Chapter 11

SYG/Nephrin/IrreC Family of Adhesion Proteins Mediate Asymmetric CellCell Adhesion in Development

Kang Shen

Abstract A hallmark of the development of the multicellular organisms is the cellcell interaction. One specific type of cellular junction structures that is essential for the function of the nervous system is the chemical synapses. Chemical synapses release chemicals from neurons to their target cells to transmit electrical signals. To establish and maintain such a junctional structure, transsynaptic adhesion molecules play important roles at different developmental stages. Indeed, a number of adhesion molecules have been implicated in the various stages of the life of synapses to bring and hold the pre- and postsynaptic partners together in developing and mature synapses. Relatively little is known about which membrane molecules mediate the initial recognition during the process of synaptogenesis. In this chapter, we will focus our discussion on the IrreC/Nephrin/SYG-1 family of adhesion molecules, whose function in synaptic target selection has been studied in much detail in the nematode Caenorhabditis elegans. Interestingly, the members of this family in Drosophila and vertebrates also play essential functions in mediating the cellular recognition in myoblast fusion, eye morphogenesis, and kidney slit membrane formation. These findings suggest that this group of adhesive molecules carry out asymmetric cellular recognition events in diverse developmental events. Keywords Adhesion molecules Immunoglobulin Superfamily Proteins synapse formation synaptic specificity

11.1 The IrreC/Nephrin/SYG-1 Family of Proteins

This family of proteins includes/encompasses two genes in Caenorhabditis elegans (syg-1 and syg-2), four genes in Drosophila (IrreC, Duf, SNS, and Hibris), and four genes in the human genome (Nephrin, NEPH1, NEPH2,
K. Shen (*) Department of Biology and Pathology, Howard Hughes Medical Institute, Stanford University, 371 Serra Mall, Gilbert 109, Stanford, CA 94305-5020, USA e-mail: kangshen@stanford.edu

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and NEPH3). syg-1 and syg-2 are two closely related genes in the C. elegans genome, suggesting that they might have derived by a gene duplication event from a common ancestoral precursor gene. Two Drosophila genes, IrreC and Duf, are homologous to syg-1 and the other two are to syg-2. In some publications, IrreC has also been referred to as Roughest, and DUF (dumbfounded) as Kirre (Kin of IrreC). The four human genes are also part of this gene family. Nephrin is the sole homolog to syg-2, while the other three human genes are most homologous to syg-1. NEPH1, 2, and 3 have also been named Kirrel1, 3, and 2, respectively (Fig. 11.1).

Fig. 11.1 Phylogenetic analysis of the IrreC/Nephrin/SYG-1 family proteins. Amino acid sequences of full-length proteins were analyzed with sequence cluster method

11.2 SYG-1 and SYG-2 Encode Synaptic Target Choice of the HSNL Neuron in C. elegans
The general specificity of neuronal connections is established through a series of developmental events, including cell migration, axon and dendrite outgrowth, and guidance, followed by target recognition and synapse assembly. Each step gradually limits the pool of possible connecting targets, eventually leading to the target choice. Although a large body of experimental data have provided us with a detailed understanding of the molecular mechanisms of axon guidance, little is known about how neurons make final decisions in selecting their synaptic partners. Based on anatomical and physiological experiments, it is well documented that synaptic connections in the brain are precise and stereotyped (Benson et al. 2001). Therefore, it is very likely that there are molecular mechanisms by which neurons select their correct synaptic partners to initiate synaptic assembly, while rejecting other contacting cells in the same target field. Naturally, one might expect that the molecules mediating the recognition event are directly or indirectly involved in the assembly of the pre- and the postsynaptic apparatus. The most intuitive model that has been generally accepted is that cell adhesion molecules (CAMs) found on pre- and postsynaptic cells mediate specific cell recognition events and that the interaction between these CAMs also initiates synaptogenesis. Surprisingly, little experimental evidence is available to support the existence of membrane molecules that can effectively perform the synapse-inducing cellcell recognition events. For any adhesion molecules to qualify for this

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job, several criteria must be fulfilled. First, these molecules need to be expressed by the synaptic partners at the time of synaptic target selection and synapse formation. Second, these molecules should be present at synapses. Third, the interaction between these adhesion molecules should trigger a molecular assembly program, which results in the construction of the pre- and the postsynaptic apparatus. The fourth and probably the most stringent criteria is that in the absence of these molecules, there should be defects in synaptic target choices. A number of CAMs, which fulfill at least some of these requirements, are likely candidates for acting as synaptic specificity determinants. For example, the neurexin and neuroligin families of membrane adhesion molecules are expressed in neurons and are localized at synapses. More importantly, when expressed in exogenous cells, a neurexin and neuroligin interaction is sufficient to trigger formation of pre- and postsynaptic specializations (Craig and Kang 2007). However, in knockout mice where most or all of the neurexins and neuroligins are deleted, little synapse development phenotypes can be detected (Missler et al. 2003, Varoqueaux et al. 2006). These results suggest that the neurexin and neuroligin molecules are synaptic localized adhesion molecules with their abilities to induce synapse formation (see Chapter 17). But whether they encode specificity is still an open question. Immuoglobulin domain family proteins, called SynCAMs, are also synaptically localized adhesion molecules that have synapse-promoting activities (Biederer et al. 2002) (see Chapter 8). However, their precise roles in synapse development have not been validated by a loss-of-function genetic analysis. EphrinB and Eph receptors are another class of molecules that qualify as prime candidates for molecules for synaptic specificity (see Chapter 16). Both ephrinB and its receptors are localized at synapses and have pre- or postsynaptic inducing activities (Aoto and Chen 2007). Forward genetic analysis using one set of motor neuron synapses in the nematode C. elegans led to the identification of two immunoglobulin super family (IgSF) proteins that fit the bill as synapse-inducing adhesion molecules. The egg-laying behavior of C. elegans is controlled by two pairs of motor neurons, HSNL/HSNR and VC4/VC5. The HSNs form en passant synapses onto vulval muscle cells and onto the VC neurons. Although HSN axons contact many other cells, they do not normally form synapses with them. Furthermore, the egg-laying synapses elaborated by HSNs are clustered in a short and stereotyped segment (about 10 mm) of the HSN axons (at least 100 mm). As expected, the position of the synapses matches the physical location of the postsynaptic targets: the VC neurons and the vulval muscles (Shen et al. 2004). Because of the simplicity of the worm nervous system and the ability of specific labeling of HSN synapses, it is possible to ask several fundamental questions about synapse formation in vivo using this system. For example, do the postsynaptic cells induce the development of presynaptic specializations directly? And what are the molecules that mediate the specificity of synapse development in vivo?

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Fig. 11.2 SYG-1 and SYG-2 determine the localization of presynaptic terminals in HSN axons. A model illustrating the cellular action of SYG-1 and SYG-2. SYG-2 is expressed in guidepost vulval epithelial cells. SYG-1 functions in the HSN axon and is recruited to future presynaptic location via a direct interaction with SYG-2. The SCFsel-10 ubiquitin E3 complex is diffusely localized throughout the HSN axon and is responsible for the degradation of the presynaptic apparatus. SYG-1 binding to Skr-1 inhibits the assembly of the SCF complex and hence locally protects synapses through the suppression of SCF activity

The first surprise that resulted from an analysis of this system was the observation that the postsynaptic cells (VC neurons and vulval muscles) are dispensable for the correct localization of the presynaptic specializations in HSN. In animals in which the postsynaptic cells were ablated by laser-assisted methods prior to the axon guidance event, HSNs still cluster presynaptic vesicles at the right locations. This suggests that the synapse-inducing signal comes from a source other than the postsynaptic cell (Shen and Bargmann 2003). Shen and Bargmann reported that a group of epithelial cells play an essential guidepost role for HSNL synaptogenesis. These guidepost cells contact the HSNL axon and induce the clustering of synaptic vesicles at the site of contact, shortly before the normal postsynaptic targets are innervated. In the absence of guidepost cells, clusters of HSNL synaptic vesicles accumulate at ectopic locations. Further analysis of the guidepost cells showed that they physically contact the HSN axons during the initial specification of the presynapse in HSN. The exact location of the contact between HSN and the guidepost cells defines the location of the synapses. These results suggest that the guidepost cells are not required for HSNs to form synapses per se, but instead, they are required to specify the location of the HSN synapses. A forward genetic screen yielded several mutants with abnormal HSN synapse localizations. In syg-1 and syg-2 mutants, HSN synapses are drastically reduced at the wild-type location and robustly form at anterior ectopic locations along the HSN axon. Interestingly, this aberrant localization pattern closely mimics the synapse localization pattern found in animals with ablated guidepost cells. Molecular cloning of the genes affected in these mutants

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revealed that both the SYG-1 and the SYG-2 genes encode transmembrane IgSF proteins. Furthermore, SYG-1 and SYG-2 are homologous to each other, and they both belong to an evolutionarily conserved family of molecules (Shen and Bargmann 2003, Shen et al. 2004). A further genetic and developmental analysis of these two genes showed that SYG-2 is expressed transiently by the guidepost cells during the early stages of HSNL synaptogenesis. SYG-1 functions in the presynaptic HSNL neuron and localizes to synapses early during synapse formation. In loss-of-function syg-1 and syg-2 mutants, the HSNL axon fails to form synaptic connections with its normal targets (VC neurons and vulval muscles) and instead forms synapses with adjacent cells that do not normally receive synaptic input from the HSNL axon (Shen and Bargmann 2003, Shen et al. 2004). When SYG-2 is expressed in the secondary vulval epithelial cells, which are located next to the guidepost cells and do not normally express SYG-2, both SYG-1 and synaptic vesicles localize to the segment of the HSNL axon that contacts these secondary vulval epithelial cells (Fig. 11.2). This gain-of-function phenotype supports the idea that interactions between SYG-1 and SYG-2 are sufficient to trigger synaptic vesicle clustering. A biochemical analysis showed that the extracellular domains of SYG-1 and SYG-2 are likely to directly interact with each other (Shen et al. 2004). Taken together, these results suggest that SYG-2 is the guidepost molecule. It binds to SYG-1 on the HSN axon and localizes SYG-1 to the future synaptic region. This interaction between SYG-1 and SYG-2 eventually leads to the localized assembly of the presynaptic machinery. These results left several questions unanswered. How does the interaction of SYG-1 and SYG-2 induce synapse formation? Why do synaptic vesicles accumulate at ectopic sites in the syg-1 and syg-2 mutants? How does SYG-1 ensure formation of presynaptic sites at the appropriate location in HSNL? Insights into these questions were obtained by studying the developmental process that leads to the specific distribution of HSN synaptic vesicles. During development, transient presynaptic sites form at multiple locations along the HSNL axon. However, by adulthood, most of these presynaptic sites are eliminated and only those that contain SYG-1 remain. As it turns out, SYG-1 helps achieve this stereotypical presynaptic pattern by playing a protective role. Ding and colleagues recently showed that an E3 ubiquitin ligase, a Skp1-Cullin-F-box (SCF) complex, acts in HSNL to eliminate unwanted presynaptic sites (Ding et al. 2007). Animals with loss-of-function mutations in components of this complex have delayed or incomplete elimination of superfluous presynaptic sites. These results argue that the SCF complex is at least in part responsible for eliminating ectopic synapses during development. However, it is still not clear how SYG-1 can protect synapses format areas where SYG-1 protein is localized. The answer to this question came from experiments examining the subcellular localization and the activity of the SCF complex. It turns out that the SCF complexes are diffusely localized throughout the entire HSN axons, implicating that active SCF can be found on the whole axon. Binding studies revealed that SYG-1 binds to the Skp1

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homolog SKR-1 and prevents it from interacting with the rest of the SCF complex. These results indicate that SYG-1 plays a protective role by locally inhibiting the SCF complex, thus preventing the degradation of presynaptic sites at locations marked by SYG-2 (Fig. 11.2). In the absence of SYG-1 or SYG-2, the activity of the synapse-degrading SCF complex becomes redistributed more toward the normal synaptic region, which leads to fewer synapses in the wild-type location and the appearance of ectopic synapses in the anterior area.

11.3 Kirre/DUF, IrreC/Roughest, SNS, and Hirbris Mediate Myoblast Fusion in Drosophila
The body wall musculature of the Drosophila embryo consists of 30 muscles in each abdominal hemisegment (see Fig. 2.2). During development, each muscle is formed by the fusion of two cell types: a founder cell and fusion-competent myoblasts. The founder cell defines the identity of a particular muscle, and the fusion-competent myoblasts are attracted by and fuse to the founder cell. The location and number of fusion events are thought to determine the shape and size of the muscles (Chen and Olson 2004). Through forward genetic analysis, a large number of mutants were isolated in which myoblast fusion is blocked (Richardson et al. 2008). Among the genes affected by these mutations are four transmembrane IgSF proteins. Dumbfounded/Kirre (Duf) and Roughest/IrreC (Rst), the orthologs of SYG-1, function in the founder cell, while Sticks and Stones (SNS) and Hibris (Hbs), which are orthologous to SYG-2, function in the fusion-competent cells. Similar to the SYG-1 and SYG-2 heterologous interaction, Duf and Rst bind directly to SNS, and these proteins are the primary mediators of myoblast adhesion. Loss-offunction genetic analysis showed that duf and rst act redundantly in the founder cells and removal of both genes leads to a complete fusion defect (Strunkelnberg et al. 2001). Interestingly, removal of SNS also causes a complete fusion defect (Bour et al. 2000). Loss of Hbs causes a mild fusion defect, and thus it is possible that Hbs regulates SNS during particular stages of fusion. Additionally, a zebrafish Kirre/Duf-like molecule is also required for myoblast fusion, suggesting that this pathway is conserved in vertebrates (Srinivas et al. 2007). It is interesting to compare HSN synapse formation and myoblast fusion. These two seemly distinct processes share certain similarities. Both processes involve asymmetric cellcell recognition. In both cases, cellular junction structures form. Interestingly, both these junctional structures are transient. The epithelialHSN junction is eventually replaced by the mature synapses between HSN and its postsynaptic targets. The myoblast fusion junction leads to the perforation of the membrane and the fusion of the two cells. Another intriguing parallel is the asymmetric expression of SYG-1 and SYG-2 and of IrreC/Rst, Kirre/DUF and SNS. In the case of HSN synapse specification, SYG-1

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functions in the HSN neurons, while SYG-2 is specifically expressed and is required in guidepost epithelial cells. SYG-1 predominantly binds to SYG-2 in a heterologous fashion. In the case of myoblast fusion, while weak homophilic interactions of IrreC/Rst and Kirre/Duf have been demonstrated in vitro, heterophilic interactions between SNS and IrreC/Rst, as well as between SNS and Kirre/Duf, are thought to be critical for myoblast fusion.

11.4 Kirre/DUF, IrreC/Roughest, SNS, and Hirbris Are Required for Proper Patterning of the Drosophila Eye
The formation of the Drosophila compound eye involves the generation and alignment of hundreds of identical eye units (ommatidia), which are organized into an ordered array. In the last step of its development, a line of pigment cells forms between neighboring ommatidia to insulate them from each other. During this process, the pool of undifferentiated cells found between the ommatidial clusters the interommatidial precursor cells (IPCs) undergoes morphogenetic movements that eventually create a precise pigment cell lattice. This final patterning process includes carefully regulated cell shape changes, cell movements, and cell death (Rusconi et al. 2000). During this process, IPCs contact other IPCs and the primary pigment cells (18), but selectively form adherent junctions with the primary pigment cells. Thus, one important aspect of this complex morphogenesis event is the cellcell recognition between IPCs and primary pigment cells. The first hint that the IrreC/Nephrin/SYG family protein might play an important role in this process came from the analysis of mutant lines. When ommatidia morphogenesis fails, the mutant eyes exhibit a rough appearance compared with wild-type controls. Interestingly, both Roughest (IrreC) and Hibris mutants exhibit a rough-eye phenotype. Expression analysis revealed that Hibris and IrreC are expressed in complementary cell types. Hibris is made by primary pigment cells, while IrreC is predominantly expressed by IPCs at the time of the adhesion event. Furthermore, both Hibris and IrreC proteins are localized to the interface between these two cell types. In vitro binding assays confirmed the specific interaction between IrreC and Hibris (Bao and Cagan 2005). Taken together, these experiments suggest that the heterologous binding between the IrreC/Nephrin/SYG family proteins across two different cell types specifies another asymmetric cellcell recognition event.

11.5 Vertebrate NEPH1 and Nephrin Are Critical Proteins in Kidney Development
While the function of the IrreC/Nephrin/SYG proteins in synapse formation and muscle fusion in vertebrate animals still awaits further confirmation, these proteins are essential for the formation of the slit diaphragm, a cellular junction

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in the kidney that functions as a molecular filter (Patrakka and Tryggvason 2007). Unlike the results on the nematode SYGs and the fly proteins, where initial mutant analysis using genetic model organisms eventually led to the understanding of the function of these proteins, our knowledge of the Nerphin/NEPH proteins started with human patients. Nephrin was discovered by positional cloning of the gene that is mutated in patients with congenital nephrotic syndrome of the Finnish type, a disease characterized by severe defects in the formation of slit diaphragm in the kidney and by massive proteinuria (Kestila et al. 1998). The slit diaphragm is the endothelial part of the glomerular filter apparatus that permits water and small molecules in the blood to pass into the urinary space, while preventing serum albumin and other larger molecules from being filtered. The slit diaphragm is highly specialized cellcell junction structure formed between the podocyte processes. Until the discovery of nephrin, a major component of this cellular junction, the biochemical nature of the slit diaphragm was elusive. The Tryggvason group identified the genetic lesion responsible for congenital nephrotic syndrome of the Finnish type, a disease characterized by the absence of slit diaphragm and severe proteinuria (Kestila et al. 1998). It turns out that mutations reside in an IgSF protein, which they termed nephrin. It is proposed that homophilic interaction between nephrin molecules across different podocyte processes is critical for the formation of the slit diaphragm. This hypothesis is supported by in vitro experiments, in which expression of full-length nephrin in HEK293 cells leads to cell aggregation (Khoshnoodi et al. 2003). Nephrin is the ortholog of SYG-2 and SNS containing the same number of Ig and fibronectin III domains in its extracellular domain. These data present a homophilic interaction-based model for nephrins function in slit diaphragm. It would be interesting to know whether the vertebrate IrreC/SYG-1 homologs are also involved in the development of the slit diaphragm. The vertebrate genome encode not just one, but three homologs for IrreC/ SYG-1. NEPH1, 2, and 3. They are also named Kirrel1, 2, and 3. NEPH1 is identical to Kirrel1; NEPH2 is the same as Kirrel3, while NEPH3 is Kirrel2. Indeed, both NEPH1 and NEPH2 are found in slit diaphragm (Barletta et al. 2003, Gerke et al. 2003). The involvement of NEPH1 in kidney development is further supported by genetic loss-of-function experiments. NEPH1-deficient mice exhibit a slit diaphragm defect (Donoviel et al. 2001). The injection of antibodies raised against nephrin and NEPH1 into kidneys causes proteinuria, further suggesting that both nephrin and NEPH1 play important roles in the formation of the slit diaphragm. In vitro binding data seem to suggest that nephrin and NEPH1 engage in predominantly homophilic interactions among themselves. However, it is currently not clear whether strict homophilic interaction or heterophilic binding between the nephrin and the NEPH is the driving force for the development of the slit diaphragm junction. Another interesting debate about NEPHs and nephrin is whether they are merely adhesion molecules that hold the two pieces of the podocyte membrane

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together, or they are signaling molecules that induce the differentiation and intracellular organization in podocyte cells. Several lines of evidence suggest that the intracellular domain of the nephrin protein is capable of assembling signal complexes and organizing cytoskeleton structures. A number of conserved tyrosine residues were found in the cytoplasmic tail of nephrin, which has been demonstrated to be the origin of signaling (Jones et al. 2006, Verma et al. 2006). Through binding to the SH2 domain of Nck1 and Nck2, the nephrin intracellular domain recruits these two adaptor proteins. Nck1 and Nck2 also contain SH3 domains, and the SH3 domains are responsible for recruiting other proteins associated with the actin cytoskeleton (Jones et al. 2006, Verma et al. 2006). Animals lacking Nck1 and Nck2 exhibit developmental defect in the formation of slit diaphragm and proteinuria. Other than the well-established role of nephrin and NEPH1 in kidney development, do the vertebrate members of the SYG family also function in other tissue such as the central nervous system, similar to the SYG proteins in C. elegans? Indeed, NEPH1 and NEPH2 are both expressed at synaptic sites in the brain, and NEPH1 and NEPH2 physically associate with CASK, a synaptic scaffolding protein, suggesting that NEPH proteins may also play a role in synapse formation in the vertebrate CNS (Gerke et al. 2006). In addition, NEPH2 and NEPH3 are expressed in olfactory glomeruli, and gain-of-function experiments suggest that SYG-1 orthologs may be involved in olfactory axon sorting and targeting (Serizawa et al. 2006). Serezawa et al. demonstrated that olfactory sensory neuron either express high level of NEPH2 and low level of NEPH3 or vice versa, but never express high level of both proteins. The ratio of these two putative adhesion molecules is one of the determinants for the topographical targeting of olfactory sensory neurons in the olfactory bulb.

11.6 Summary
The IrreC/Nephrin/SYG family of IgSF proteins are a group of evolutionarily conserved membrane proteins. The heterophilic and homophilic interactions between the family members are involved in different cellcell recognition events during development. In the cases of HSN synapse formation in C. elegans, and myoblast fusion and the patterning of the compound eye in flies, the asymmetry of the cellcell recognition is mediated by the specific expression of particular members of this protein family in one of the two interacting cell types. These data hint that the expression of IrreC/Nephrin/SYG proteins determines the fate of these cells in the asymmetric recognition events. In the case of the slit diaphragm formation in vertebrates, symmetric adhesion might be provided by homophilic interaction between nephrin and NEPH1 molecules. Another common feature of these four different recognition events is that all of them lead to intracellular signaling processes, although with diverse cellular outcomes. SYG-2/SYG-1 interactions lead to the assembly of organelles and

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presynaptic proteins adjacent to SYG-1 and the disassembly of presynaptic structures away from SYG-1. DUF and IrreC/SNS interactions recruit multiple structural and signaling proteins to the site of contact and lead to the formation of the perfusion complex between the muscle founder cell and the fusioncompetent cells, which eventually leads to the fusion of two cells. IrreC/Hibris interactions position the interommatidial precursor cells against primary pigment cells and induce the formation of adherent junctions. Finally, nephrin/ nephrin interactions recruit Nck1 and Nck2 and rearrange the actin cytoskeleton in the development of the slit diaphragm. Many interesting questions about this family of proteins still need to be answered. What we have learned so far indicates that specific developmental events in different organisms and tissues depend on IrreC/Nephrin/SYG genes. A systematic analysis of the neuronal wiring in vertebrates and invertebrates will shed light on how important this family of genes is for the establishment of neural circuits. It will also be of interest to examine nephrin- and NEPHsknockout mice for possible muscle phenotypes. The structural basis of the heterophilic binding between SYG-1 and SYG-2, and IrreC and SNS will also be an interesting subject since many of the known Ig domain interactions are homophilic.

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