Synapses can undergo rapid changes in size as well as in their vesicle release function during both plasticity processes and development. This fundamental property of neuronal cells requires the coordinated rearrangement of synaptic membranes and their associated cytoskeleton, yet remarkably little is known of how this coupling is achieved. In a GFP exon-trap screen, Drosophila Basigin (Bsg) was identified as an immunoglobulin domain-containing transmembrane protein accumulating at periactive zones of neuromuscular junctions. Bsg is required pre- and post-synaptically to restrict synaptic bouton size, its juxtamembrane cytoplasmic residues being important for that function. Bsg controls different aspects of synaptic structure, including distribution of synaptic vesicles and organization of the presynaptic cortical actin cytoskeleton. Strikingly, bsg function is also required specifically within the presynaptic terminal to inhibit nonsynchronized evoked vesicle release. It is thus proposed that Bsg is part of a transsynaptic complex regulating synaptic compartmentalization and strength, and coordinating plasma membrane and cortical organization (Besse, 2007).

Synapses are highly specialized and asymmetric intercellular junctions organized into morphologically, biochemically, and physiologically distinct subdomains. At the presynaptic terminal membrane, active zones mediate Ca²⁺-dependent synaptic vesicle fusion, whereas the surrounding periactive zones are essential for synaptic vesicle endocytosis and the control of synaptic terminal growth. Definition of distinct synaptic subdomains is not restricted to the plasma membrane but is also clearly visible within the presynaptic terminal cytoplasm. Notably, synaptic vesicles are clustered at the cell cortex, in the vicinity of active zones. In addition, they seem organized into functional subpools displaying distinct release and recycling properties. Such an organization requires the precise trafficking and targeting of vesicles to their appropriate location and the specific recruitment and release of subsets of vesicles, depending on the stimulation conditions. One of the main challenges synapses have to face is maintaining such a highly organized structure while constantly adapting their morphology and strength in response to developmental programs and/or external stimuli. Indeed, synaptic terminals can adjust their size. the number, size, and composition of their pre- and post-synaptic membrane specializations; and the availability and release competence of cytoplasmic synaptic vesicles. These dynamic changes require the maintenance of precise physical and functional connections between pre- and post-synaptic compartments, as well as between cytoplasmic and plasma membrane subdomains (Besse, 2007).

To date, the mechanisms allowing such a dynamic reorganization are still poorly understood. However, using the Drosophila neuromuscular junction (NMJ) as a genetic model, different components of periactive zones, including transmembrane proteins and adaptor molecules, have been implicated in the control of terminal outgrowth. Cell adhesion molecules (CAMs) of the Ig superfamily seem particularly important in maintaining the integrity of synaptic terminals but also in transmitting signals to the cell interior, thereby promoting differentiation of pre- and postsynaptic specializations and regulating synaptic structure and function. Moreover, the actin-rich presynaptic cytoskeleton is important for rearranging synaptic domains and for controlling synaptic vesicle distribution and release ability. How the linkage between cortical cytoskeleton, cytoplasmic vesicle pools, and specialized membrane domains is mediated and, more generally, how plasma membrane and cytoplasmic membranes are spatially and functionally connected largely remain to be elucidated (Besse, 2007).

This study identified the transmembrane Ig CAM Basigin (Bsg) as a new component of periactive zones at D. melanogaster NMJ synapses. Bsg is the only Drosophila member of the Basigin/Embigin/Neuroplastin family of glycoproteins, of which mammalian Bsg has been shown to have multiple functions, including in tumor progression (Nabeshima, 2006). It seems to regulate cell architecture and cell-cell recognition (Fadool, 1993; Curtin, 2005), act in signaling (Guo, 1997; Tang, 2006), and act as a chaperone for transmembrane proteins (Kirk, 2000; Zhou, 2005). By analogy to other mammalian cell surface glycoproteins, and in particular to the CD44 transmembrane protein family (Ponta, 2003), Bsg may be essential for establishment of transmembrane complexes and for organization of cell structure and signal transduction cascades. Interestingly, mammalian Bsg and Neuroplastin have been suggested to play a role in memory functions and long-term potentiation (Naruhashi, 1997; Smalla, 2000), respectively, although their precise function has not been determined (Besse, 2007).

Drosophila Bsg is required in both pre- and postsynaptic compartments to control formation and growth of synaptic varicosities (or boutons) at larval NMJs. Bsg is a bona fide Ig CAM because (1) it can promote cell-cell adhesion and (2) its transmembrane and/or juxtamembrane cytoplasmic domains are critical for its function in vivo. Furthermore, down-regulation of bsg affects the size of postsynaptic receptor fields, as well as the distribution of synaptic vesicles within neuronal terminals. These defects are associated with alterations of the actin/Spectrin network, suggesting that Bsg accumulation at the plasma membrane regulates synaptic compartmentalization and architecture. Strikingly, it was found that Bsg function is also essential within the presynaptic compartment for the restriction of neurotransmitter release. Based on these in vivo data, it is proposed that Bsg may be part of a transsynaptic complex surrounding active zones and involved in the coordinated development of pre- and post-synaptic membranes, as well as in the functional coupling of plasma membrane and cortical subdomains (Besse, 2007).

The transmembrane Ig CAM Bsg has been identified as a new component of perisynaptic zones of Drosophila NMJs. Bsg function is required in pre- and post-synaptic compartments for the formation and growth of synaptic boutons, and Bsg controls different aspects of synapse structure, including distribution of synaptic vesicles and organization of the presynaptic terminal cortical actin network. Bsg behaves as a canonical Ig CAM, since it promotes cell-cell adhesion and has a conserved motif in its cytoplasmic tail essential for its function in vivo. It is proposed that Bsg is part of a transsynaptic complex regulating synaptic growth and structural organization. Moreover, and very originally for an Ig CAM, it was found that Bsg is essential for inhibiting transmitter release, and this function is restricted to the presynaptic compartment (Besse, 2007).

In Drosophila, the final pattern of larval motoneuron connections and the establishment of synapses are complete by the end of embryogenesis, yet NMJs are highly dynamic during larval development, expanding through sprouting of new branches and addition of new synaptic boutons. This study has shown that down-regulation of Bsg levels at the Drosophila NMJ strongly affects bouton growth and budding, resulting in a decrease in bouton number. This effect is probably independent of the increased transmitter release observed in bsg larvae because (1) it is already observed in early second instar larvae and (2), in contrast to the increased neurotransmission phenotype, it corresponds to a requirement for bsg function in both pre- and postsynaptic compartments. This may reflect a role of Bsg in regulating adhesion between pre- and postsynaptic membranes, as described for the Ig CAM Fas II. Bsg function is, however, not restricted to modulation of synaptic membrane adhesiveness; mutant forms lacking the transmembrane and/or juxtamembrane cytoplasmic domains can promote cell-cell aggregation but function poorly in vivo. Bsg may thus also signal toward the cell cytoplasm and/or regulate the actin cytoskeleton (Besse, 2007).

In addition, Bsg controls synaptic architecture: it modulates the size of postsynaptic glutamate receptor fields and, more strikingly, is required for the anchoring of synaptic vesicles to the presynaptic terminal cortex. This suggests that Bsg could be a key component coupling organization of the plasma membrane and cytoplasmic vesicular compartments. Consistent with such a role, defects were observed in 'plasma membrane versus internal membrane' sorting of presynaptic transmembrane components. It is thus proposed that Bsg may be part of a transsynaptic complex surrounding active zones and involved in the coordinated development of pre- and postsynaptic membranes, as well as in the functional coupling of plasma membrane and cytoplasmic vesicles. Notably, Bsg recently has been identified (Takamori, 2006) within synaptic vesicle preparations (Besse, 2007).

Bsg might act directly, or through interaction with transmembrane and cytoplasmic partners. Consistent with this latter hypothesis, it has been shown that conserved amino acids found in the cytoplasmic tail of Bsg are crucial for the function of the protein in vivo and that they may thus mediate transduction of a signal toward the cell cytoplasm and/or interaction with the cortical cytoskeleton (Besse, 2007).

The F-actin/Spectrin cytoskeleton underlying pre- and postsynaptic membranes seems essential for different aspects of synaptic terminal growth and plasticity, including terminal expansion, organization of presynaptic vesicle pools, and postsynaptic receptor clustering. This study has shown that Bsg modulates the organization of the presynaptic actin cytoskeleton, as revealed by the presence of ectopic aggregates of F-actin and actin-associated proteins within the lumen of bsg synaptic boutons. Although no obvious alterations of the postsynaptic actin cytoskeleton, which is intermingled with the dense membraneous network of the SSR, was detected it is nonetheless possible that Bsg also regulates this cytoskeleton. These observations further support previous reports (Schlosshauer, 1995; Curtin, 2005) showing that Bsg colocalizes with the actin cytoskeleton specifically at cell-cell contacts and that expression of Bsg in cultured cells results in reorganization of the F-actin network and consequent formation of lamellipodia (Besse, 2007).

At the NMJ, Bsg may modulate actin cytoskeleton organization indirectly, by interacting with integrin subunits at the plasma membrane. Indeed, mammalian Bsg has been found in a complex with ß1-integrin (Berditchevski, 1997; Xu, 2005), and both α- and ß-integrin subunits colocalize with Drosophila Bsg at larval NMJs. Furthermore, although no genetic interaction was observed between bsg and myspheroid (mys, which encodes the main Drosophila ß-integrin subunit) during larval NMJ development, several mys missense mutations were observed displaying a junction undergrowth phenotype very similar to that of bsg mutants. Another attractive hypothesis is that Bsg, through its juxtamembrane cytoplasmic motif, recruits Spectrin or other actin-associated proteins and thereby directly participates in organizing a cortical actin network. Interestingly, different members of the FERM domain protein family have been shown to link cell surface glycoproteins and the actin cytoskeleton by directly binding to both the intracellular region of transmembrane proteins and to actin or Spectrin. In particular, Moesin directly interacts with the intracytoplasmic domains of mammalian CD43, CD44, and intercellular adhesion molecule 2, through a positively charged amino acid cluster found in the juxtamembrane region of these proteins. The striking conservation and functional importance of the KRR juxtamembrane motif of Bsg suggests that such cytoplasmic proteins may physically link cell-surface Bsg to the underlying F-actin network and mediate organization of cortical domains at the NMJ (Besse, 2007).

Down-regulation of bsg at Drosophila NMJ terminals causes a dramatic increase in transmitter release, which, is unique among Ig CAM mutants. This phenotype corresponds to a specific presynaptic function of Bsg and may be explained by (1) an alteration of the excitability of the synaptic terminal or (2) an altered definition of the different functional synaptic vesicle pools (Besse, 2007).

Mammalian Bsg has been shown to promote translocation of transporter proteins to the plasma membrane, as well as regulate the activity of multiprotein transmembrane complexes (Kirk, 2000; Zhou, 2005). At the Drosophila NMJ, Bsg may thus be required for the proper distribution and/or clustering of ion channels regulating Ca²⁺ dynamics. In this context, it was recently demonstrated that the presynaptic scaffolding protein BRP is required for the clustering of Ca²⁺ channels and for their spatial coupling with synaptic vesicles at the Drosophila active zone. This process appears to be required for the rapid evoked component of synaptic vesicle release but not for spontaneous release (Kittel, 2006). Therefore, both the additional spontaneous and delayed evoked component of transmitter release in bsg mutants might correspond to the fusion of vesicles lacking a tight association with Ca²⁺ channels. An elevated contribution of asynchronous release has also been reported to occur naturally at particular synapses of the mammalian central nervous system and is thought to reflect long-lasting Ca²⁺ transients and a loose coupling between Ca²⁺ sources and vesicles. It is thus conceivable that down-regulation of Bsg alters Ca²⁺ dynamics, leading to an abnormal recruitment of vesicles distant from Ca²⁺ sources (Besse, 2007).

Alternatively, the observed transmitter release phenotype may not be associated with an alteration of Ca²⁺ signals, but rather reflects a role of Bsg in organizing synaptic vesicle populations. It has been suggested that synaptic vesicles are organized into functionally distinct pools (readily releasable pool, recycling pool, and reserve pool) with specific recycling and mobilization properties. This study has shown that down-regulation of bsg leads to an abnormal distribution of vesicles in resting terminals, as well as an aberrant trafficking of vesicles to the center of boutons (where reserve pool vesicles are thought to reside) under conditions where synaptic vesicle recycling is normally restricted to the periphery (and to the recycling pool). These data suggest that the definition of different synaptic vesicle populations may be altered in bsg mutants, which in turn may explain the observed defects in precise recruitment and release of vesicles. This is of particular interest given that mammalian Bsg has been suggested to physically associate with synaptic vesicles (Takamori, 2006). Additionally, presynaptic actin filaments have been proposed to provide a physical barrier impeding vesicle dispersion and, in particular, to regulate the availability of the reserve pool. They have also been suggested to participate in a mechanism restraining fusion of synaptic vesicles in cultured hippocampal neurons. An attractive possibility is therefore that Bsg controls synaptic vesicle organization and retention through its effect on the cortical actin cytoskeleton (Besse, 2007).

The gene for the Drosophila homologue of mammalian basigin was discovered in a screen for genes that act in the eye. This gene maps to cytological band 28E3, spans 25 kb and had been previously designated gelded (gel) (Castrillon, 1993), but has been renamed basigin (bsg). All three mammalian basigin protein sequences were used to search the Drosophila genome for predicted proteins related to basigin, and it was found that bsg is the only Drosophila gene that encodes a basigin family member in flies. According to data from the Drosophila genome project, the bsg gene encodes nine distinct transcripts that appear to fall into three structural classes. These transcripts encode two predicted protein isoforms of D-basigin, 265 and 298 amino acids, each with two IgG-C2 domains, a transmembrane domain and a short internal tail. Class 1 and 2 bsg transcripts contain distinct 5' non-coding exons that splice onto common coding exons to code for D-basigin 265. The class 3 bsg transcript encodes D-basigin 298. The two isoforms are identical over 240 amino acids, differing only at the N- and C-termini (Curtin, 2005).

A Northern blot showed that bsg was expressed in all stages of Drosophila development tested. Only two sizes of bsg message were seen. The larger, more diffuse band corresponded to class 1 and 2 transcripts that have a nearly identical predicted size of ~2.5 kb. Around embryonic stage 5 a slightly smaller transcript was expressed that corresponded more closely to the predicted size of ~2 kb for the class 3 transcript. Since no other transcript sizes were seen, it is concluded that the Drosophila bsg gene probably encodes only the two predicted isoforms of D-basigin. The expression of this smaller transcript at very early stages may mean that it is maternally contributed (Curtin, 2005).

Mouse basigin showed 26% identity and 34% similarity with Drosophila basigin protein. The extracellular domains showed 20% identical residues and 28% similar residues, whereas there was 80% identity in or near the transmembrane domains. Indeed, the transmembrane domains of basigin, neuroplastin and embigin from many different species show very high identity (Ochrietor, 2003), including spaced leucines, as well as conserved proline and glutamic acid residues. The presence of a charged residue in the transmembrane domain is consistent with the fact that basigin forms complexes (Fadool, 1996), possibly within the plane of the membrane. There was no homology in the short internal tail between mouse and D-basigin with the exception of the first five cytoplasmic residues. D-basigin showed 30% similarity to both rat neuroplastin and rat basigin (Curtin, 2005).

To identify new proteins controlling synapse development, proteins specifically accumulating at developing NMJs of Drosophila larvae were sought. A protein-trap screen was performed in which GFP fusion proteins expressed from their endogenous promoters are randomly generated and the expression pattern of ~350 GFP⁺ lines was analyzed. Thereby, ten lines exhibiting GFP expression at the larval NMJ were identified and focus was placed on three independent lines showing strong GFP accumulation at larval NMJs, but only low GFP levels along the motoneuron axons, and at the surface of muscle fibers. In these lines, a strong GFP signal is also observed in different neuropil structures of the larval brain (Besse, 2007).

Using inverse PCR, it was found that in each of these three GFP⁺ protein trap lines the protein-trap cassette was inserted in the gene CG31605, encoding the Drosophila homologue of the mammalian protein CD147/EMMPRIN/Basigin, Basigin (Bsg; Curtin, 2005). According to predictions, the artificial GFP exon should be incorporated upon splicing into mature transcripts whose transcription starts upstream of the insertion, resulting in the in-frame incorporation of GFP. This was confirmed by RT-PCR and Western blot analysis using anti-Bsg antibodies raised against the Drosophila protein (Besse, 2007).

Drosophila Bsg is a small transmembrane protein composed of two extracellular Ig-like domains, a highly conserved transmembrane domain, and a short cytoplasmic tail. Mammalian Basigin has been described as a multifunctional protein regulating different processes, including tumor invasion, reproduction, and sensory and memory functions (Muramatsu, 2003; Toole, 2003). Interestingly, bsg is highly expressed in the mouse nervous system (Fan, 1998), and Bsg protein is present in purified postsynaptic densities (PSDs) of mouse central nervous system synapses (Collins, 2006). In Drosophila, Bsg has been proposed to regulate cellular architecture during eye morphogenesis (Curtin, 2005). The cellular mechanisms underlying its functions, however, are still poorly understood. Bsg is the only Drosophila member of a mammalian protein family including Basigin, Embigin, and Neuroplastin/gp65/gp55. All the members of this family have been suggested to regulate cell-substratum adhesion and/or cell-cell adhesion, and are therefore proposed to belong to the Ig CAM family (Fadool, 1993; Huang, 1993; Kasinrerk, 1999; Smalla, 2000; Besse, 2007 and references therein).

According to data from the Drosophila genome project, bsg encodes nine distinct putative transcripts, of which eight encode the same protein. One of the predicted transcripts, RG, encodes a slightly different protein. However, this transcript is barely detected after quantitative RT-PCR on larval fillet extracts (Besse, 2007).