acts upstream of

FasII, CaMKII itself has been suggested to operate downstream of another class

of adhesion molecules, the Integrins (Beumer et al., 2002). Integrins, which may

mediate both cell-to-cell and cell-to-matrix adhesion, have been implicated in

synaptogenesis and plasticity (Clegg et al., 2003). Two �-Integrin subunits, �position-specific1 (�PS1; encoded by multiple edematous wings, mew) and �PS2(encoded by inflated, if ), and the �-Integrin subunit �PS (encoded by myospheroid,

mys), are colocalized with Dlg at the SSR (Beumer et al., 2002). In addition, a

third �-Integrin, �PS3 (encoded by scab, scb, also known as Volado, Vol ) is likely to

colocalize with �PS at presynaptic terminals (Beumer et al., 2002; Rohrbough

et al., 2000). Coimmunoprecipitation of �PS together with Dlg from adult fly

head homogenates suggests that both proteins may also be associated at NMJs.

Hypomorphic mys mutant larvae usually display overgrown NMJs. However, in a

way reminiscent to FasII, an unbalanced decrease in postsynaptic �PS in mysb9

mutants goes along with reduced NMJ expansion. In both cases elevated FasII

expression levels were observed (Beumer et al., 2002), but it may be speculated

that the opposing phenotypes result from symmetric versus asymmetric pre- and

postsynaptic increase of FasII levels. The NMJ growth phenotypes of mysmutants

could be rescued by overexpression of a wild-type isoform of CaMKII, suggesting

that Integrins regulate FasII levels and hence NMJ structure through CaMKII

and Dlg. In line with this hypothesis, Integrins have been reported to signal

through CaMKII in diVerent cellular systems including hippocampal neurons

(Illario et al., 2003; Kramar et al., 2003; Lu et al., 2005; Shi and Ethell, 2006).

3. Dlg and Ion Channels: Direct and Indirect Interactions

Probably the most clear-cut physical interaction of Dlg involves the Sh, which

binds to both the first or second PDZ domain of Dlg with high aYnity (Tejedor

et al., 1997; Zito et al., 1997). Mutational analyses have revealed that Dlg is

essential for the NMJ localization of Sh. As in the case of Dlg and FasII, this

dependency is unidirectional. The robust interaction has been exploited to gener-

ate green fluorescent protein (GFP)-tagged transmembrane reporter constructs

192 ATAMAN et al.

with an Sh-type carboxyl terminus which allow the in vivo visualization of NMJs in

intact larvae (Zito et al., 1997).

Pioneering studies have documented several cases of direct linkage between

mammalian Dlg-like MAGUKs and various types of ionotropic GluRs, mostly

mediated by canonical interactions between PDZ domains and appropriate

binding motifs within individual GluR subunits (Funke et al., 2005). To date,

however, no physical linkage between Dlg and any of the five GluR subunits

known to be expressed at larval NMJs has been reported. In fact, none of these

GluR subunits carries a type-I PDZ-binding motif and Dlg is barely detectable

within the postsynaptic receptor fields (Qin et al., 2005) (Chapter 8 by DiAntonio

and Chapter 11 by GriYth and Budnik for further details on GluR), although

GluRIIC contains a type-II PDZ-binding motif (Marrus and DiAntonio, 2004).

Nevertheless, studies have revealed that in dlgXI-2 mutant embryos the size of

receptor fields bearing the GluRIIB subunits is specifically reduced at newly

formed NMJs (Chen and Featherstone, 2005). This implies that Dlg is involved

in regulating the relative amount of GluRIIA-containing receptors versus

GluRIIB-containing receptors, which constitutes a critical parameter for synaptic

function and plasticity (DiAntonio et al., 1999; Marrus et al., 2004; Petersen et al.,

1997; Sigrist et al., 2002). The underlying regulatory mechanism remains elusive.

SAP97, the closest mammalian homologue of Dlg, has been implicated in the

transport and synaptic delivery of a specific subset of �-amino-3-hydroxy-5-

methyl-4-isoxazolepropionic acid (AMPA)-type GluRs (Sans et al., 2001). In that

case, however, a direct interaction between the C-terminal of the GluRI subunit

and a PDZ domain of SAP97 is crucial (Leonard et al., 1998). It may thus be

assumed that Dlg aVects GluR composition indirectly at the Drosophila NMJ.

4. Dlg-S97-Specific Interaction: DLin-7

The L27-type domain within the amino terminal region of Dlg-S97 (S97N)

provides a potent interface for homo- and heterodimerization and thus provides a

means to expand Dlg-based scaVolds. Dimerization of Dlg-S97 is inferred from

in vitro interaction studies (Bachmann, A., Timmer, M., Knust; E., Sierralta, J.,

and Thomas, U., unpublished data) and from parallel observations on mam-

malian hDlg/SAP97 (Marfatia et al., 2000; Nakagawa et al., 2004). In addition,

the S97N region is important for Dlg-dependent postsynaptic recruitment of

DLin-7, an evolutionary conserved small scaVolding protein with one L27 and

one PDZ domain (Bachmann et al., 2004). Like its homologues in nematodes and

mammals, DLin-7 can bind to the carboxyl terminus of EGF receptors in vitro

(Bachmann, A. and Thomas, U., unpublished data), but the in vivo significance

of this interaction with regard to NMJ function is unknown. The interaction

between Dlg-S97 and DLin-7 is largely indirect and requires a linker protein,

which is expressed in muscles but not in epithelial cells (Fig. 1B) (Bachmann et al.,

2004). In mammalian epithelial cells, Cask (mLin-2), which belongs to a distinct


subfamily of MAGUKs, has been shown to link SAP97 and mLin-7 (Veli, MALS)

by means of two L27 domains which give rise to pair-wise interactions with the

single L27 domains in SAP97 and mLin-7, respectively. However, mutations

aVecting the Drosophila homologue of Cask, Camguk (Cmg; also known as Caki,

DLin-2, and dCask), have no obvious eVect on the postsynaptic localization of

DLin-7 (Bachmann et al., 2004). Moreover, endogenous Cmg, which is highly

enriched in neuropil regions of the CNS, is otherwise barely detectable at NMJs

(Lu et al., 2003). Neuronal overexpression of Cmg, however, results in clear

localization of the protein to motor nerve terminals, suggesting that Cmg acts

as a presynaptic scaVolding protein (Lu et al., 2003). In line with this assumption,

both Cmg and Cask have been implicated in the control of neurotransmitter

release at distinct synapses (Butz et al., 1998; Zordan et al., 2005). Several other

MAGUKs with two L27 domains are encoded in the Drosophila genome and it

is thus conceivable that one of them serves as a linkage between Dlg-S97 and

DLin-7 at NMJs.

Knockout mice lacking all isoforms of mLin-7 were reported to die perina-

tally, exhibiting a severe impairment in neurotransmitter release at excitatory

synapses. In line with this observation, mLin-7 was found to be associated with

the presynaptic scaVold proteins Cask and Liprin-�2 (Olsen et al., 2005). DLin-7,

however, is hardly detectable within presynaptic terminals of motoneurons, even

upon targeted overexpression (Bachmann et al., 2004). On the other hand, the

role of DLin-7 within the postsynaptic Dlg-based scaVold remains elusive, as no

mutants have been reported to date.

5. Scribble and Dlg: A Synaptic Pas-de-Deux of Tumor Suppressors

Very much alike mutations in dlg, loss-of-function alleles of scrib cause severe

neoplastic tumors in imaginal disk epithelia and brain hemispheres (Bilder et al.,

2000). The close interaction of both genes together with yet another tumor

suppressor gene, lethal(1) giant larvae (lgl-1), is essential for the establishment of

apicobasal polarity in developing epithelia and for proper asymmetric division

of neuroblasts (Albertson and Doe, 2003; Bilder and Perrimon, 2000; Bilder et al.,

2000, 2003; Peng et al., 2000). The longest isoform of Scrib comprises 16 leucine-

rich repeats (LRR) and 4 PDZ domains and hence belongs to the family of LRR

and PDZ domain scaVolding proteins (LAPs; Fig. 1A).

At NMJs, Scrib colocalizes precisely with Dlg and like in epithelia, Scrib

localization clearly depends on Dlg (Bilder and Perrimon, 2000; Mathew et al.,

2002). Genetic and biochemical evidence implies that at NMJs both molecules

are physically linked through GUK-holder (GUKH), a protein that binds to the

GUK domain of Dlg as well as to PDZ domains of Scrib (see later). Dlg-specific

immunoreactivity remains unaVected in scrib-mutant NMJs (Mathew et al., 2002).

This situation diVers from embryonic epithelia where Scrib is required to restrict

Dlg localization to a distinct region of the plasma membrane (Bilder et al., 2000).

194 ATAMAN et al.

According to the clear epistatic relationship between both proteins at NMJs,

Scrib might be expected to act downstream of Dlg. Surprisingly, however, scrib

mutants exhibit ultrastructural and electrophysiological phenotypes which are

poorly related to those previously described for dlg mutants. While the number

of boutons at scrib-mutant NMJs is normal (but see later), the reserve pool of

synaptic vesicles was found to be enlarged, extending into the core region of

type-I boutons, which usually harbors only few vesicles. In contrast the concen-

tration of synaptic vesicles next to AZs, that is, the ready releasable pool (RRP),

was not aVected (Roche et al., 2002). Consistently, evoked excitatory junctional

currents (EJCs) are normal at low-frequency stimulation (1 Hz). Nonetheless a

failure to resupply the RRP became evident on high-frequency stimulation

(10 Hz), causing loss of both facilitation and posttetanic potentiation, and in turn

leading to faster synaptic depression (Roche et al., 2002). This interpretation was

confirmed by imaging the stimulation-dependent vesicular uptake of the fluorescent

dye FM1–43, which revealed that vesicle dynamics is impaired in scrib mutants

(Roche et al., 2002). Scrib seems to localize at low levels to synaptic vesicles

(Fuentes Medel, Y. F., Marfatia, S., and Budnik, V., in preparation). However,

presynaptic overexpression of Scrib dramatically enhances the localization of

Scrib to synaptic vesicles and alters the distribution of synaptic vesicles in

presynaptic terminals, as determined by visualization of the synaptic vesicle

markers Synapsin and Synaptotagmin (Syt). Study in Drosophila epithelial cells

also reported that mutations in the Syntaxin avalanche (avl), shown to be required

for the initial membrane fusion events in the endocytic pathway, phenocopy the

polarity and overproliferation defects observed in scribmutants, strengthening the

proposed role of Scrib in the regulation of vesicle dynamics (Lu and Bilder, 2005).

Scrib mutant boutons further display a moderately reduced number of T-bar

containing AZs, accompanied by a slightly reduced frequency of mEJCs. This

phenotype, which can be reversed by overexpression of Scrib, contrasts with

increased numbers of AZs in dlgXI-2 mutants (Roche et al., 2002; Thomas et al.,

1997b). Thus, Scrib appears to counteract the limiting eVect of Dlg on the

number of AZs.

A precisely controlled dosage of Scrib expression appears to be crucial to achieve

normal growth of NMJs (Fuentes Medel, Y. F., Marfatia, S., and Budnik, V.,

in preparation). Scrib loss-of-function mutants display increased synaptic bouton

budding, a phenotype that can be rescued by presynaptic expression of Scrib.

Neuronal overexpression of Scrib in a wild-type background, however, again

results in supernumerary bouton buds. A structure–function analysis revealed that

the LRR domain of Scrib accounts for this kind of dominant-negative activity. In

fact, expression of the LRR domain in motoneurons was found to alter the

localization of endogenous Scrib both pre- and postsynaptically, suggesting a

coordinated regulation of Scrib complexes on either side of the synaptic cleft.


6. Clients of the GUK Domain: GUK-Holder and G-Taxin

The GUK domain of Dlg-like MAGUKs does not display guanylate kinase

activity, but instead has emerged as a module for protein–protein interactions. A

number of vertebrate GUK domain-binding partners, including guanylate

kinase-associated protein (GKAP), microtubule associated protein 1A (MAP1A),

the kinesin GAKIN, and the Rap62-specific GTPase-activating protein SPAR,

have been identified (Brenman et al., 1998; Hanada et al., 2000; Kim et al., 1997;

Pak et al., 2001). Although these proteins are structurally quite diverse, a common

theme appears to be their association with the cytoskeleton. A yeast-two hybrid

screen using the GUK domain of Dlg as bait led to the identification of two novel

binding partners referred to as GUKH and G-taxin (GTX), respectively (Gorzcyca,

D., Ashley, J., Speese, S., Thomas, U., Gundelfinger, E., Gramates, S., and

Budnik, V., in preparation; Mathew et al., 2002).

GUKH carries a putative type-I Wiskott-Aldrich syndrome protein (WASP)

homology domain (WH1, also known as EVH1 domain) within its amino terminal

half but no other well-defined domains. WH1 domains have been implicated in

binding to Actin or Actin cytoskeleton-interacting proteins such as Zyxin, Ankyrin

(Prehoda et al., 1999); and the Spectrin-bound scaVolding protein Shank/ProSAP

(Boeckers et al., 2002; Tu et al., 1999). It may, therefore, be speculated that GUKH

could provide a link between Dlg and the synaptic Actin cytoskeleton. GUKH

interacts with the GUK domain of Dlg via a C-terminal domain of about 200

amino acid residues, which is specific for a subset of predicted splice isoforms of

GUKH (891 and 1044 amino acid residues, respectively). These isoforms further

display a C-terminal motif (ETALCOOH) which can bind to the second PDZ

domain of Scrib (Mathew et al., 2002). The existence of Dlg/GUKH/Scrib trimers

at NMJs is strongly supported by coimmunoprecipitations and by considerable

although not perfect overlap in their synaptic immunoreactivities. Most notably,

Scrib was found to be reduced to roughly the same extent (~40%) as GUKH at

NMJs of hypomorphic gukh mutants. No changes in the level of GUKH at type-I

boutons were detected in dlg or scrib mutants (Mathew et al., 2002).

The interaction of GUKHwith two multidomain scaVolding proteins that havevarious interacting partners suggests that GUKH may coordinate the functions of

two distinct multiprotein complexes within the developing nervous system. To date,

however, the persistent unavailability of more severe gukh alleles has precluded a

more detailed analysis on the synaptic function of GUKH. In particular, it remains

elusive whether the interaction between Dlg and GUKH is related to the proposed

role of the GUK domain for proper traYcking and localization of Dlg to NMJs.

Careful immunofluorescence studies suggest that the role of GUKH at NMJs goes

beyond the linkage of Scrib and Dlg. GUKH is highly enriched at presynaptic

boutons even at regions whereDlg is not detected.Moreover, GUKH is dynamically

regulated during synaptic bouton development, thereby being enriched at the

196 ATAMAN et al.

initiation sites of bouton budding. This is complementary to the localization of Dlg,

which otherwise colocalizes with GUKH at the periphery of mature boutons

(Mathew et al., 2002). The association of GUKHwith bouton buds could be related

tothe budding defectsobservedinscribmutants,possibly reflecting aDlg-independent

interaction (Fuentes Medel, Y. F., Marfatia, S., and Budnik, V., in preparation).

Two mammalian genes with similarity to gukh have been identified. Muta-

tions in one of them (GUKH 1) account for the human X-linked Nance-Horan

disease, a syndrome that involves craniofacial abnormalities and mental retarda-

tion (Katoh, 2004). Thus, studies on GUKH in Drosophilamay once again provide

insights on cellular and molecular mechanisms underlying human diseases.

The second identified binding partner of the GUK domain of Dlg, GTX,

belongs to the family of soluble N-ethylmaleimide-sensitive factor attachment

receptor (SNARE) proteins. It shares particular sequence similarities with Ufe1

from yeast and with human Syntaxin-18, two SNAREs which are unusual

in promoting homotypic fusion events and which have both been implicated

in Golgi–ER traYcking (Lewis and Pelham, 1996; Patel et al., 1998; Spang and

Schekman, 1998). At larval NMJs, GTX colocalizes with Dlg postsynaptically and

both proteins coimmunoprecipitate from body wall muscle extracts, leaving little

doubt that Dlg and Gtx interact in vivo. Mutations in dlg suppress the synaptic

localization of GTX (Gorczyca et al., in preparation). Biochemical assays further

revealed that the SNARE-typical formation of sodium dodecyl sulfate (SDS)-

resistant G-Taxin (GTX) complexes is impaired in dlg mutants. Hypomorphic

mutations in gtx cause a decrease in SSR surface to a similar extent as observed in

dlg mutants (Fig. 3). Strikingly, overexpression of GTX in muscles results in the

formation of ectopic SSR-like structures bothwithin the cytoplasm and at themuscle

surface. All these findings are in line with the idea that GTX acts downstream of Dlg

to shape the SSR. A working model proposes that Dlg recruits GTX-containing

FIG. 3. gtx mutants have a reduced SSR similar to dlg mutants. Panels show electron micrographs

of a type-Ib bouton in (A) wild-type mutant and (B) gtxex6 mutant. Arrows point to active zones (AZs);

m, muscle; b, bouton; SSR, subsynaptic reticulum. (Images from Gorczyca et al., in preparation.)


vesicles to the postsynaptic area, where homotypic fusion of the vesicles with the SSR

may serve as a means to expand the surface of the latter (Gorzcyca et al., in

preparation).

V. Dystrophin

Dystrophin and its closely related paralog Utrophin are members of the

Spectrin superfamily, which have been identified as postsynaptic scaVolding pro-teins at cholinergic NMJs in vertebrates.Mutations aVectingDystrophin are causal

to Duchenne muscular dystrophy, a lethal muscle-wasting disease. It remains to

be clarified, however, to which extent loss of synaptic functions of Dystrophin

contributes to the fatal degeneration process (Blake et al., 2002). Whereas Dystro-

phin and Utrophin are likely to exert partially redundant functions in mammals,

only a single dystrophin-like gene, dys, is present in the Drosophila genome (Fig. 1A).

van der Plas et al. (2006) have provided an extensive analysis on the role of

Dystrophin at larval NMJs. The dys locus encodes four major protein isoforms,

which exhibit diVerential expression. Three isoforms (DLP1–3), each of enor-

mous size (~400 kDa), share a domain organization comprising an amino

terminal Actin-binding region followed by 11 Spectrin repeats with interspersed

coiled-coil regions and a cysteine-rich domain. The latter has been implicated in

the binding of �-Dystroglycan (see later) and includes a WW domain, a tandem

of calcium-binding EF-hand motifs and a zinc finger domain. A fourth variant,

Dp186 (186 kDa), originates from the diVerential usage of a transcriptional start

site further downstream within the dys locus. Among all isoforms, DLP2 appears

to be the only one present at NMJs. The protein is enriched postsynaptically

within the SSR but was also found to colocalize with Actin within the sarcomeric

I-bands. The absence of DLP2 from presynaptic terminals could be inferred from

the lack of respective transcripts in the CNS.

True dys null mutants have not yet been described, but the DLP2-specific

mRNA is virtually absent in dysE6 mutants and none of the large isoforms is

detectable in dysGE20705 mutant larvae. At the light microscopical level, dys

mutants do not display any obvious abnormalities with regard to expansion of

NMJs, shape of boutons, or localization of GluRs. Ultrastructurally, however, the

number of T-bar containing AZs was found to be doubled as compared to wild

type. Strikingly, this phenotype is paralleled by a substantial increase in quantal

content, which was shown to reflect an increased release probability rather than

an enlarged readily releasable pool of synaptic vesicles. In light of the pure

postsynaptic localization of Dystrophin it must be concluded that DLP2 partici-

pates in retrograde signaling to control synaptic activity. This view was further

supported by genetic interaction studies, which demonstrated that in comparison

198 ATAMAN et al.

to wild type, quantal content is reduced if both DLP2 and the presynaptic BMP-

receptor encoded by wishful thinking (wit) are diminished. This finding is consistent

with the idea that DLP2 counteracts retrograde BMP signaling, which has

previously been shown to be required for the proper formation of T-bar contain-

ing AZs (Aber le et al ., 2002). Postsynap tic activity of CaMKII exe rts a similar

eVect on retrograde BMP signaling (Haghighi et al., 2003) (Chapter 12 byMarques

and Zhang). However, a more direct link between DLP2 and CaMKII in this

pathway remains elusive.

The muscular Dystrophin-associated protein complex of vertebrates has been

characterized in detail (Ehmsen et al., 2002). Briefly, Dystrophin provides a direct

link between F-Actin and the transmembrane protein �-Dystroglycan, which in

turn is associated directly or indirectly with other membrane proteins and with

secreted proteins of the extracellular matrix such as Laminin. Intracellularly,

Dystrophin binds to Syntrophin, another scaVolding protein with two PDZ do-

mains. Many components of the Dystrophin-based complex are conserved in flies

and several of them including orthologues of �-Dystroglycan and Syntrophin-2 are

expressed in muscles, suggesting that at larval NMJs Dystrophin might be embed-

ded in a similar complex as in vertebrate muscle membranes and NMJs (Dekkers

et al., 2004; Greener and Roberts, 2000).

VI. dGRIP

Various AMPA-type GluRs from vertebrates have been reported to interact

directly with PDZ domain scaVolding proteins including SAP97/hDlg, protein

interacting with C kinase 1 (PICK1), and glutamate receptor interacting protein

(GRIP)/AMPA receptor-binding protein (ABP) (Dong et al., 1997; Leonard

et al., 1998; Xia et al., 1999). Well-conserved homologues of the aforementioned

scaVolding molecules exist in Drosophila, but their involvement in GluR function

at NMJs is unclear. Studies have addressed the role of the single GRIP homo-

logue in flies, dGRIP, during muscle development and NMJ diVerentiation(Ataman et al., 2006; Swan et al., 2004).

Like mammalian GRIP1, dGRIP consists of seven PDZ domains (Fig. 1A).

Loss-of-function mutants are, for the most part, lethal and display impaired

guidance of the ventral longitudinal muscles resulting in muscle patterning

defects. Ectopic expression of dGRIP in lateral transverse muscles causes mis-

oriented muscle pioneer extensions, supporting the idea that dGRIP is required

for the processing of guiding cues that direct muscles to attachment sites at

segmental borders (Swan et al., 2004).

A striking association of dGRIP with vesicular structures is evident at presyn-

aptic terminals and within muscles later in development (Ataman et al., 2006).


Presynaptically dGRIP-carrying vesicles are closely associated withmicrotubules. In

muscles, dGRIP is present in Golgi bodies and on a subset of traYcking vesicles that

are highly concentrated within the postsynaptic area (Ataman et al., 2006). Insights

into the synaptic function of dGRIP during larval NMJ development have been

obtained from analyses of dgrip hypomorphic alleles and from RNAi experiments

(Ataman et al., 2006). Decrease of dGRIP levels by targeted expression of DNA-

based siRNAs hampers synaptic bouton formation and hence the growth of NMJs.

In particular, postsynaptic elimination of dGRIP leads to the formation of atypical

boutons, which clearly express presynaptic markers, such as the horseradish peroxi-

dase (HRP) neuronal membranes epitope, Synapsin, and cysteine-string protein

(CSP), but which otherwise lack the active zone marker nc82 (identified as Bruchpi-

lot, Brp; see later). In addition, these boutons are devoid of all hitherto tested

postsynaptic proteins, that is, Dlg, GluRIII (Fig. 4), Bazooka, Scribble, and Spectrin.

Consistent with these findings EM analyses revealed the presence of boutons which

are filled with synaptic vesicles but lack AZs as well as postsynaptic specializations,

that is, PSDs and SSR. These characteristics are strikingly similar to those previously

observed in conditional wingless (wg) mutants (Packard et al., 2002), suggesting a

potential relationship between dGRIP and the Wg pathway. Wg is secreted by

motorneuron terminals and presumably binds to postsynapticDFrizzled-2 receptors

(DFz2). DFz2 in turn becomes endocytosed and transported to perinuclear areas,

where its cytoplasmic tail is cleaved and enters the nucleus (Mathew et al., 2005;

Packard et al., 2002). dGRIP and DFz2 colocalize on traYcking vesicles and, most

notably, a reduction of dGRIP impairs the transport of DFz2 from synapses to the

perinuclear zone. In vitro binding assays further imply that the PDZ4 and five

domains of dGRIP bind directly to the C-terminal PDZ-binding motif of DFz2

(Ataman et al., 2006). It may therefore be assumed that dGRIP exerts its postsynaptic

function by organizing the traYcking of DFz2 in response to Wg signaling.

VII. dX11/dMint/dLin-10

Mammalian and nematode X11/Mint/Lin-10 scaVolding proteins have been

implicated in the exocytosis of synaptic vesicles (Biederer and Sudhof, 2000; Butz

et al., 1998; Okamoto and Sudhof, 1997; Setou et al., 2000), the metabolism of

Amyloid precursor protein (APP) (Miller et al., 2006) and the regulated traYcking

and localization of epidermal growth factor (EGF) and GluRs (Rongo et al., 1998;

Setou et al., 2000; Stricker and Huganir, 2003). X11/Mint family members share a

central phosphotyrosine-binding domain (PTB) and a C-terminal tandem of PDZ

domains (Fig. 1A). The amino terminal region is less conserved across the protein

family. InDrosophila, two closely relatedX11/Mint proteins, dX11� and dX11�, areencoded by separate genes and both are expressed in the nervous system (Hase

FIG. 4. Postsynaptic elimination of dGRIP results in the formation of ghost boutons, which lack

postsynaptic proteins and most AZs. Third instar larval NMJs in (A, B, G–I) wild type and in (C–F

and J–L) larvae expressing dGRIP-RNAi postsynaptically in preparations labeled with (A–F) anti-

HRP (green) and anti-DLG (red), and (G–L) anti-HRP (blue), anti-GluRIII (red), and nc82 (green).

Arrows, ghost boutons. Asterisk in G–I highlights an uncommon ghost bouton observed in wild type.

Arrowhead in D points to an HRP-labeled process connecting a ghost bouton with the main arbor.

Asterisk in J–L marks a ghost bouton containing nc82 immunoreactivity. Scale bar (mm): 15 in A–F,

12 in G–L. (Figure, with permission, reproduced from Ataman et al., 2006#2006 National Academy

of Sciences, USA.)

200 ATAMAN et al.


et al., 2002; Vishnu et al., 2006). A study identified both dX11� and dX11� as

interacting partners for the cell adhesion molecule Roughest (Rst) and suggested a

role for dX11� in cell specification and sorting during pupal eye development

(Vishnu et al., 2006).

Direct evidence for a role of dX11� in synapse development was provided by an

extensive analysis on its interaction with FasII and the APP-like protein (APPL)

(Ashley et al., 2005). Reduced levels of either dX11� or APPL interfere with NMJ

expansion, whereas neuronal overexpression of either protein strongly promotes the

proliferation of synaptic boutons. Many of the surplus boutons remain unusually

small and closely attached to normal sized boutons and have therefore been referred

to as satellite boutons. Nonetheless, these satellites are clearly distinct from nascent

bouton buds as they contain AZs and are surrounded by SSR (Ashley et al., 2005;

Torroja et al., 1999). Overexpression of APPL or dX11� is thus suYcient to induce

the formation of new synaptic boutons, but a yet unidentified factor seems to be

limiting to the subsequent enlargement of these boutons. In addition to the common

phenotypical characteristics, several findings support the idea that APPL and

dX11� are intimately linked. Both proteins are detectable at presynaptic terminals

and have been shown to coimmunoprecipitate from body wall muscle extracts. In

fact, the PTB domain of dX11� can bind to the evolutionary conserved

GYENPTY-sequence motif within the cytoplasmic domain of APPL (Ashley et al.,

2005; Hase et al., 2002). Notably, the APPL overexpression phenotype was shown to

depend on the GYENPTY motif, suggesting that binding to dX11� is required for

enhanced bouton proliferation. In turn, deletion of the PTB domain in dX11� also

abolishes the respective overexpression phenotype. Overexpression of dX11� in the

absence of APPL, however, still results in supernumerary boutons, not only implying

that dX11� acts downstream of APPL but also pointing to a role of the PTB domain

beyond its interaction with APPL.

Simultaneous pre- and postsynaptic overexpression of FasII results in a phe-

notype very similar to those observed on presynaptic overexpression of APPL or

dX11�, suggesting that the three proteins act in the same pathway. The existence

of a tripartite FasII/APPL/dX11 complex was supported by coimmunoprecipita-

tions from both body walls and transfected Drosophila S2 cells, and it was further

demonstrated that the proteins are able to interact in a pair-wise manner in any

combination. The increase in bouton number either due to symmetric increase or

symmetric decrease of FasII were completely suppressed by null mutations in appl.

Thus, FasII perhaps acts upstream of both dX11 and APPL. However, the exact

mechanisms and the functional significance of the individual interactions within

the dX11/APPL/FasII complex are yet to be determined. A speculative model

postulates that on transhomophilic activation of FasII, APPL facilitates the fusion

of dX11 containing vesicles with the presynaptic membrane, thus, leading to

the local delivery of new dX11-associated cargo as well as membrane material

required for the formation of buds (Ashley et al., 2005).

202 ATAMAN et al.

VIII. Dliprin-a

A. DLIPRIN REGULATES ACTIVE ZONE MORPHOLOGY AND SYNAPTIC PHYSIOLOGY

LAR-interacting protein-� (Liprin-�) refers to a family of scaVolding proteinswhich were initially identified as interaction partners of leukocyte antigen-

related (LAR) receptor tyrosine phosphatases (Fig. 1A). Liprin-� was further

proposed to recruit LAR to specialized cell–substrate interaction domains of the

plasma membrane (Serra-Pages et al., 1998, 2005). The domain architecture of the

Liprin-� family members is highly conserved, comprising a coiled-coil homodi-

merization domain at the N-terminal, a LAR-binding region, as well as three

putative steryl alpha motifs (SAMs) at the C-terminal (Serra-Pages et al., 1998).

A pivotal role for Liprin-� in presynaptic diVerentiation was initially unraveled

by mutational analysis in Caenorhabditis elegans, where Syd-2, the nematode Liprin-�orthologue, was found to control the expansion ofAZs and to ensure normal levels of

synaptic transmission (Zhen and Jin, 1999). Ultrastructural and electrophysiological

analyses revealed a very similar phenotype at larval NMJs of hypomorphicDliprin-�mutants (Kaufmann et al., 2002). The gross morphology of pre- and postsynaptic

structures in these mutants appears normal, but serial electron microscopy (EM)

reconstructions revealed that AZs atDliprin-�mutant type-I boutons are of irregular

shape and size, being enlarged by more than twofold at average. In addition, the

overall expansion and branching of Dliprin-� mutant NMJs is diminished, and the

reduced number of synaptic boutons (30–50%) is correlated with a decrease in

budding at terminal boutons (Kaufmann et al., 2002).

Electrophysiological studies showed that Dliprin-� mutants have significantly

lower (36%) evoked excitatory junctional potentials (EJPs) and reduced quantal

content (50%), while the mean amplitudes and the frequency of spontaneous excit-

atory junctional potentials (mEJPs) are not changed. These findings indicate that the

neurotransmitter content of synaptic vesicles as well as the postsynaptic sensitivity to

synaptic vesicle release are not altered. The evoked synaptic vesicle release, however,

is reduced, consistent with both the decrease in bouton number and the abnormal

active zone morphology in Dliprin-�mutants (Kaufmann et al., 2002).

B. DLIPRIN-� INTERACTS WITH DROSOPHILA LEUKOCYTE ANTIGEN-RELATED

RECEPTOR TYROSINE PHOSPHATASES (DLAR) TO CONTROL

SYNAPTIC DEVELOPMENT

Like their counterparts in other organisms, Dliprin-� and DLAR can interact

directly, at least in vitro. Moreover, both proteins are coexpressed in the nervous

system during embryogenesis, suggesting that they may also interact in vivo.

In fact, genetic interaction studies strongly suggest that both proteins act in the


same pathway to control NMJ expansion. The morphological, ultrastructural,

and electrophysiological phenotypes observed at Dlar-mutant NMJs were re-

markably similar to those observed in Dliprin-� mutants. An epistasis analysis,

in which an increased growth of NMJs due to presynaptic Dlar overexpression

was found to be suppressed in a Dliprin-� background, clearly suggests that

Dliprin functions genetically downstream of Dlar during synapse development

(Kaufmann et al., 2002).

C. DLIPRIN-� AS A TARGET FOR REGULATED DEGRADATION BY

THE APC/C COMPLEX

Growth control of larval NMJs involves a tight regulation of local protein

turnover by enzymes that determine the level of ubiquitination of selected

proteins (Di Paolo et al., 2002). A study demonstrated that the anaphase-

promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase best known for

its role in cell-cycle regulation, controls NMJ growth and synaptic transmission

through ubiquitination and degradation of target proteins (van Roessel et al.,

2004). In this study, Dliprin-� was identified as a potential target for APC/

C-mediated degradation (van Roessel et al., 2004). Dliprin-� contains three

putative destruction box motifs (RxxLxxxxN), which have previously been

described as consensus target sites for APC/C substrates (Ayad et al., 2005; King

et al., 1996). Consistently, YFP-tagged Dliprin-� was shown to be ubiquitinated

on expression in the larval CNS (van Roessel et al., 2004). NMJs of APC2/mr3

mutant larvae exhibit a dramatic NMJ overgrowth phenotype accompanied by a

significant increase in synaptic levels of Dliprin-�. Removal of Dliprin-�suppressed the APC2/mr3 mutant phenotype, thus, supporting the idea that

Dliprin-� is a downstream eVector of the APC/C complex. Noteworthy, how-

ever, overexpression of Dliprin-� is not suYcient to induce considerable

NMJ overgrowth, that is, Dliprin-� is necessary but not suYcient for APC/

C-dependent growth control (van Roessel et al., 2004).

D. NOT JUST CARGO: DLIPRIN-� PROMOTES PROPER TRAFFICKING OF

SYNAPTIC VESICLES

The pivotal role of Liprin-� in neurotransmitter release appears to be

evolutionarily conserved in nematodes, arthropods, and vertebrates (Kaufmann

et al., 2002; Olsen et al., 2005; Zhen and Jin, 1999). A postsynaptic function for

Liprin-�, however, has been described for mammals only, where it was shown to

be involved in AMPA receptor traYcking, primarily via its interactions with

GRIP (Wyszynski et al., 2002), GTPase-activating protein GIT1 (Ko et al.,

204 ATAMAN et al.

2003) and the neuron-specific kinesin motor KIF1A (Shin et al., 2003). Despite its

considerable abundance within the postsynaptic SSR and aside from the fact that

a possible role for Dliprin-� in retrograde signaling has not been ruled out

explicitly, none of the aberrations observed at Dliprin-� mutant NMJs has been

attributed to a postsynaptic requirement for the protein (Kaufmann et al., 2002).

Thus, presynaptic Dliprin-�, albeit weak in terms of immunodetection, probably

accounts for all the structural and physiological functions described to date.

This view is strongly supported by the observation that anterograde axonal

transport of the synaptic vesicle associated proteins Syt and GFP-tagged Synap-

tobrevin (nSyb-GFP) is hampered in Dliprin-� mutants as reflected by unusual

accumulations of both markers within axons of segmental nerves (Miller et al.,

2005). In contrast, retrograde movements were found to be increased in the

mutants, suggesting that the balance between antero- and retrograde motor

activities on synaptic vesicles is shifted. In fact, the impaired transport is most

probably related to the association of Dliprin-� with the anterograde motor

protein Kinesin-1 (Khc), and it has been proposed that Dliprin-� activates

Khc allowing it to counteract Dynein-mediated retrograde motor activity (Miller

et al., 2005).

The active role of Dliprin-� in synaptic vesicle traYcking constitutes another

case in point within the growing concept that scaVolding proteins are not only

passively transported on vesicles but also they may actually be involved in the

regulation of vesicle traYcking.

IX. Bruchpilot: A Crash Pilot Targets the Active Zone

As described earlier, a number of scaVolding proteins exert presynaptic func-tions related to the structural and/or physiological properties of NMJs. None of

them, however, exhibits a particular enrichment at the electron-dense AZs. A

pioneering study byWagh et al. (2006) has led to the identification of Brp as a major

scaVolding protein that accumulates at AZs, as detectable by means of the Brp-

specific monoclonal antibody nc82. An N-terminal region of roughly 480 amino

acid residues displays striking sequence homology with previously described syn-

aptic proteins inmammals and C. elegans, alternatively referred to as ELKS, CAST,

or ERC. Brp isoforms, however, include a much longer C-terminal region than

ELKS/CAST/ERC. The presence of multiple coiled-coil regions suggests that

Brp may form hetero- and homooligomers. In fact, mammalian ELKS/CAST/

ERCs oligomerize with other scaVolding proteins of the so-called ‘‘cytoskeletal

matrix associated with AZs’’ (CAZ) (Dresbach et al., 2001; Zhai and Bellen, 2004),

for example, Piccolo, Bassoon, Liprin-� Munc-13, and RIM1 (Ko et al., 2003;

Ohtsuka et al., 2002; Takao-Rikitsu et al., 2004).


The function of Brp was addressed by neuronal expression of DNA-based

siRNAs, which caused a dramatic reduction of the gene product, in some cases

leading to late embryonic or early larval lethality. Expression of brp-specific siRNA

in photoreceptors led to a severe impairment of neurotransmission in the synaptic

target area within the optic lobe. Most strikingly, EM analyses revealed a

complete lack of T-bar–like structures in the photoreceptor terminals. A very

similar, yet somewhat less dramatic eVect was observed at type-I boutons on

motorneuronal reduction of Brp. In this case, the relative abundance of

presynaptic densities carrying T-bars was reduced to about 7% as compared

to 40% in control animals. Notably the overall number of presynaptic densities

appeared largely unchanged. The reduced number of T-bars goes along with

a substantial decrease in quantal content, which is due to impaired evoked

responses rather than alterations in miniature amplitudes. In fact, neither the

number nor the expansion of PSDs, as monitored by GluRIIC-specific immuno-

fluorescence, was significantly altered. Similarly, presynaptic markers such as

Synapsin, Syntaxin, and Dynamin appeared largely unaVected, suggesting

that synaptic vesicles and the vesicle recycling machinery remain properly loca-

lized under these brp-hypomorphic conditions. An emerging concept, however,

strongly suggests that Brp is required for the localrecruitment of a presynaptic

calcium channel encoded by cacophony (cac) (Kawasaki et al., 2004).

X. Bazooka (Par-3)/Par-6/aPKC

Atypical protein kinase C (aPKC) has been implicated in the maintenance of

long-term potentiation in mammals (Ling et al., 2002) and in prolonging memory in

the fly (Drier et al., 2002), suggesting a general role in synaptic plasticity. The enzyme

has otherwise been found together with the PDZ domain proteins Par-3 [known as

Bazooka (Baz) in flies] and Par-6 in an evolutionarily conserved complex, which

controls the establishment and maintenance of polarity in many cell types including

Drosophila neuroblasts, oocytes and epithelial cells, mammalian epithelial cells and

hippocampal neurons, C. elegans zygotes, and Xenopus oocytes (Fig. 1A) (Klein and

Mlodzik, 2005; Wodarz, 2002, 2005). The same trio of proteins has been shown to

localize at NMJs ofDrosophila and has been suggested to regulate cytoskeletal dynam-

ics during NMJ development and plasticity (Ruiz-Canada et al., 2004). In line with

this notion, mutations in par-6, baz, and dapkc all lead to a reduced number of synaptic

boutons. Although the three proteins interact genetically and biochemically during

NMJdevelopment, it should be stressed that they are not coassembled persistently. As

in other cell types, activity of aPKCmay instead be assumed to regulate the assembly

of the complex, while Par-3/Baz and Par-6 in turn control the activity of aPKC. Both

Par-3/Baz and Par-6 bind to aPKC, yet at distinct sites. Par-6 contains a single PDZ

206 ATAMAN et al.

domain and a semi-CRIBdomainwhich binds the smallG-proteinsCdc42andRac1

in their activated, that is, GTP-bound form (Fig. 1C) (Joberty et al., 2000; Lin et al.,

2000). In association with activated Cdc42 or Rac1, Par-6 activates aPKC. Par-3/

Baz comprises three PDZ domains and an N-terminal multimerization domain. In

contrast to Par-6, Par-3/Baz exerts an inhibitory eVect on aPKC when bound to the

regulatory region of the enzyme. This inhibition, however, is released when aPKC

phosphorylates a conserved serine residue in Par-3/Baz (Fig. 1C) (Lin et al., 2000).

Within presynaptic terminals aPKC regulates microtubule (MT) dynamics

during bouton formation and branch extension by promoting the association

of the MT-stabilizing protein Futsch to MTs. Inside boutons Par-6 colocalizes

with the MT bundle and with aPKC but in addition displays some diVusedistribution. Baz-specific immunoreactivity appears enriched in postsynaptic mus-

cles near the presynaptic bouton membrane and is also diVusely distributed withinboutons. In contrast to aPKC and Par-6, however, Baz is not associated with the

MT bundle. In accordance to the aforementioned inhibitory influence of Baz on

aPKC, this finding suggests that the MT-associated fraction of presynaptic aPKC

is in its active state, that is, enabled to exert its stabilizing eVect on MT bundles.

In the postsynaptic muscle cell, Baz and Par-6 are largely colocalized with

Spectrin in the peribouton area, which covers the SSR and a definedMT-free space

around it (Ruiz-Canada et al., 2004). Hypomorphic baz mutants display a reduced

peribouton area and reduced Spectrin immunoreactivity. Notably, aPKC is absent

from the peribouton area. Nonetheless, both the expansion of the peribouton area

and Baz-specific immunoreactivity are reduced in aPKC loss-of-function mutants.

Expression of a constitutively active form of aPKC (PKM) results in the opposite

phenotype. These results suggest that aPKC controls the boundaries between MT-

rich and Actin/Spectrin-rich areas at the peribouton area through Baz (Chapter 11

by GriYth and Budnik). This idea is supported by previous work in which Baz was

implicated in the maintenance of the zonula adherens, an Actin-rich belt encircling

epithelial cells just below the apical membrane compartment (Muller and

Wieschaus, 1996).

XI. Missing Prominents: Homer/Vesl, Shank/ProSAP, and GKAP/SAPAP

Fly homologues of a number of mammalian PSD scaVolding proteins have notyet been detected at NMJs. This notion applies, for instance, to Shank proteins

(also known as ProSAPs), abundantly expressed PSD proteins which link core PSD

scaVolding proteins to the Actin–Spectrin cytoskeleton and to proteins flanking thePSDs, most notably Homer (see later). Aside from some truncated splice variants,

all family members share a ProSAP-specific homology domain at the N-terminal,

one SH3 and one PDZ domain followed by a long, probably rodlike C-terminal


region with a number of proline-rich motifs and coiled-coil domains. Numerous

synaptic interaction partners have been described for Shank/ProSAP (Boeckers

et al., 2002), including the so-calledGKAP/SAPAP proteins which are able to bind

to the GUK domain of Dlg-like MAGUKs and, simultaneously, to the PDZ

domain of Shank/ProSAP, a mode of interaction that is reminiscent to the linkage

of Dlg and Scrib by GUKH (see earlier). A single orthologue of Shank/ProSAP,

dProSAP, exists in Drosophila, however, repeated attempts to detect this protein at

NMJs using specific antibodies or on targeted expression of EGFP-tagged dPro-

SAP failed (Thomas, U., unpublished results). It is tempting to speculate that the

absence of any obvious GKAP homologue in Drosophila may account for this

negative result. Another binding partner of Shank/ProSAP, Homer, is well

conserved in flies. The domain structure of Homer proteins is bipartite. An

amino terminal Homer-specific EVH1/WH1 domain mediates binding to

PPxxF motifs, whereas an extensive coiled-coil region in the C-terminal half

allows multimerization. In the PSD of dendritic spines, Homer is thus able to

link the EVH1-binding proteins Shank/ProSAP, type-I metabotropic GluRs

(mGluRs) and IP3-receptor (Thomas, 2002). The principal binding properties

appear to be conserved in dHomer (Diagana et al., 2002). However, neither

the fly mGluRs nor the IP3 receptor carries the canonical PPxxF motif.

dHomer is strongly enriched in neuropil areas of the CNS throughout devel-

opment and null mutant adults are severely impaired in associative learning

(Diagana et al., 2002). Despite expression in both motoneurons and muscles no

enrichment of endogeneous or overexpressed dHomer at NMJs is evident. The

protein instead tends to localize to intracellular compartments, most likely ER,

and to distinct extrasynaptic microdomains underneath the muscle surface

(Diagana et al., 2002). Somewhat unexpectedly, dHomer has been implicated

in the posterior localization of the pole cell determinant Oskar during oocyte

development (Diagana et al., 2002). Oskar and some other determinants of

oocyte polarity have emerged from a screen for memory mutants (Dubnau

et al., 2003), suggesting that the interaction between dHomer and Oskar might

be involved in memory formation. A role for dHomer as a scaVolding protein

at NMJs, however, remains questionable.

XII. Perspectives

Without doubt, the Drosophila NMJ has greatly contributed to the analysis of

the building blocks responsible for the precision with which synaptic proteins are

organized. The use of a genetic strategy to investigate the role of scaVoldingproteins, combined with the tractability of larval synapses, aVords an analysis of

their in vivo function, in ways that are still cumbersome in other organisms.

208 ATAMAN et al.

In addition, the highly conserved nature of the synaptic scaVold renders the studiesin Drosophila far reaching across phylogenies. Several challenges, however, will

have to be addressed in the future, beyond the identification of the full complement

of scaVolding proteins at the synapse. For example, although many synaptic

scaVolding proteins have been identified in Drosophila, the discovery of module-

specific binding partners lags behind. True understanding of scaVolding functioncan become fully apparent only once binding partners are recognized. A second

area in which much research is needed in order to truly understand scaVoldingprotein function is the analysis of the dynamics of scaVolding proteins. It is now

clear that the synaptic scaVold is not static but likely to undergo changes that

may result in traYcking through diVerent synaptic compartments, and in the

dynamic exchange of protein-binding partners. This dynamics is required

not only to transport synaptic complexes from their site of synthesis but also as a

regulatory mechanism during synaptic plasticity. Progress in elucidating these

problems is likely to emerge for the study of the larval NMJ in the near future.

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[International Review of Neurobiology] The Fly Neuromuscular Junction: Structure and Function Second Edition Volume 75 || Scaffolding Proteins at the Drosophila Neuromuscular Junction