InteractiveFly: GeneBrief

sticks and stones: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - sticks and stones

Synonyms - rost

Cytological map position - 44F1

Function - surface receptor

Keywords - mesoderm, myoblasts, fusion competent cells

Symbol - sns

FlyBase ID: FBgn0024189

Genetic map position - 2R

Classification - Immunoglobulin C2 type domain, Fibronectin type III domain

Cellular location - surface transmembrane

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Na, J., Sweetwyne, M. T., Park, A. S., Susztak, K. and Cagan, R. L. (2015). Diet-induced podocyte dysfunction in Drosophila and Mammals. Cell Rep 12: 636-647. PubMed ID: 26190114
Diabetic nephropathy is a major cause of end-stage kidney disease. Characterized by progressive microvascular disease, most efforts have focused on injury to the glomerular endothelium. Recent work has suggested a role for the podocyte, a highly specialized component of the glomerular filtration barrier. This study demonstrates that the Drosophila nephrocyte, a cell analogous to the mammalian podocyte, displays defects that phenocopy aspects of diabetic nephropathy in animals fed chronic high dietary sucrose. Through functional studies, an OGT-Polycomb-Knot-Sns pathway was identified that links dietary sucrose to loss of the Nephrin ortholog Sticks and stones (Sns). Reducing OGT through genetic or drug means is sufficient to rescue loss of Sns, leading to overall extension of lifespan. Upregulation of the Knot ortholog EBF2 is demonstrated in glomeruli of human diabetic nephropathy patients and a mouse ob/ob diabetes model. Furthermore, rescue was demonstrated of Nephrin expression and cell viability in ebf2-/- primary podocytes cultured in high glucose. Therefore, this study provides evidence for a pathway that includes flux through the hexosamine biosynthetic pathway and the Polycomb gene complex, which in turn regulates the transcription factor Knot to regulate Sns expression. In cultured mouse primary podocytes, the Knot ortholog EBF2 similarly mediated response by Nephrin to high dietary sucrose. Finally, it was demonstrated how a chemical inhibitor of hexosamine flux can improve the whole-animal response to high dietary sucrose, providing a guideline for candidate therapeutics.

Ivy, J. R., Drechsler, M., Catterson, J. H., Bodmer, R., Ocorr, K., Paululat, A. and Hartley, P. S. (2015). Klf15 is critical for the development and differentiation of Drosophila nephrocytes. PLoS One 10: e0134620. PubMed ID: 26301956
Insect nephrocytes are highly endocytic scavenger cells that represent the only invertebrate model for the study of human kidney podocytes. Despite their importance, nephrocyte development is largely uncharacterised. This work tested whether the insect ortholog of mammalian Kidney Kruppel-Like Factor (Klf15), a transcription factor required for mammalian podocyte differentiation, was required for insect nephrocyte development. Expression of Drosophila Klf15 (dKlf15, previously known as Bteb2) was found to be restricted to the only two nephrocyte populations in Drosophila, the garland cells and pericardial nephrocytes. Loss of dKlf15 function led to attrition of both nephrocyte populations and sensitised larvae to the xenotoxin silver nitrate. Although pericardial nephrocytes in dKlf15 loss of function mutants were specified during embryogenesis, they failed to express the slit diaphragm gene sticks and stones and did not form slit diaphragms. Conditional silencing of dKlf15 in adults led to reduced surface expression of the endocytic receptor Amnionless and loss of in vivo scavenger function. Over-expression of dKlf15 increased nephrocyte numbers and rescued age-dependent decline in nephrocyte function. The data place dKlf15 upstream of sns and Amnionless in a nephrocyte-restricted differentiation pathway and suggest dKlf15 expression is both necessary and sufficient to sustain nephrocyte differentiation. These findings explain the physiological relevance of dKlf15 in Drosophila and imply that the role of KLF15 in human podocytes is evolutionarily conserved.

Kuckwa, J., Fritzen, K., Buttgereit, D., Rothenbusch-Fender, S. and Renkawitz-Pohl, R. (2015). A new level of plasticity: Drosophila smooth-like testes muscles compensate failure of myoblast fusion. Development [Epub ahead of print]. PubMed ID: 26657767
The testis of Drosophila resembles an individual testis tubule of mammals. Both are surrounded by a sheath of smooth muscles, which in Drosophila are multinuclear and originate from a pool of myoblasts that are set aside in the embryo and accumulate on the genital disc later in development. These muscle stem cells start to differentiate early during metamorphosis and give rise to all muscles of the inner male reproductive system. Shortly before the genital disc and the developing testes connect to each other, multinuclear nascent myotubes appear on the anterior tips of the seminal vesicles. This study shows that adhesion molecules were distinctly localized on the seminal vesicles; founder cell-like myoblasts (FC-like) expressed Dumbfounded (Duf) and Roughest (Rst), and fusion-competent myoblast-like cells (FCM-like) mainly expressed Sticks and stones (Sns). The smooth but multinuclear myotubes of the testes arose by myoblast fusion. RNAi-mediated attenuation of Sns or both Duf and Rst severely reduced the number of nuclei in the testes muscles. Duf and Rst likely acted independently in this context. Despite reduced fusion, myotubes migrated onto the testes, testes were shaped and coiled, muscle filaments arranged as in the wild-type, and spermatogenesis proceeded normally. Hence, the testes muscles compensated for fusion defects so that the myofibres encircling the adult testes are indistinguishable from those of the wild- type and male fertility is guaranteed.

The body wall muscles in the Drosophila larva arise from interactions between Dumbfounded/Kirre and Irregular chiasm C-roughest (IrreC-rst)-expressing founder myoblasts and Sticks-and-stones (Sns)-expressing fusion competent myoblasts in the embryo. Sns, a member of the immunoglobulin superfamily that is essential for myoblast fusion (Bour, 2000), mediates heterotypic adhesion of S2 cells with Duf/Kirre and IrreC-rst-expressing S2 cells, and colocalizes with these proteins at points of cell contact. These properties are independent of their transmembrane and cytoplasmic domains, and are observed quite readily with GPI-anchored forms of the ectodomains. Heterotypic interactions between Duf/Kirre and Sns-expressing S2 cells occur more rapidly and to a greater extent than homotypic interactions with other Duf/Kirre-expressing cells. In addition, Duf/Kirre and Sns are present in an immunoprecipitable complex from S2 cells. In the embryo, Duf/Kirre and Sns are present at points of contact between founder and fusion competent cells. Moreover, Sns clustering on the cell surface is dependent on Duf/Kirre and/or IrreC-rst. Finally, although the cytoplasmic and transmembrane domains of Sns are expendable for interactions in culture, they are essential for fusion of embryonic myoblasts (Galletta, 2004).

In most organisms, muscle fibers are composed of large multinucleate cells generated by the fusion of committed myoblasts. Prerequisites to this process include recognition by, migration toward, and adhesion to, other myoblasts. In Drosophila, formation of the larval body wall muscles involves two distinct myoblast populations -- founder cells and fusion competent cells, each of which is essential for formation of multinucleate fibers. Founder cells control the unique identities of each muscle fiber, and seed the fusion process. In contrast, the fusion competent myoblasts, which comprise a much larger population, recognize, migrate to, and adhere to a founder cell. Coincident with and subsequent to myoblast adhesion, intracellular events include the appearance of electron dense vesicles at sites of cell-cell contact, resolution of these vesicles into 'fusion plaques', and vesiculation of the cell membrane. The fusion competent myoblasts are then thought to take on the identity of the founder cell with which they fuse (Galletta, 2004 and references therein).

Implicit in the above mechanism is an apparent requirement that founder and fusion competent myoblasts recognize each other, and studies have revealed that members of the immunoglobulin superfamily (IgSF) serve this purpose. Sticks and stones (Sns), which is found exclusively on the surface of the fusion competent myoblasts, appears in cells just prior to fusion and decreases rapidly thereafter. It is essential for fusion of both somatic and visceral muscles (Bour, 2000 and Klapper, 2002), and the fusion competent myoblasts of sns mutant embryos do not migrate to or associate with the founder cells (reviewed in Abmayr, 2003). Sns shares significant homology with Hibris (Hbs), which appears to play a nonessential regulatory role in myoblast fusion, since both ectopic expression and loss of Hbs cause subtle defects in myoblast fusion that do not result in lethality. Two additional IgSF members, Dumbfounded/Kin of IrreC (Duf/Kirre) and Irregular chiasm C-roughest (IrreC-rst), serve redundant functions in the embryonic founder cells. Specifically, removal of both genes results in a complete absence of myoblast fusion, and targeted expression of either Duf/Kirre or IrreC-rst is sufficient to rescue the muscle loss. Fusion competent cells fail to migrate toward their founder cell partners in embryos lacking both duf/kirre and irreC-rst, and both can serve as attractants for the fusion competent myoblasts when ectopically expressed. While Duf/Kirre expression is limited to the founder cells, IrreC-rst is likely expressed in some fusion competent cells as well as the founder cells (Galletta, 2004 and references therein).

The differential expression of Sns in the fusion competent myoblasts and either Duf/Kirre or IrreC-rst in the founder cells, along with their respective loss-of-function phenotypes, supports a model in which these molecules mediate recognition and adhesion between founder and fusion competent cells. Consistent with this suggestion, Schneider line 2 (S2) cells transiently transfected with Sns adhere to cells that express Duf/Kirre (Dworak, 2001). While the in vivo relevance remains unclear, studies have also demonstrated that Duf/Kirre and IrreC-rst can direct homotypic aggregation when expressed in S2 cells, and accumulate at points of cell–cell contact (Galletta, 2004).

To better understand the roles of Duf/Kirre, IrreC-rst, and Sns, their behavior was examined in both cultured S2 cells and intact embryos. Consistent with a direct role in cell adhesion, it was demonstrated that all three proteins are enriched and colocalize at points of cell-cell contact within S2 cell clusters. Moreover, Duf/Kirre and Sns colocalize at points of contact between fusion competent and founder-cells in the embryo, and are present in a complex that can be immunoprecipitated from heterotypic S2 cell clusters. In both S2 cells and in embryos, localization of Sns is dependent on the presence of Duf/Kirre and/or IrreC-rst-expressing cells, and requires only the extracellular domain. Although the cytoplasmic and transmembrane domains are absolutely essential for myoblast fusion, neither aggregation nor colocalization is dependent on their presence. Thus, these data support a model in which Sns mediates cell adhesion through its extracellular domain and plays a role in other aspects of myoblast fusion through its cytoplasmic domain and/or transmembrane region (Galletta, 2004).

Previous studies have demonstrated that Duf/Kirre and IrreC-rst, but not Sns, can mediate homotypic aggregation of S2 cells. The behavior of Duf/Kirre and IrreC-rst is similar to that of their vertebrate ortholog Neph1. Unlike Sns, however, its vertebrate ortholog nephrin interacts homotypically (Khoshnoodi, 2003). In any case, Sns can clearly direct aggregation with cells that express Duf/Kirre (Dworak, 2001) or IrreC-rst. These data, in combination with the Sns, Duf/Kirre and IrreC-rst loss of function phenotypes and embryonic patterns of expression, suggest that these molecules mediate interaction between founder and fusion competent myoblasts (Galletta, 2004).

Using indirect immunofluorescence to identify the cells expressing Duf/Kirre, IrreC-rst and Sns, as well as the specific location of these proteins within the cell, it has been established that cells expressing any one of these proteins do not adhere to untransfected S2 cells. Thus, cell adhesion requires the presence of these proteins on opposing cells, and cell surface molecules endogenous to S2 cells are not sufficient to direct aggregation with Duf/Kirre, IrreC-rst or Sns-expressing cells. In addition, examination of subcellular localization revealed enrichment of IrreC-rst, Duf/Kirre and Sns at points of contact between S2 cells in an adherent pair. Such sites of co-localization are observed in embryonic cells in a segmentally repeated pattern reminiscent of the spatial distribution of founder cells. Most convincingly, Duf/Kirre and Sns co-localization is observed at points of contact between elongated founder cells and associated fusion competent myoblasts. Thus, these molecules are present at the appropriate time and subcellular location to mediate a direct cell-cell interaction. Finally, Sns is closely associated with Duf/Kirre, as evidenced by their co-immunoprecipitation from S2 cell aggregates (Galletta, 2004).

While these data strongly support the direct association of Sns with Duf/Kirre, they do not eliminate the possibility that either protein requires interaction in cis with a molecule endogenous to S2 cells to interact in trans. However, two additional observations argue against such a mechanism. These findings relate to the behavior of Sns and Duf/Kirre ectodomains when localized to the cell membrane with a GPI-anchor. These truncated molecules are able to direct cell adhesion quite effectively. Thus neither the transmembrane nor the cytoplasmic domains, or any proteins that associate in cis with these domains, are necessary for cell adhesion. Moreover, GPI-linked Sns does not function as a dominant inhibitor when overexpressed in the embryonic musculature, as one might anticipate if the extracellular domain normally mediates an essential interaction in cis. Though indirect, and not definitive, these data strongly support a direct interaction between Sns and Duf/Kirre (Galletta, 2004).

Although Duf/Kirre and IrreC-rst are capable of directing homotypic adhesion when expressed in S2 cells, the biological relevance of these interactions in the embryonic musculature is unclear. Numerous studies suggest that Duf/Kirre, IrreC-rst-expressing founder cells seed the fusion process, and pattern the resulting muscle fibers through information transferred to the naïve fusion competent cells as fusion progresses. Moreover, founder cells do not appear to interact with each other in the embryo. The data demonstrates that Duf/Kirre-expressing S2 cells have a greater affinity for cells expressing Sns than they do for other cells expressing Duf/Kirre. This observation may reflect a difference that also occurs in vivo, such that founders have a higher affinity for fusion competent cells than for other founder cells. This difference in affinity, coupled with the fact that fusion competent myoblasts outnumber founder cells by at least 10 to 1, may underlie preferential founder:fusion competent cell association (Galletta, 2004).

The absence of the transmembrane and cytoplasmic domains of Duf/Kirre has a significant effect on formation and/or stability of homotypic cell interactions. By contrast, the cytoplasmic/transmembrane domains of Duf/Kirre and Sns play little, if any, role in their ability to direct heterotypic cell adhesion. One potential explanation for this effect is that the apparently weaker homotypic interaction of Duf/Kirre relies more heavily on stabilization provided by association with the cytoskeleton, and is therefore more sensitive to removal of the cytoplasmic domain. Interestingly, the cytoplasmic domain of Duf/Kirre can interact with Anti-social/Rolling pebbles (Ants/Rols), which recruits the cytoskeletal protein D-titin to distinct points between founders and fusion competent cells. The Duf/Kirre interaction with Sns, which appears to form more efficiently or stably, may be less sensitive to the presence of the corresponding cytoplasmic domains (Galletta, 2004).

Examination of cell adhesion driven by other IgSF members has revealed examples in which the cytodomain is important, such as PECAM, and examples in which it is dispensable, such as L1-CAM and neuroglian. Interestingly, several IgSF proteins associate with components of the cytoskeleton, including L1-CAM, ICAM-1, ICAM-2, and CEACAM1-L, possibly to stabilize interactions with other proteins (Galletta, 2004).

Although not necessary to promote adhesion of S2 cells, the transmembrane and/or cytoplasmic domains of Sns are absolutely essential in the embryo. Since Sns is necessary for migration of fusion competent cells toward the founder myoblasts (Abmayr, 2003), and this behavior can be rescued by full length, but not GPI-anchored Sns, the cytodomain/transmembrane requirement likely reflects a role for Sns in directing migration of the fusion competent cells (Galletta, 2004).

There is already ample evidence implicating other IgSF proteins in cell signaling through their cytoplasmic tails. For example, migratory responses to axon guidance cues are controlled by the cytoplasmic tails of Roundabout (Robo) and Frazzled. Moreover, the Robo cytoplasmic domain interacts with such signaling molecules as Enabled and Abelson. It remains to be determined whether the Sns domain also functions in events subsequent to cell migration and adhesion. Intracellular events critical to myoblast fusion include the recruitment of organelles to points of contact between myoblasts. Binding to the cytoplasmic domains of Duf/Kirre or Sns may provide a mechanism by which proteins or vesicles can be recruited to sites of membrane fusion. For example Ants/Rols, which interacts with the Duf/Kirre cytodomain, can also interact physically with MBC, a molecule implicated in the Rac1 pathway. Ants/Rols, MBC, and the small GTPases Rac1 and Rac2 are all required for myoblast fusion, suggesting that the Duf/Kirre cytodomain may recruit molecules essential to fusion. In addition, Loner, an ARF guanine nucleotide exchange factor that is essential for myoblast fusion, also co-localizes with Duf/Kirre in the founder myoblasts. Whether its cytodomain is associated solely with migration or with both migration and fusion, Sns appears to act as a signaling molecule that likely mediates its effects through interaction with intracellular components (Galletta, 2004).

Sns and Kirre, the Drosophila orthologs of Nephrin and Neph1, direct adhesion, fusion and formation of a slit diaphragm-like structure in insect nephrocytes

The Immunoglobulin superfamily (IgSF) proteins Neph1 and Nephrin are co-expressed within podocytes in the kidney glomerulus, where they localize to the slit diaphragm (SD) and contribute to filtration between blood and urine. Their Drosophila orthologs Kirre (Duf) and Sns are co-expressed within binucleate garland cell nephrocytes (GCNs) that contribute to detoxification of the insect hemolymph by uptake of molecules through an SD-like nephrocyte diaphragm (ND) into labyrinthine channels that are active sites of endocytosis. The functions of Kirre and Sns in the embryonic musculature, to mediate adhesion and fusion between myoblasts to form multinucleate muscle fibers, have been conserved in the GCNs, where they contribute to adhesion of GCNs in the 'garland' and to their fusion into binucleate cells. Sns and Kirre proteins localize to the ND at the entry point into the labyrinthine channels and, like their vertebrate counterparts, are essential for its formation. Knockdown of Kirre or Sns drastically reduces the number of NDs at the cell surface. These defects are associated with a decrease in uptake of large proteins, suggesting that the ND distinguishes molecules of different sizes and controls access to the channels. Moreover, mutations in the Sns fibronectin-binding or immunoglobulin domains lead to morphologically abnormal NDs and to reduced passage of proteins into the labyrinthine channels for uptake by endocytosis, suggesting a crucial and direct role for Sns in ND formation and function. These data reveal significant similarities between the insect ND and the SD in mammalian podocytes at the level of structure and function (Zhuang, 2009).

In Drosophila, the Immunoglobulin superfamily (IgSF) proteins encoded by kin of irre [kirre; also known as dumbfounded (duf)], roughest (rst), sticks and stones (sns) and hibris (hbs) function as ligand-receptor pairs on the surface of founder cells and fusion competent myoblasts. These proteins mediate recognition, adhesion and fusion to form multinucleate syncitia through direct interaction at sites of myoblast contact. However, neither their action nor their expression is exclusive to the musculature, and previous studies noted their role in cell recognition and adhesion in the Drosophila eye. Moreover, multiple studies have confirmed the presence of the kirre transcript and sns transcript in the binucleate garland cell nephrocytes (GCNs). These nephrocytes possess a structure visible by transmission electron microscopy (TEM) reminiscent of the slit diaphragm (SD) in the vertebrate kidney, and process waste products from the hemolymph. It is therefore compelling that the fly detoxification machinery may have similarities to that in mammals, and that Sns and Kirre play roles similar to those of their vertebrate counterparts (Zhuang, 2009).

Removal of waste products from the closed circulatory system of vertebrates takes place in the kidney glomerulus. Podocytes, kidney epithelial cells that surround the capillary blood vessels, extend foot processes that contact the surface of these vessels. Filtration then occurs as molecules flow out of the bloodstream through slits between adjacent foot processes into the urine. Neph1, vertebrate orthologs of the above Drosophila IgSF proteins, localize to this filter and appear to be an important determinant of glomerular permeability (Hamano, 2002; Liu, 2003). Mutations in nephrin and neph1 are associated with congenital nephrotic syndrome as a consequence of defects in this filtration diaphragm. Lack of either nephrin or neph1 leads to podocyte foot process effacement and detachment of podocytes from the glomerular basement membrane, loss of SDs, and proteinuria (Donoviel, 2001; Putaala, 2001; Zhuang, 2009 and references therein).

In addition to their high degree of homology, Nephrin and Neph1 have other features in common with Sns and Kirre. Heterophilic interactions occur in trans between the extracellular domains of Nephrin and Neph1, and Sns and Kirre. Studies have suggested that, in addition to serving as a scaffold onto which other proteins in the SD assemble, Nephrin and Neph1 function as signaling molecules to direct downstream cytoplasmic events (Benzing, 2004). They cooperate to transduce a signal that directs actin polymerization (Garg, 2007), and activation of this pathway occurs through interaction of phosphorylated tyrosines in the cytoplasmic domains of Nephrin and Neph1 to adaptor proteins (Jones, 2006; Verma, 2006). These adaptor proteins recruit components of the actin polymerization machinery that include N-WASp and Arp2/3. Similar phosphotyrosine modifications are important for Sns function and studies have shown that the WASp and Arp2/3 actin polymerization machinery functions in Drosophila myoblast fusion, probably downstream of Sns and Kirre (Zhuang, 2009).

The pericardial cells and garland cells comprise two subpopulations of Drosophila nephrocytes that, along with Malpighian tubules, form the excretory system. Approximately 25-30 tightly associated binucleate GCNs encircle the anterior end of the proventriculus in a 'garland' at its junction with the esophagus. The cortical region of the cytoplasm includes elaborate channels that are generated by invagination of the plasma membrane during embryogenesis and early larval instar stages. The initial invagination is associated with formation of a junction between two sites on the plasma membrane that are visible by TEM. Through a mechanism that is not entirely clear, this initial invagination expands into an extensive array of labyrinthine channels by the third-instar larval stage. The GCNs are very active in endocytosis via coated vesicles at sites deep within these labyrinthine channels. Thus, molecules to be eliminated must gain access to the endocytic machinery deep in these channels. These studies also identified a thin bridge spanning the channel opening that is visually similar to the vertebrate SD. The presence of Sns and Kirre and a slit diaphragm-like structure in these binucleate cells raised the possibility that these IgSF proteins might function in GCN fusion and/or in formation of this structure (Zhuang, 2009).

This study, along with that of Weavers (2009) demonstrates that Sns and Kirre are present in, and crucial for, the nephrocyte diaphragm (ND). Knockdown of Kirre or Sns results in a severely diminished number of NDs and smoothening of ND-associated furrows on the GCN surface, implicating Sns and Kirre in their formation. Mutations in the extracellular domain of Sns cause major perturbations in the ND, establishing that Sns also dictates fundamental aspects of its structure. Similar smoothening of the GCN surface occurs upon knockdown of Polychaetoid (Pyd), the Drosophila ortholog of the zonula occludens (ZO-1) tight junction protein that interacts with Neph1, providing strong support for functional conservation of these molecules. The ND controls access of molecules to the labyrinthine channels for uptake by endocytosis, and can discriminate between molecules of different sizes in a rate-dependent manner. Finally, in contrast to that reported by Weavers (2009) and reminiscent of their action in the embryonic musculature, Sns and Kirre contribute to the adhesion of the GCNs into an organized garland and their fusion into binucleate cells (Zhuang, 2009).

These data those of Weavers (2009) demonstrate that the GCNs have significant structural and functional similarities to podocytes in the mammalian kidney. Sns and Kirre are instrumental in directing and/or stabilizing interactions at sites of membrane invagination that become the NDs. These proteins parallel the role of their mammalian orthologs Nephrin and Neph1 in the SD that forms between podocyte foot processes in the kidney glomerulus. In addition, Sns and Kirre mediate tight adhesion between GCNs in the embryo, and, in contrast to Weavers this study notes that these proteins also direct GCN fusion. Both proteins are expressed during larval life and significant cell death occurs in their absence. Sns clearly plays a specific structural role in the ND that is perturbed by mutations in its extracellular domain. Finally, the SD and ND both mediate the flow of molecules between the circulatory system and the excretory system, and appear to discriminate between molecules on the basis of size and rate of passage (Zhuang, 2009).

The GCNs are thought to process waste material and detoxify the insect hemolymph, its open circulatory system, through a process of endocytosis and degradation. Endocytosis occurs from sites deep within labyrinthine channels that form by invagination of the plasma membrane, and proteins associated with endocytosis localize to the cortical region of the cytoplasm in membranes associated within these channels. The channels and associated membranes expand in mutants that block endocytosis, and compounds such as horseradish peroxidase, dye-conjugated BSA or avidin, and various dextrans, readily pass through the plasma membrane into these channels. Access appears to occur through a structure that is reminiscent of the SD in vertebrates. This study has shown that this nephrocyte diaphragm is dependent on the presence of Sns and Kirre, and that perturbation of the Sns extracellular domain causes obvious defects in the ND. Thus, IgSF homologs appear to be a structural component of this access point in both insects and vertebrates (Zhuang, 2009).

The number of NDs decreases significantly upon knockdown of Sns or Kirre, but a small number still remain. The uptake of large molecular tracers is severely diminished under these conditions, suggesting that the NDs are a major route of access to the endocytic machinery within the labryinthine channels. Perhaps more revealing relative to the initial findings of Weavers, it was found that the uptake of small molecules is slower under conditions of Sns or Kirre knockdown but ultimately achieves normal levels. Thus, like the SD, the ND appears to be more permeable to small molecules. Interestingly, studies in vertebrates have addressed the relative contributions of the podocyte basement membrane and the slit diaphragm to glomerular permeability, and Nephrin and Neph1 were found to be crucial. Moreover, electron tomography has identified Nephrin as a decisive determinant for filtration of molecules larger than BSA (Zhuang, 2009).

Nephrin and Neph1 are capable of forming both homodimers and heterodimers, and these abilities could reflect interactions that occur in vivo in cis and/or in trans. The diameter of the vertebrate SD is consistent with a model in which this distance could be spanned by homophilic interaction of Nephrin or heterophilic interaction between Neph1 and Nephrin in trans. The similar diameter of the Drosophila ND therefore supports a model in which interactions between the Kirre and Sns ectodomains determine this distance. The exact molecular interactions remain to be determined, however, and may differ in vertebrates and Drosophila. For example, Nephrin is capable of homophilic interactions in trans, a property that Sns does not appear to have. Thus, it seems unlikely that Sns spans this distance, as suggested for Nephrin. Homophilic interactions of Kirre, which can occur, could serve this purpose. One might then predict the spacing to be decreased from the observed 30-35 nm due to the shorter extracellular domain of Kirre. Of note, kinetic studies in Drosophila S2 cells indicate a strong preference for interaction with Sns. Moreover significant levels of Sns or Kirre remain in GCNs from second instar larvae upon knockdown of the corresponding partner, yet the number of NDs is diminished. Localization of each protein by immunoEM analysis under these conditions may prove to be illustrative in this regard. Given the above interaction studies and fact that both proteins are continuously present in the GCN, a model is favoed in which heterotypic interactions are preferred as in the embryonic musculature. One fundamental difference between Sns and Kirre in the embryonic musculature and the GCNs is that they are expressed in different myoblast cell types but co-expressed within individual garland cells. However, their co-expression in GCNs is another feature in common with Nephrin and Neph1 in vertebrate podocytes (Zhuang, 2009).

It is unclear whether Sns and Kirre function through interactions with signal transduction components that parallel those of Nephrin and Neph1 in the GCNs. Signaling molecules thought to be downstream of Sns and/or Kirre in the musculature, and known to be downstream of Nephrin, include N-WASp and components of the Arp2/3 pathway. One other functional parallel between the SD and ND is that of the tight junction protein Pyd, which contributes to formation of ND-associated furrows on the surface of the GCN. Although Pyd interacts biochemically with two different forms of Kirre, it remains to be shown whether this interaction occurs through postsynaptic density-95/disks large/zonula occludens-1 (PDZ)-binding sites in Kirre, as observed for binding of its vertebrate counterpart ZO-1 to Neph1 (Zhuang, 2009).

GCNs become binucleate before or immediately after their assimilation into the garland of cells that surrounds the esophagus at its junction with the proventriculus. This binucleate nature seems almost invariant, with cells rarely remaining mononucleate or having more than two nuclei. Although an explanation for this invariance is not apparent, the cell appears to accommodate multiple processes to ensure it. Quantitation of cells and nuclei over time, the absence of dying GCNs, and time-lapse imaging suggest that cell fusion is the primary mechanism utilized by wild-type GCNs, and that the IgSF proteins contribute to this process. Some mutant cells are still binucleate, but the possibility cannot be eliminate that other molecules contribute to GCN fusion or that these IgSF proteins function in yet more redundant ways to drive this fusion. Perhaps a drive to become binucleate has forced the cell to compensate for defects in fusion in other ways, such as cell division without cytokinesis. Although all efforts to address such a mechanism have yielded negative results, behavior of this type may be another common property between insect garland cell nephrocytes and mammalian podocytes (Zhuang, 2009).


Notch controls cell adhesion in the Drosophila eye

Sporadic evidence suggests Notch is involved in cell adhesion. However, the underlying mechanism is unknown. This study has investigated an epithelial remodeling process in the Drosophila eye in which two primary pigment cells (PPCs) with a characteristic 'kidney' shape enwrap and eventually isolate a group of cone cells from inter-ommatidial cells (IOCs). This paper shows that in the developing Drosophila eye the ligand Delta is transcribed in cone cells and Notch is activated in the adjacent PPC precursors. In the absence of Notch, emerging PPCs fail to enwrap cone cells, and hibris (hbs) and sns, two genes coding for adhesion molecules of the Nephrin group that mediate preferential adhesion, are not transcribed in PPC precursors. Conversely, activation of Notch in single IOCs leads to ectopic expression of hbs and sns. By contrast, in a single IOC that normally transcribes rst, a gene coding for an adhesion molecule of the Neph1 group that binds Hbs and Sns, activation of Notch leads to a loss of rst transcription. In addition, in a Notch mutant where two emerging PPCs fail to enwrap cone cells, expression of hbs in PPC precursors restores the ability of these cells to surround cone cells. Further, expression of hbs or rst in a single rst- or hbs-expressing cell, respectively, leads to removal of the counterpart from the membrane within the same cell through cis-interaction and forced expression of Rst in all hbs-expressing PPCs strongly disrupts the remodeling process. Finally, a loss of both hbs and sns in single PPC precursors leads to constriction of the apical surface that compromises the 'kidney' shape of PPCs. Taken together, these results indicate that cone cells utilize Notch signaling to instruct neighboring PPC precursors to surround them and Notch controls the remodeling process by differentially regulating four adhesion genes (Bao, 2014).

Protein Interactions

Proteins belonging to the Ig superfamily are frequently implicated in cell-cell adhesion. The ability of Hbs, Sns, Kirre, IrreC-Rst and Sidestep (Side) to bind homotypically was tested with the S2 cell aggregation assay. As a negative control, S2 cells were transfected with RmHa3 vector, and as a positive control, S2 cells were transfected with Fasciclin II-RmHa3. Homotypic aggregation was observed for Fasciclin II and Kirre. To test for heterotypic interactions, the S2 cells were labeled with either DiI (red) or DiO (green), and the aggregates were examined using confocal microscopy. When Fasciclin II-transfected cells (red) were mixed with RmHa3-transfected cells (green), all aggregates formed contained only red cells. Similarly, when Kirre-transfected cells (red) were mixed with RmHa3-transfected cells (green), aggregates were again all comprised of only red cells. When Kirre-transfected cells (red) were mixed with Hbs- or Sns-transfected cells (green), the resultant aggregates all had both red and green cells, but when Kirre-transfected cells (red) were mixed with RmaHa3- or Irrec-transfected (green) cells, all the resultant aggregates contained only red fluorescent cells. This is the first evidence suggesting that Nephrin proteins interact heterophilically in trans with other potentially non-Nephrin extracellular partners (Dworak, 2001).

Sns interacts with Duf/Kirre and IrreC-rst to mediate myoblast cell-cell adhesion

The ability of Sns, Duf/Kirre and IrreC-rst to mediate cell–cell adhesion was examined using Drosophila S2 cells, which are predominantly non-adherent under normal conditions. As a prelude to examining the behavior of these molecules in combination, each was examined individually to evaluate their ability to direct homotypic aggregation. S2 cells were transiently transfected with Duf/Kirre, IrreC-rst, or Sns under the control of the copper inducible metallothionein promoter and allowed to aggregate. Following aggregation, the cells were fixed and examined by indirect immunofluorescence using anti-sera directed against specific domains or tags within each protein. As anticipated from previous studies (Dworak, 2001), Duf/Kirre-expressing S2 cells were frequently found in aggregates. Duf/Kirre protein accumulates at points of cell-cell contact in aggregates but is uniformly distributed on the surface of non-aggregated S2 cells. Similar to the behavior of Duf/Kirre, IrreC-rst mediates homotypic aggregation of S2 cells, and becomes enriched at points of cell-cell contact in the resulting cell clusters. Duf/Kirre and IrreC-rst enrichment is occasionally observed in regions where cell-cell contact is not apparent, possibly as a consequence of processes, visible by transmission electron microscopy, that extend around neighboring cells. In contrast to the behavior of Duf/Kirre or IrreC-rst, expression of Sns protein on the surface of S2 cells does not lead to homotypic cell adhesion (Dworak, 2001). A lower magnification view emphasizes the presence of many unassociated Sns-expressing cells. As anticipated, Sns is distributed uniformly on the surface in the absence of aggregation (Galletta, 2004).

To ensure that the Duf/Kirre and IrreC-rst clusters were the consequence of aggregation rather than cell division, the number of cells in aggregates of three cells or more were counted. Since cells should only divide at most once during the course of the experiment, clusters of three cells must represent those formed from adhesive events. In a survey of 4171 Duf/Kirre-expressing cells, 40% (1697) were found in aggregates of three or more. In a survey of 1002 IrreC-rst-expressing cells, 19% (192) were in aggregates of three or more cells. These results suggest that while Duf/Kirre may be more efficient in mediating homotypic aggregation than IrreC-rst, clearly both are capable of mediating such interactions. By contrast, a survey of 3275 Sns-expressing cells revealed only 26 cells in aggregates of three or more cells (Galletta, 2004).

While Sns-expressing cells do not aggregate homophilically, studies have indicated that these cells aggregate with cells expressing Duf/Kirre (Dworak, 2001). It was of interest to determine whether cells expressing Sns would interact with cells expressing IrreC-rst, and whether Sns and Duf/Kirre or IrreC-rst co-localize at points of cell-cell contact. To this end, S2 cells were independently, transiently transfected and the ability of Duf/Kirre and IrreC-rst-expressing cells to form aggregates and direct membrane co-localization of Sns in these aggregates was examined. All of these proteins were uniformly distributed on the cell surface in unaggregated cells. In contrast to their behavior in isolation, Sns-expressing cells readily associated in large clusters when combined with cells expressing Duf/Kirre. The Sns-expressing cells also associated with cells expressing IrreC-rst, with a similar efficiency. At least one of these IgSF members must be expressed on the cell surface for it to cluster, since no untransfected cells were observed in an analysis of 1109 small clusters of either Duf/Kirre:Sns or IrreC-rst:Sns-expressing cells. While the biological significance of such an interaction remains unclear, Duf/Kirre and IrreC-rst-expressing cells are capable of forming heterotypic aggregates with each other when expressed in S2 cells under similar conditions (Galletta, 2004).

Examination of individual proteins in small aggregates revealed clustering of Sns with either Duf/Kirre or IrreC-rst at points of cell contact. Thus, either Duf/Kirre or IrreC-rst can direct cells to associate with Sns-expressing cells, and co-localize with Sns at points of cell contact. Frequently much of the Sns protein in the cell accumulates at the points of cell-cell contact, leaving little if any protein on the rest of the cell surface. In rare cases, both proteins are observed in regions outside of obvious cell contacts. However, this pattern may reflect cell membranes that are extending around neighboring cells, mentioned earlier. Since Duf/Kirre and IrreC-rst serve redundant functions in the founder myoblasts, and behave similarly in the assays described above, subsequent experiments focused on Duf/Kirre (Galletta, 2004).

In some cases, the cytoplasmic domains of cell adhesion molecules play no role in their ability to direct cell interactions, while this domain can be critical in other cases. It was therefore of interest to determine whether these regions of Sns or Duf/Kirre were required for the S2 cell interactions. For these studies, the extracellular domains of Duf/Kirre and Sns were fused in frame to the GPI-anchor sequence of Fasciclin I. These constructs were separately, transiently transfected into S2 cells, and aggregation was examined. In the case of Duf/Kirre-GPI, the efficiency of homotypic aggregation was severely reduced compared to that of cells expressing full length Duf/Kirre. Since the relevance of Duf/Kirre homotypic aggregates in vivo is unclear, the role of the Duf/Kirre and Sns cytoplasmic and transmembrane domains in heterotypic aggregation was also examined. In an analysis similar to that done for Duf/Kirre homotypic aggregates, the ability of cells expressing the GPI-anchored or full length forms of Duf/Kirre to mediate heterotypic adhesion with cells expressing full-length or GPI-anchored forms of Sns was examined in pairwise comparisons. The influence of the Sns cytoplasmic and transmembrane domains was examined on adhesion with cells expressing full length Duf/Kirre. Within the limits of statistical significance, GPI-anchored Sns mediate aggregation at a level comparable to that of full length Sns. A similar analysis was carried out to examine the influence of the Sns cytoplasmic and transmembrane regions on adhesion with cells expressing Duf/Kirre-GPI. Again, Sns-GPI mediates adhesion with the Duf/Kirre-GPI-expressing cells at a level comparable to that of full length Sns. Thus, the Sns cytodomain and membrane spanning region appear to play no role in its ability to direct aggregation with Duf/Kirre-expressing cells (Galletta, 2004).

Since the Duf/Kirre-expressing cells were in excess in the above experiments, these data could not be used to determine the relative contribution of the Duf/Kirre cytodomain and transmembrane region. Therefore additional assays were carried out in which Sns or Sns-GPI-expressing cells were in a five-fold excess over either Duf/Kirre or Duf/Kirre-GPI-expressing cells to determine whether there was a requirement for the Duf/Kirre cytodomain or membrane spanning region in interactions with cells expressing Sns. These experiments were also set up as pairwise comparisons, and demonstrated that Duf/Kirre-GPI mediate aggregation at a level comparable to that of full length Duf/Kirre. These data suggest that there is no significant difference between the ability of Duf/Kirre or Duf/Kirre-GPI to aggregate with cells expressing full-length Sns. Lastly, the cytoplasmic and transmembrane domain of Duf/Kirre have a modest affect on its ability to direct aggregation with GPI-anchored Sns. However, the effect of the cytoplasmic or transmembrane domain on Duf/Kirre's ability to mediate heterotypic aggregation with Sns-GPI was not as great as its effect on the ability of Duf/Kirre to mediate homotypic cell adhesion. Of note, Duf/Kirre-GPI was enriched at points of cell-cell contact in both homotypic aggregates and in heterotypic aggregates with Sns and Sns-GPI. Thus, neither the cytoplasmic nor transmembrane domains of Sns or Duf/Kirre are essential for recruitment to cell-cell contacts (Galletta, 2004).

In summary, these results indicate that the cytoplasmic/transmembrane domains of Sns and Duf/Kirre do not influence the efficacy with which they direct heterotypic cell-cell adhesion. This observation is in contrast to that seen for Duf/Kirre, in which the cytodomain or membrane spanning region of Duf/Kirre plays a critical role in its ability to direct homotypic aggregation. One possible explanation for these results is that heterotypic association of Sns and Duf/Kirre is stronger, and does not require stabilization of the receptor through cytoplasmic or intramembrane interactions. Since the affinity of Duf/Kirre for homotypic versus heterotypic interactions could play a critical role in myoblast interactions in the embryo, the S2 cell aggregation assay was used to examine this preference (Galletta, 2004).

In the embryonic musculature, founder cells appear to fuse only with fusion competent myoblasts, and never fuse with each other. In principle, this directional fusion could be attributed to the differential expression of Duf/Kirre and Sns by these two cell types, and inability of these molecules to associate homotypically. However, results reported in this study and by Dworak (2001) demonstrate that Duf/Kirre-expressing cells do associate with each other in culture. It was therefore of interest to determine whether the affinity of Duf/Kirre-expressing cells for cells expressing Sns was greater than the affinity of Duf/Kirre-expressing cells for each other. To address this question, Duf/Kirre-expressing cells were aggregated in isolation or in the presence of an equal number of Sns-expressing cells. This analysis utilized stable cell lines in which approximately 30% of the corresponding population expressed Duf/Kirre and approximately 8% expressed Sns. Aggregation of Duf/Kirre-expressing cells was examined in three different conditions, all with the same total cell number. The goal was to ensure that any change in aggregation of Duf/Kirre cells was due to the specific addition of Sns-expressing cells rather than a consequence of doubling the number of adherent cells. For each time point, the number of Duf/Kirre-expressing cells free in solution was counted and the number that had been incorporated into aggregates. Duf/Kirre-expressing cells were incorporated into aggregates that included Sns-expressing cells at a faster rate and to a greater extent than those containing only Duf/Kirre-expressing cells. This behavior was not a simple consequence of the number of adherent cells present, since a two-fold increase in the number of Duf/Kirre-expressing cells did not have a dramatic effect on the rate or extent of aggregation. Thus, Duf/Kirre-expressing cells associate more readily into heterotypic aggregates with Sns-expressing cells than into homotypic aggregates with only Duf/Kirre-expressing cells (Galletta, 2004).

The striking colocalization of Duf/Kirre and Sns described earlier suggested the possibility that these proteins might physically associate in trans. To address this possibility, aggregates of stably transfected, Sns and Duf/Kirre-expressing cells were subjected to reversible protein cross-linking and lysed. HA-tagged Duf/Kirre was immunoprecipitated from the cell lysate using anti-HA resin, and the resulting immunoprecipitate examined by Western blot for the presence of Sns. HA-tagged Duf/Kirre was efficiently precipitated from both Duf/Kirre-only and Duf/Kirre-Sns mixed cell populations. As expected, Sns was not present in the anti-HA immunoprecipitate from cells expressing only Duf/Kirre or only Sns. However, it was clearly detected in immunoprecipitates from the mixed population of cells expressing Duf/Kirre-HA and Sns. Thus, Duf/Kirre and Sns are closely associated in an immunoprecipitable protein complex, possibly through a direct protein interaction (Galletta, 2004).

In the embryonic musculature, Duf/Kirre, IrreC-rst and Sns are necessary, either directly or indirectly, for the association of founder and fusion competent myoblasts. The striking co-localization of Sns with either Duf/Kirre or IrreC-rst in S2 cells prompted an examination of whether similar co-localization could be observed between embryonic myoblasts. First it was examined whether punctate clustering of Sns on the surface of embryonic myoblasts, previously described by Bour (2000), was dependent on the presence of Duf/Kirre or IrreC-rst. The distribution of Sns protein was examined in embryos deficient for both Duf/Kirre and IrreC-rst, and compared to that seen in wild-type embryos. As anticipated, Sns becomes localized to discrete sites in wild-type myoblasts (Bour, 2000). In contrast, Sns is distributed more uniformly on the myoblast surface in embryos lacking Duf/Kirre and IrreC-rst. Thus in embryos, as in S2 cells, the localization of Sns is dependent on the presence of Duf/Kirre or IrreC-rst (Galletta, 2004).

To determine whether Sns and Duf/Kirre co-localize in embryonic myoblasts in a manner similar to that observed in S2 cells, stage 13 embryos were examined by indirect immunofluorescence using polyclonal antisera directed against the Duf/Kirre and Sns proteins. As previously described for Sns (Bour, 2000), Duf/Kirre is expressed in a dynamic pattern that is restricted to discrete sites on the surface and in the cytoplasm of expressing cells. The pattern of Sns expression intersects that of Duf/Kirre, and is in close proximity to rP298-lacZ positive founder cell nuclei in the somatic mesoderm. Of note, punctate Sns expression is apparent at some sites in which Duf/Kirre expression is not detected. To address whether these sites might intersect points of IrreC-rst protein, which can interact with Sns-expressing cells and can substitute for Duf/Kirre in vivo, embryos were triple labeled with Duf/Kirre, IrreC-rst and Sns. IrreC-rst is readily detected at many sites of Sns enrichment that do not appear to colocalize with Duf/Kirre. In fact, examination of 204 discrete sites of Sns protein, derived from eight stage 13 embryos, revealed that 97% were colocalized with either Duf/Kirre and/or IrreC-rst (Galletta, 2004).

To determine whether sites of Duf/Kirre and Sns colocalization occur, as expected, on the cell surface, mesodermally expressed CD2 was used to visualize the cell membrane. CD2 staining revealed the surface of a growing myofiber and associated myoblasts. Duf/Kirre and Sns co-localize to points of contact between the fiber and a myoblast. Since the expression of both Duf/Kirre and Sns is dynamic and rapidly decreases upon fusion (Bour, 2000), co-localization of Duf/Kirre and Sns was examined in myoblast city (mbc) mutant embryos in which the myoblasts associate but remain unfused. By stage 14, the founder cells of these mutant embryos become morphologically distinct from the fusion competent cells, elongating and extending processes. As an apparent consequence of this fusion block, Duf/Kirre and Sns are stabilized at points of contact between the extended founder cell and several fusion competent cells. These data clearly show that Sns and Duf/Kirre co-localize in the embryo at critical contact points between founder cells and fusion competent myoblasts (Galletta, 2004).

WIP/WASp-based actin-polymerization machinery interacts Dumbfounded and Sticks and Stones to facilitate myoblast fusion in Drosophila

Formation of syncytial muscle fibers involves repeated rounds of cell fusion between growing myotubes and neighboring myoblasts. Wsp, the Drosophila homolog of the WASp family of microfilament nucleation-promoting factors, is an essential facilitator of myoblast fusion in Drosophila embryos. D-WIP (termed Verprolin 1 in FlyBase), a homolog of the conserved Verprolin/WASp Interacting Protein family of WASp-binding proteins, performs a key mediating role in this context. D-WIP, which is expressed specifically in myoblasts, associates with both the WASp-Arp2/3 system and with the myoblast adhesion molecules Dumbfounded and Sticks and Stones, thereby recruiting the actin-polymerization machinery to sites of myoblast attachment and fusion. This analysis demonstrates that D-WIP recruitment is normally required late in the fusion process, for enlargement of nascent fusion pores and breakdown of the apposed cell membranes. These observations identify cellular and developmental roles for the WASp-Arp2/3 pathway, and provide a link between force-generating actin polymerization and cell fusion (Massarwa, 2007).

The evolutionarily conserved Arp2/3 protein complex is the primary microfilament-nucleating machinery in eukaryotic cells. To perform its diverse cellular roles, the complex must first be activated by nucleation-promoting factors (NPFs), such as members of the WASp and WAVE/SCAR protein families. These elements serve as essential mediators, linking signal-transduction pathways and Arp2/3-based actin polymerization. Actin polymerization triggered by this system is translated into forces that drive a variety of key cellular functions, including cell locomotion, motility of membrane-bound particles within cells, and formation of endocytic vesicles (Massarwa, 2007).

A major challenge in the field is the assignment of physiological roles to this potent cellular machinery during the development of multicellular organisms. While genetic approaches in model organisms have shown promise in this regard, the numerous and sometimes overlapping roles assigned to the Arp2/3 system often prove difficult to separate. Previous work has shown that Wsp, the Drosophila WASp homolog, acts as an Arp2/3 activator in restricted developmental contexts, thus allowing for characterization of Arp2/3 function in vivo. This approach was used to reveal an unexpected involvement of the WASp-Arp2/3 system in myogenesis. Specifically, this system is shown to play a distinct role in myoblast fusion during Drosophila embryogenesis (Massarwa, 2007).

Somatic muscle fibers in the mature Drosophila embryo are comprised of multinucleated cells that form by multiple rounds of fusion between two distinct myoblast subpopulations. After the initial specification of the mesoderm, each embryonic trunk hemi-segment contains ~30 'founder cell' myoblasts, which will direct muscle formation and differentiation, and a large number of fusion-competent myoblasts (FCMs). Founder cells possess the information necessary for determining the identity and size of the individual somatic muscles, while the FCMs serve as a repository that will add cytoplasmic bulk to each muscle fiber (Massarwa, 2007).

Recognition and association of founder cells and FCMs are based on heterotypic interactions between differentially expressed immunoglobulin superfamily cell-surface proteins. Founder cells express Dumbfounded (Duf) and the closely related Roughest (Rst), which serve as attractants for FCMs. Physical association between Duf/Rst and the FCM-specific protein Sticks and Stones (SNS) provides a key step in myoblast adhesion and alignment of the myoblast cell membranes. Founder cells initially fuse with one or two FCMs, leading to the formation of bi-/trinuclear muscle precursors. A second, major phase of muscle growth then ensues, in which the precursor myotubes undergo successive rounds of fusion with multiple FCMs. In addition to the cell-adhesion molecules, genetic approaches have revealed a number of elements that contribute to various steps of the fusion process, including transcription factors, signaling molecules, and cytoskeleton-associated proteins (Massarwa, 2007).

This study demonstrates that function of the WASp-Arp2/3 system is essential for the second phase of myoblast fusions, between maturing myotubes and FCMs, and acts after formation of fusion pores in the double membrane of the apposed cells. Recruitment of the WASp-Arp2/3 system to founder cell-FCM attachment sites is achieved via D-WIP, a Drosophila homolog of the Verprolin/WASp Interacting Protein (Vrp/WIP) family. Functional associations with members of this protein family constitute an evolutionarily conserved feature of WASp activity. D-WIP is specifically expressed in myoblasts and associates with the cell-surface proteins that mediate adhesion between founder cells and FCMs, thereby establishing a critical link between the cellular machineries that govern fusion and microfilament dynamics. These findings present a novel tissue context for the involvement of the Arp2/3 system in physiological events and extend the functional applications of the forces generated by actin polymerization to a central process of tissue morphogenesis (Massarwa, 2007).

This study has identified an exceptional and highly cell-type-specific mode for regulating the Arp2/3 system. Functional selectivity in this system is usually achieved via spatial and temporal control over the operation of signal-transduction pathways and the resulting production of potent activating elements for the relevant Arp2/3 nucleation-promoting factor. In contrast, it is the restricted expression of D-WIP in the FCMs that confines Wsp-mediated triggering of Arp2/3 activity to the fusing myoblasts of Drosophila embryos. Transcriptional control over D-WIP expression, governed directly or indirectly by the Lame Duck (Lmd) transcription factor, thus provides a means for translating embryonic patterning schemes into distinct and specific cellular activities, which can profoundly influence cell morphology (Massarwa, 2007).

The structural basis for the interaction between D-WIP and Wsp is consistent with the established principles of Vrp/WIP-WASp protein association, which rely on an interaction between an ~25 residue long peptide from the extreme C-terminal region of Vrp/WIP proteins and the WH1/EVH1 N-terminal region of WASp proteins. Most critical residues within these domains are conserved in the Drosophila homologs. Moreover, genetic data and S2 cell localization observations strongly implicate these domains in mediating physical association between the two proteins (Massarwa, 2007).

By virtue of its association with the cell-surface adhesion proteins Duf and SNS, expressed in founder cells and FCMs, respectively, D-WIP may impose a common functionality on these distinct myoblast types. Yet to be determined, however, is the nature of the interaction between D-WIP and the myoblast-attachment machinery, and whether this interaction is constitutive or is dependent upon founder cell-FCM contact. Colocalization in both developing embryonic muscles and aggregated S2 cells, as well as the coimmunoprecipitation of D-WIP and Duf, underlies the suggestion of a physical association, but whether this association is direct requires further investigation (Massarwa, 2007).

The lack of significant sequence homology between the cytoplasmic portions of the Duf and SNS proteins, and the comparatively tighter correspondence between D-WIP and SNS localizations, may be indicative of distinct modes of association between D-WIP and the two types of adhesion proteins. It is interesting to note in this context that mammalian Nephrin, which shares structural and sequence similarities with SNS, employs direct binding of its cytoplasmic portion to the adaptor protein Nck, as a means of establishing a functional link to the actin-based cytoskeleton (Massarwa, 2007).

WASp-family proteins are thought to reside in an auto-inhibited conformation, which prevents productive interaction with Arp2/3 and is alleviated only by binding of signaling molecules. Scenarios consistent with a recruiting role for Vrp/WIP proteins have been described, including involvement of WASp in actin-based motility of intracellular pathogens and in cytoskeletal remodeling of the immune synapse. However, Vrp/WIP proteins on their own fail to stimulate, or may even inhibit, WASP-based Arp2/3 activation (Martinez-Quiles, 2001: Ho, 2004), implying a requirement for additional activating elements. The observation that WspMyr, a membrane-tethered form of Wsp, can partially compensate for loss of D-WIP function is consistent with an exclusive recruitment role for D-WIP. However, it should be born in mind that an additional step of Wsp activation may be required after its recruitment. Since the results of phenotypic rescue experiments further imply that established activators of WASp-type proteins such as CDC42 and PIP2 do not operate in this context, the identity of an independent Wsp activator during myoblast fusion, if one indeed exists, is currently unknown (Massarwa, 2007).

Activation of the Arp2/3 complex promotes the generation of branched networks of polymerizing actin filaments, in close proximity to both the cell surface and to internal cell membranes. The physical force liberated by this energetically favorable process can be harnessed to push against, or otherwise influence, membrane behavior. A key challenge stemming from the experimental observations is to identify the mechanism by which Arp2/3-based force production contributes to the progress of myoblast fusion (Massarwa, 2007).

The detailed TEM-level description of Drosophila myoblast fusion has stipulated a series of events, including formation of pores next to sites of accumulated electron-dense material along the apposed myoblast membranes, vesiculation/fragmentation of the membranes between the pores, and removal of the residual membrane material. Analysis of the D-WIP and Wsp mutant phenotypes demonstrates a requirement for the Arp2/3 system at a relatively late stage of the fusion process, after formation of the initial fusion pores (Massarwa, 2007).

Much of what is known about the mechanisms driving cell-cell (including myoblast) fusion relates to recognition and adhesion between pairs of cells and construction of initial fusion pores, while the more advanced processes of pore enlargement and the eventual establishment of full cytoplasmic continuity between the fusing cells remain mostly unexplored. The demonstration of a requirement for the cellular actin-polymerization machinery at these stages holds the promise of establishing a mechanistic basis for these late events (Massarwa, 2007).

Several possible mechanisms can be proposed for the manner by which polymerization-based forces drive fusion to completion, after initial pore formation. Pore enlargement during membrane fusion poses considerable energy requirements, which Arp2/3-based polymerization seems well suited to satisfy. The 'pushing' forces inherent in this cellular machinery can be applied to the contours of nascent fusion pores, thereby ensuring their continuous expansion. Alternatively, myoblast membranes may be broken down by vesiculation, akin to endocytosis. Detailed genetic and cellular studies have demonstrated essential roles for the Vrp/WIP-WASp-Arp2/3 machinery during endocytosis of clathrin-coated vesicles in budding yeast, and mechanistic interpretations of the forces involved have been put forward. In keeping with previous discussions of these issues, it is tempting to suggest that electron-dense structures, common to the contact sites of myoblasts in both Drosophila and vertebrate species, may provide a structural framework through which polymerization-based forces exert their influence. Finally, a role for the Arp2/3 machinery can be invisioned in an even more advanced step in the fusion process, namely, the final removal of residual, vesiculated membrane material from the disrupted sites of membrane contact to create full cytoplasmic continuity (Massarwa, 2007).

In summary, these observations linking myoblast cell-surface adhesion proteins in Drosophila embryos with the WIP/WASp module suggest a mechanism through which the conserved cellular machinery promoting force production via microfilament nucleation can be harnessed to drive muscle fiber formation to completion. Future studies will determine the finer mechanistic details of the cellular mechanism employed in this instance, and the degree to which this link can be generalized to myogenesis in vertebrate species, as well as other processes of cell fusion (Massarwa, 2007).

A critical function for the actin cytoskeleton in targeted exocytosis of prefusion vesicles during myoblast fusion

Myoblast fusion is an essential step during muscle differentiation. Previous studies in Drosophila have revealed a signaling pathway that relays the fusion signal from the plasma membrane to the actin cytoskeleton. However, the function for the actin cytoskeleton in myoblast fusion remains unclear. This study describes the characterization of solitary (sltr), a component of the myoblast fusion signaling cascade. sltr encodes the Drosophila ortholog of the mammalian WASP-interacting protein. Sltr is recruited to sites of fusion by the fusion-competent cell-specific receptor Sns and acts as a positive regulator for actin polymerization at these sites. Electron microscopy analysis suggests that formation of F-actin-enriched foci at sites of fusion is involved in the proper targeting and coating of prefusion vesicles. These studies reveal a surprising cell-type specificity of Sltr-mediated actin polymerization in myoblast fusion, and demonstrate that targeted exocytosis of prefusion vesicles is a critical step prior to plasma membrane fusion (Kim, 2007).

Characterization of Sltr provides a number of novel, and somewhat unexpected, findings concerning the role of the actin cytoskeleton in myoblast fusion. In contrast to the widespread expression of WIP in mammals, Sltr is specifically expressed in developing muscles and, moreover, only in fusion-competent myoblasts. Such cell-type specificity is unexpected, given that Sltr is the only WIP homolog in Drosophila. As a positive regulator of actin polymerization, Sltr is recruited to sites of fusion by the fusion receptor Sns and is required for the formation of F-actin-enriched foci at these sites. EM studies suggest that these actin-rich foci may provide directionality for the trafficking of prefusion vesicles, which are routed to ectopic membrane sites in the fusion-competent cells in sltr mutant embryos. It is suggested that targeted exocytosis of prefusion vesicles represents a critical step leading to plasma membrane fusion (Kim, 2007).

The identification of Antisocial/Rolling pebbles as a founder cell-specific protein that mediates signaling from the fusion receptor Duf to the actin cytoskeleton suggests the existence of a fusion-competent cell-specific protein(s) with an analogous function. The current work suggests that Sltr represents such a molecule. Not only is Sltr recruited to sites of fusion by the fusion-competent cell-specific receptor Sns, likely mediated by the small adaptor protein Crk, it also brings the actin polymerization machinery to these sites by binding to WASP and G-actin. As a result, Sltr colocalizes with F-actin-rich foci at sites of fusion, and is required for the formation of these actin foci in fusion-competent cells. Thus, like Ants/Rols7 in founder cells, Sltr is a fusion-competent cell-specific protein that links the fusion receptor with the actin cytoskeleton (Kim, 2007).

How does Sltr regulate actin polymerization? In vitro and in vivo assays demonstrate that this activity is mediated by the WH2 and WBD domains of Sltr, which bind to actin and WASP, respectively. In Drosophila S2 cells, overexpression of Sltr, but not mutant forms lacking the WH2 or WBD domains, leads to profound changes in actin cytoskeleton organization characterized by the formation of F-actin-filled microspikes. Likewise, the ability of Sltr to rescue sltr mutant embryos requires the WH2 and WBD domains. These observations suggest that both actin and WASP binding contribute to Sltr function. Interestingly, while the first WH2 domain of Sltr binds to G-actin, the second WH2 domain and its flanking region interact with F-actin. Thus, the actin-binding activity of Sltr serves a dual role -- it not only provides a pool of monomeric G-actin (in addition to the G-actin recruited by WASP) at sites of fusion but also stabilizes the newly formed actin filaments. The importance of the WASP-binding activity in Sltr function is further supported by the observation that RNAi knockdown of WASP abolished the ability of Sltr to induce microspikes in S2 cells, and that WASP itself is required for myoblast fusion in Drosophila. How WASP activity is regulated in myoblast fusion remains to be determined. Although mammalian WASP is known to be activated by the small GTPase Cdc42, this is unlikely the case in Drosophila myoblast fusion, as expression of a dominant-negative Cdc42 does not cause any fusion defects. Future experiments are required to identify the specific WASP activating factor(s) in myoblast fusion (Kim, 2007).

Myoblast fusion is a multistep process that includes cell recognition, adhesion, alignment, and membrane merger. To initiate the fusion process, fusion-competent cells that reside in a deeper mesodermal layer in the embryo need to migrate and extend filopodia toward founder cells that are in close contact with the ectoderm. Because the actin cytoskeleton is required for both cell migration and filopodia formation, one may predict that the actin cytoskeleton plays a role in these early events of myoblast fusion. Indeed, mutations in mbc and rac, components of the Mbc --> Rac --> WAVE pathway, produce a large number of round-shaped fusion-competent cells, suggesting a potential defect in myoblast migration and/or filopodia formation (Kim, 2007).

This study has identified a novel function of the actin cytoskeleton in a later step during myoblast fusion. This function is mediated by the Crk --> Sltr --> WASP pathway, which is independent of that of Mbc --> Rac --> WAVE; the recruitment of Sltr by the fusion receptor Sns is not affected by either mbc or rac. Using light microscopy, F-actin-rich foci have been observed localized to sites of fusion during myoblast adhesion, raising the possibility that localized actin polymerization at these sites may be functionally important for fusion. It was also demonstrated that the F-actin-rich foci are organized by the fusion receptor and actin cytoskeleton regulators, since they are absent in sns and sltr mutant fusion-competent cells. Taken together, these observations suggest a potential link between the fusion receptor, actin polymerization, and the membrane fusion machinery (Kim, 2007).

EM studies provide further insights into how localized actin polymerization may contribute to myoblast fusion by uncovering a relationship between the actin-rich foci and prefusion vesicles. The prefusion vesicles are of exocytic origin and are transported to the plasma membrane via the microtubule network. Immuno-EM analyses further revealed two classes of vesicles, one coated with and the other devoid of actin, at different subcellular locations. While naked vesicles are farther away from the actin foci, actin-coated vesicles are either within the foci or have reached the plasma membrane. That all vesicles at the membrane are actin-coated suggests that they have transited through actin foci and thus become distinct from the naked vesicles. Although the trafficking of these vesicles was not followed in real time due to the lack of vesicle-specific markers, the 'snapshots' provided by the EM analyses are most consistent with a model that actin foci at sites of fusion provide cortical capturing sites for the prefusion vesicles. This model is further supported by the transient nature of the actin-rich foci, which disintegrate in the cortical region after the prefusion vesicles have paired along the membrane in mature myoblasts. This model provides a plausible explanation for the mistargeting of vesicles in sltr mutant embryos - in the absence of actin foci at the prospective fusion sites, prefusion vesicles are randomly routed to the plasma membrane, resulting in their accumulation between adjacent founder and fusion-competent cells as well as between neighboring fusion-competent cells (Kim, 2007).

It is intriguing that in mature myoblasts, the prefusion vesicles that have aligned at the plasma membrane are all actin-coated, concurrent with an absence of the actin-rich patches at the cell cortex. Could actin coating of the prefusion vesicles, as well as disintegration of the actin-rich foci in mature myoblasts, play a role in vesicle-membrane fusion? The failure of vesicle-plasma membrane fusion in sltr mutant myoblasts with either too little (in fusion-competent cells) or prolonged accumulation of (in founder cells) actin is consistent with this possibility. While definitive answers to these questions await future investigation, it is worth noting that actin is required for yeast vacuole fusion and that actin has recently been identified as a component of neuronal synaptic vesicles (Kim, 2007).

An important future direction is to identify the biochemical composition of the prefusion vesicles. The involvement of such vesicles in myoblast fusion is not unique to Drosophila, as similar vesicles with electron-dense material have been reported in quail myoblast cultures and the L6 rat muscle cells. It is conceivable that these vesicles may deliver an unknown fusogen, or proteins/chemicals that stimulate fusogen activity, to the fusion sites, which ultimately leads to the fusion of apposing cell membranes. Because the actin cytoskeleton has also been implicated in other types of cell-cell fusion events, including fusion of human macrophages and viral-induced cell-cell fusion, it is speculated that targeted exocytosis of prefusion vesicles might represent a general step in myoblast fusion from Drosophila to mammals and perhaps in other cell-cell fusion events as well (Kim, 2007).

Analysis of the cell adhesion molecule sticks-and-stones reveals multiple redundant functional domains, protein-interaction motifs and phosphorylated tyrosines that direct myoblast fusion in Drosophila melanogaster

The larval body wall muscles of Drosophila arise by fusion of founder myoblasts (FMs) and fusion-competent myoblasts (FCMs). Sticks-and-Stones (SNS) is expressed on the surface of all FCMs and mediates adhesion with FMs and developing syncytia. Intracellular components essential for myoblast fusion are then recruited to these adhesive contacts. This study, a functional analysis of the SNS cytodomain using the GAL4/UAS system, identified sequences that direct myoblast fusion, presumably through recruitment of intracellular components. An extensive series of deletion and site-directed mutations were evaluated for their ability to rescue the myoblast fusion defects of sns mutant embryos. Deletion studies revealed redundant functional domains within SNS. Surprisingly, highly conserved consensus sites for binding PDZ proteins and serines with a high probability of phosphorylation play no significant role in myoblast fusion. Biochemical studies establish that the SNS cytodomain is phosphorylated at multiple tyrosines and their site-directed mutagenesis compromises the ability of the corresponding transgenes to rescue myoblast fusion. Similar mutagenesis revealed a requirement for conserved proline-rich regions. This complexity and redundancy of multiple critical sequences within the SNS cytodomain suggest that it functions through a complex array of interactions that likely includes both phosphotyrosine-binding and SH3-domain-containing proteins (Kocherlakota, 2008).

The process of myoblast fusion in Drosophila embryos requires intracellular events that include the formation of F-actin-rich foci and recruitment of electron-dense vesicles to sites of cell-cell contact. In the fusion-competent myoblast, actin polymerization occurs coincident with transport of fusion-related electron-dense vesicles and is suggested to aid in the targeting of the latter. The vesicles appear to become actin coated as they move through the actin-rich foci close to the cell surface, where they undergo exocytosis. Separate studies have identified discrete actin-rich complexes at the cell surface that are composed of proteins essential for myoblast fusion, including SNS. These structures, termed FuRMAS, have been implicated in determining the exact site of fusion and the position and size of the fusion pore (Kesper. 2007). Thus, signal transduction pathways that direct actin polymerization and mediate cytoskeletal reorganization appear to be guided by cell-adhesion molecules to points of cell contact prior to membrane breakdown at these sites. The cytodomains of these cell-adhesion molecules therefore likely serve critical roles in signaling pathways that lead to myoblast fusion (Kocherlakota, 2008).

These studies establish the importance of a highly conserved membrane proximal region of 166 amino acids in the SNS cytodomain. Surprisingly, smaller deletions within this region do not have a profound impact on SNS function, with the deletions A1113-I1163, Y1233-Y1263, and Y1263-H1278 each restoring the wild-type muscle pattern in sns mutants. However, a combination of these deletions represented by UAS-δA1113-I1163 + Y1233-H1278 is unable to rescue the fusion defects in sns mutant embryos. It is concluded from these results that the SNS cytodomain houses significant functional redundancy and is rendered nonfunctional only when multiple sites that serve the same purpose are removed. Examination of the sequences within each of these regions reveals a high number of predicted and conserved sites for phosphorylation on both serine and tyrosine. It cannot be concluded that serines are unimportant in SNS-mediated signal transduction since all sites have not been mutated. Nevertheless it can be concluded that loss of the 17 most conserved, best candidate sites for phosphorylation on serine does not affect SNS function. Moreover, the remaining serines must be capable of fulfilling any role that these serines play in SNS-mediated signal transduction. Phosphotyrosines can also be critical intermediates in signal transduction pathways and mediate interactions with proteins containing SH2, WW, or phosphotyrosine-binding (PTB) domains. Tyrosine phosphorylation is a key modification in the SNS ortholog, nephrin (Li, 2004; Jones, 2006), and regulates signaling to two downstream pathways: one that activates Akt by phosphoinositide 3-kinase (PI3K) and another that directs actin rearrangements via Nck binding. Both the Akt signaling and Nck pathways are critical for nephrin function (Kocherlakota, 2008).

This study has demonstrated that SNS is phosphorylated on tyrosine and that mutagenesis of these sites impairs SNS function during myoblast fusion. Surprisingly, even residues with a low probability of modification in SNS are nonetheless phosphorylated in the SNS cytodomain. It is not possible with the methods available to distinguish between residues that are phosphorylated in the wild-type protein and residues that are phosphorylated in the mutant proteins in the absence of preferred targets. Nevertheless, all sites must be removed to affect SNS function. Proteins that may interact with these phosphotyrosines include SH2-SH3 domain-containing adaptor proteins that have similar roles downstream of other membrane receptors. Candidates include Crk, Dreadlocks (Dock)/Nck, and Downstream of receptor kinase (Drk), the Drosophila ortholog of Grb2. SNS has been shown to recruit Sltr/D-Wip to the membrane of cotransfected S2 cells via Crk, supporting it as a pathway intermediate in the FCMs (Kim, 2007), and Nck plays a critical role downstream of nephrin as cited above (Kocherlakota, 2008).

While the importance of phosphorylated tyrosines is clear, removal of all tyrosines does not render SNS completely nonfunctional. The data also demonstrate a requirement for two proline-rich regions with core PXXP motifs. These sites are common to transmembrane receptors and, for example, the IgSF proteins Robo and DSCAM utilize such sites for interactions that lead to directional migration in the Drosophila nervous system. PXXP sites frequently interact with SH3 domains, which are found in signaling proteins that include adaptors, kinases, and GTPase activator proteins. In SNS, the simultaneous mutation of both PXXP sites (as in UAS-sns2xPXXP) impairs the ability of SNS to function during myoblast fusion. Although a direct requirement for a conserved motif in nephrin has not been described, the nephrin cytodomain interacts with the third SH3 domain of CD2AP to activate Akt signaling and for association with the actin cytoskeleton in mouse kidney cells. Moreover, the loss of CD2AP in the mouse is associated with kidney defects reminiscent of those observed in the absence of nephrin, suggesting that CD2AP-nephrin interactions are critical to maintaining slit diaphragm function (Kocherlakota, 2008).

One muscle-specific candidate for interaction with the SNS PXXP site is MBC, which is essential in both FMs and FCMs and requires its SH3 domain for function during myoblast fusion. While this SH3 domain seems more likely to mediate interaction of MBC with ELMO to form a bipartite guanine nucleotide exchange factor, it is a formal possibility that it interacts directly with SNS. Alternatively, if the interaction is indirect, one might anticipate the presence of factors in the FCMs that recruit MBC to SNS in a manner reminiscent of the relationship between MBC and Ants/Rols downstream of Duf/Kirre in the FMs. Other candidates for interaction with the SNS PXXP sites that are ubiquitous during embryogenesis or expressed specifically during myogenesis are D-Crk and CG31012, the apparent ortholog of , as well as the SH2-SH3-domain-containing adaptor proteins previously mentioned (Kocherlakota, 2008).

This study has revealed two critical sequences in the SNS cytodomain. However, neither phosphotyrosines nor consensus PXXP motifs alone can account for the full repertoire of interactions necessary for SNS function. It is unclear if removal of both signaling motifs might account for the more drastic effect of the double deletion on the ability of SNS to direct myoblast fusion. It also remains to be determined whether these two motifs function in related interactions that converge on a single pathway or reflect independent interactions that direct distinct downstream events. Each motif seems most likely to bind a different target protein, as observed for domains within the neuronal cell adhesion molecules NCAM, L1CAM, Robo, and DSCAM. Multiple protein interaction domains are also found in the more closely related nephrin and Duf/Kirre proteins. Another formal possibility is that both the phosphotyrosines and the PXXP motifs serve the same function by actually binding to the same protein. This type of mechanism, albeit unlikely and unprecedented, seems feasible given the large number of adaptor proteins with multiple SH2 and/or SH3 domains. In this case, interaction of SNS with its target adaptor might be compromised but not completely lost by mutagenesis of either phosphotyrosine- or proline-rich regions, as observed in the results herein. In summary, current models support the presence of at least two signaling pathways downstream of SNS, one leading from SNS via Crk and Sltr/D-Wip to Wasp and another involving MBC-mediated activation of Rac and its subsequent activation of Arp2/3 via Kette/WAVE. Other SNS-associated pathways may yet be identified that play distinct roles in directional migration of FCMs to FMs, accurate alignment prior to fusion, and cytoskeletal or membrane turnover. The SNS cytodomain seems a likely site for interactions with proteins that mediate many of these events (Kocherlakota, 2008).



The embryonic expression pattern of the sns transcript was examined using a digoxigenin labeled cDNA fragment. The earliest expression of sns is seen during stage 11 in the visceral mesoderm, and in the somatic mesoderm prior to the onset of myoblast fusion. Although expression in the visceral mesoderm diminishes as germ band retraction proceeds, expression persists in the somatic mesoderm at high levels until stage 14, during which time myoblast fusion is taking place. No expression was evident in the visceral musculature or in the dorsal vessel at this time. By stage 15, transcript levels have also declined in the somatic musculature, and by stage 17 only faint expression could be detected. During stage 17, weak expression is also seen in the muscle attachment sites (Bour, 2000).

Antisera generated against the carboxy-terminal portion of the Sns protein confirmed that the pattern of protein expression is similar to that of the transcript. Confocal microscopy confirmed the co-expression of Sns and FASIII in the visceral musculature. As anticipated for a putative cell adhesion molecule, both colorimetric and immunofluorescent confocal staining revealed the enrichment of the Sns protein at the cell membrane. In addition, Sns appears to become localized to discrete sites in the membrane as fusion progresses . This membrane localization of Sns clusters has been confirmed by examination of serial confocal sections through the embryo. Like its transcript, Sns also appears to decline significantly as fusion progresses, such that little expression is observed in multinucleate syncitia (Bour, 2000).

Three independent experimental approaches have been used to address whether Sns is expressed in both the founder cells and putative fusion competent cells of the somatic mesoderm. In the first approach, wild-type embryos were double-labeled with antisera against Sns and various founder cell markers, and examined by confocal microscopy. Markers included the enhancer trap rP298-lacZ and even-skipped. In brief, Sns expression is not detected in isolated cells that express rP298-lacZ. These isolated cells were observed in many confocal sections, in embryos in several orientations. Because most of these are not near the ventral nerve cord, it is inferred that these are unfused founder cells rather than rP298-lacZ expressing glial cells. A similar analysis addressed co-expression of the Eve founder cell marker and Sns. At the earliest appearance of the Eve-expressing founder cell, no Sns expressing cells are observed in its vicinity, consistent with the pattern of expression observed with rP298-lacZ. After a brief period of time, Sns-expressing cells are observed in close proximity, and begin to fuse almost immediately. These data suggest that Sns marks the fusion-competent cells shortly before fusion, and is not expressed in the founder cells (Bour, 2000).

Two additional analyses addressed whether Sns expression in the somatic musculature might be exclusive to the fusion competent cells. Both a non-null allele of sns and a null allele of mbc were used to determine whether Sns is expressed in morphologically distinct founder cells. Elongated cells were observed in both snsXS5 and mbcD11.2 mutant embryos immunostained with MHC but not with Sns, consistent with the interpretation that Sns is not expressed in the founder cells. A second analysis relied on the hypothesis that Notch mediates a cell-fate decision between muscle progenitors (from which the founder cells arise) and the putative fusion competent cells. Notch NXK11 mutant embryos were double-labeled with vestigial (Vg) another founder cell marker, and Sns. Wild-type embryos exhibit normal distribution patterns of both markers. By comparison, one observes a dramatic expansion in the number of Vg-expressing founder cells in NXK11 mutant embryos, in agreement with studies using other founder cell markers. Of most significance is a dramatic reduction in the number of Sns-expressing cells in these mutant embryos. This observation supports the hypothesis that Notch mediates a cell-fate decision between muscle progenitors and fusion-competent cells, that Notch is necessary for the selection of fusion-competent myoblasts, and that Sns specifically marks this population of myoblasts (Bour, 2000).

Live imaging provides new insights on dynamic F-Actin filopodia and differential endocytosis during myoblast fusion in Drosophila

The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 (see Drosophila Arp2/3 component Arpc1) pathways. This study utilized the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. Differential endocytosis of cell surface components was observed at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the myotube (Haralalka, 2014: PubMed).


The sns locus, which is essential for myoblast fusion, was uncovered during an F2 lethal screen for EMS-induced point mutations in cytological region 95A on the third chromosome. In this screen, the original mutagenized fly was later found to have contained two recessive lethal mutations, one in the region of interest on the third chromosome and one on the second chromosome. Genetic mapping revealed that the muscle defect segregated with the second chromosome, and the recovered mutant locus was named sticks and stones (sns). Examination of the developing body wall muscles in snsA3.24 mutant embryos revealed an almost complete block in myoblast fusion (Bour, 2000).

The muscle phenotype of embryos homozygous for the original snsA3.24 allele includes a large number of unfused myosin-expressing cells and a corresponding absence of differentiated muscle fibers. Embryos transheterozygous for this sns allele and Df(2R)BB1, which deletes the entire sns region, exhibit the same mutant phenotype. Thus snsA3.24 behaves as a null allele by genetic criteria. The presence of founder cells was then assessed using an antibody directed against Nautilus. Nau-expressing cells are detected in their correct positions in sns mutant embryos, but do not appear to fuse. These cells are capable of producing myosin heavy chain (MHC) protein, and appear to be analogous to the muscle founders described in mbc mutant embryos. The entire myoblast population was also examined using a polyclonal antibody directed against Mef2, an early marker for most if not all cells of the somatic musculature. Mef2 expression is detected in the myoblasts of sns mutant embryos, in numbers approximately equivalent to those observed in wild-type embryos. These results imply that the precursors of the somatic musculature begin their differentiation program in sns mutant embryos, but become blocked at the point of myoblast fusion. In contrast to the severe defects in the somatic musculature, only subtle defects were observed in constrictions of the visceral musculature (Bour, 2000).

The rost locus, located at cytological position 30, encodes another protein that was reported to be essential for myoblast fusion (Paululat, 1995; Paululat, 1997). However, the original rostP20-containing chromosome was recently shown to contain two mutations that affect muscle development, a P-element insertion in the rost locus and a second mutation that maps to genetic position 43-49 (Paululat, 1999). Because the doubly-mutant chromosome was used in an EMS mutagenesis screen to isolate noncomplementing mutations (Paululat, 1995), the resulting alleles could be in either locus. Due to the localization of this second mutation to cytological region 43-49, it was of interest to determine whether any of the EMS alleles obtained in this screen were, in fact, allelic to sns. Indeed, all sns mutant alleles fail to complement the 43-49 mutation. Recombinants of rost2023 that separated cytological regions 30 and 44 were also isolated and crossed to sns6.1. Recombinants that retained region 44 did not complement sns6.1, whereas recombinants that retained region 30 complemented sns6.1. These data strongly suggest that the rost2023 mutation is allelic to sns. In addition, the sns sequence was examined in several putative rost alleles by NIRCA. Through this analysis, rost202 was found to contain a C to T transition that results in a nonsense mutation at amino acid residue 367 of the sns gene (Bour, 2000).

The presence of the transmembrane and/or cytoplasmic domain of Sns is essential for Sns to direct myoblast fusion in the embryo

Although the cytoplasmic and transmembrane domains of neither Duf/Kirre nor Sns play a significant role in the ability of these proteins to direct interaction of cultured S2 cells or become localized to points of cell-cell contact between the associated cells, the cytoplasmic domains of other members of the IgSF have been implicated in critical cell signaling events. It was therefore of interest to determine whether the cytoplasmic or transmembrane domain of Sns is required in the embryo to direct events more complex than those observed in cultured cells. To determine if the cytoplasmic/transmembrane domains of Sns are essential for myoblast fusion, the full length sns cDNA and sns-GPI were placed under UAS control, and transformed into flies. These transgenes were then introduced into an sns mutant background and their expression directed by the mesodermal expression of GAL4. Interestingly, mesodermal expression of the full length Sns is sufficient to rescue myoblast fusion in an sns mutant background, and these embryos grow into viable adults. However, the GPI-anchored form of Sns, which lacks both the transmembrane and cytoplasmic domains, is unable to rescue muscle formation in sns mutant embryos even when expressed from multiple copies of the transgene. The UAS-sns and UAS-sns-GPI transgenes have been sequenced in their entirety, express similar levels of protein in membrane preparations from embryos, as detected by Western blot, and do not exhibit dominant effects when expressed in wild-type embryos. Moreover, the fusion competent myoblasts did not appear to migrate toward the founder cells in these embryos. These data suggest that, in contrast to the ability to direct aggregation of S2 cells, the presence of the transmembrane and/or cytoplasmic domain of Sns is essential for Sns to direct myoblast fusion in the embryo (Galletta, 2004).

The formation of syncytia within the visceral musculature of the Drosophila midgut is dependent on duf, sns and mbc

The visceral musculature of the Drosophila midgut consists of an inner layer of circular and an outer layer of longitudinal muscles. The circular muscles are organized as binucleate syncytia that persist through metamorphosis. At stage 11, prior to the onset of the fusion processes, two classes of myoblasts are detected within the visceral trunk mesoderm. One class expresses the founder-cell marker rP298-LacZ in a one- to two-cells-wide strip along the ventralmost part of the visceral mesoderm, whereas the adjacent two to three cell rows are characterised by the expression of Sticks-and-stones. During the process of cell fusion at stage 12, SNS expression decreases within the newly formed syncytia that spread out dorsally over the midgut. At both margins of the visceral band several cells remain unfused and continue to express SNS. Additional rP298-LacZ-expressing cells arise from the posterior tip of the mesoderm, migrate anteriorly and eventually fuse with the remaining SNS-expressing cells, generating the longitudinal muscles. Thus, although previous studies have proposed a separate primordium for the longitudinal musculature located at the posteriormost part of the mesoderm anlage, cell lineage analyses as well as morphological observations reveal that a second population of cells originates from the trunk mesoderm. Mutations of genes that are involved in somatic myoblast fusion, such as sns, dumbfounded (duf or kirre) or myoblast city (mbc), also cause severe defects within the visceral musculature. The circular muscles are highly unorganized while the longitudinal muscles are almost absent. Thus the fusion process seems to be essential for a proper visceral myogenesis. These results provide strong evidence that the founder-cell hypothesis also applies to visceral myogenesis, employing the same genetic components as are used in the somatic myoblast fusion processes (Klapper, 2002).

The gene mbc, a homolog of human DOCK180, is expressed in all somatic myoblasts during the fusion processes. In embryos mutant for mbc, syncytia within somatic muscles are almost absent, presumably due to defects in the rearrangement of the cytoskeleton during preceding myoblast fusion. Mbc is also expressed in the visceral mesoderm from stage 12 onward. Embryos mutant for mbc not only exhibit defects in the formation of midgut constrictions but also show severe abnormalities in the formation of visceral muscles. At stage 12 the visceral band is randomly interrupted and the elongated FAS III-expressing cells seem to be disoriented. During further development, parts of the visceral band either stretch out in the dorsoventral direction, as in the wild type, or form disarranged patches. The status of the founder cells in an mbc mutant background was analyzed by expressing rP298-LacZ in the strain mbcC1. At stage 14 the number of rP298-LacZ-expressing cells within the remaining population of myoblasts appears to be reduced and large gaps within the visceral band are visible that are not present in the wild type. Many globular cells are still observed at the margins of the visceral band that appear to be unfused myoblasts. Antibody staining reveals a prolonged expression of SNS within a corresponding subpopulation of myoblasts. As described for duf and sns mutant embryos, no myoblast fusions were detectable. While the circular musculature shows severe defects and is apparently reduced, the longitudinal musculature is completely absent at the end of embryogenesis (Klapper, 2002).

By using the GAL4/UAS transplantation system for cell lineage analyses of the mesoderm anlage, syncytia were detected not only within somatic muscles but also in the visceral musculature. Since the visceral muscles have classically been described as mononuclear, this surprising observation led an examination of syncytia formation within this tissue. Evidence is provided that the circular visceral muscles of the midgut are likewise organized as syncytia. The first signs of GFP expression (driven by daGAL4) within these muscles were observed in embryos at stage 15. Since there is a considerable delay of about 2-4 h between the activation of the UAS-GFP construct and the formation of the fluorescent product, it is assumed that the formation of syncytia begins at stage 12. This is consistent with the observation of the first fusion processes within the visceral band. Using GFP expression as an in vivo marker, individual syncytia could be followed throughout development. In contrast to longitudinal muscle fibers, which have been found to contain up to six nuclei, the circular muscles of hindgut as well as midgut always comprise two nuclei each. Thus, fusion processes within this tissue stop after the formation of binucleate minimal syncytia that each cover one-half of the gut tube. This is a curious finding since in other muscle types of Drosophila many nuclei share a common cytoplasm to generate a large structural and functional unit (Klapper, 2002).

Following labelled syncytia through all stages of development, it has been shown that the visceral musculature is not replaced by a newly formed imaginal tissue but persists through metamorphosis. Since the visceral musculature plays a crucial role for the proper formation of the midgut during embryonic development, the persisting visceral musculature might again serve as a template for remodelling of the gut tube during metamorphosis (Klapper, 2002).

Since myoblast fusions were discovered within the visceral musculature, whether these syncytia are also formed by Duf-expressing founder and Sns-expressing fusion-competent cells was examined. duf can be detected in muscle precursors as long as they incorporate further fusion-competent cells into the syncytia. The expression of Duf and Sns within the visceral mesoderm suggests a function of specifying founder and fusion-competent cells similar to that observed for the somatic musculature. To clarify whether these genes indeed play a functional role in visceral myoblast fusion, the phenotypes of sns and duf mutant embryos was examined with respect to syncytia formation within the developing visceral musculature. In wild type embryos the ventrally located cells of the visceral band exhibit all characteristics of muscle founder cells, in that they express Duf, become elongated and fuse specifically with Sns-expressing myoblasts. Without duf function no signs of visceral muscle fusion are detectable. It is therefore proposed that duf is the key component in the visceral muscle founder cells. This observation is in agreement with the postulated role of Duf as an attractant for fusion-competent cells in the somatic musculature (Klapper, 2002).

These results indicate that loss of duf function leads to a complete loss of fusion even though the two populations of founders and fusion-competent myoblasts are already aligned within the visceral band at stage 11. Therefore, in the visceral mesoderm, Duf expression seems not only to attract the fusion-competent cells but also to play a crucial role in the fusion process itself. Probably the gaps within the visceral band of duf mutant embryos may indicate an early Duf adhesion function necessary for a proper anterior-posterior alignment of the visceral myoblasts. Furthermore, despite initial cell alignment at stage 11, this contact between founder cells and fusion-competent myoblasts is lost during further development, so that additional gaps appear between these cell layers. In sns mutant embryos, again, no myoblast fusions were detectable. The phenotype closely resembles that of duf mutant embryos, since the two populations of founders and fusion-competent cells seem to reject one another. However, the large gaps within the visceral band of duf embryos were never observed in sns mutants. Whereas in the anterior part of the midgut severe defects of the visceral musculature are obvious at later stages of embryogenesis, the posterior part looks quite normal. This regional difference is somewhat difficult to explain, since at earlier stages no such regional distinctions were observed. It is thought that either in the posterior visceral band sns function can be mimicked or bypassed later on by another spatially restricted gene product, or that even unfused myoblasts can form a seemingly normal musculature in the posterior part. The stronger phenotype at later stages in duf mutant embryos may indicate that the founder cells play a more crucial role in visceral myogenesis than the fusion-competent cells. In somatic myogenesis, founder and fusion-competent cells play specific roles in the recognition process, while in mbc mutants the fundamental capability to fuse is lost in both cell types. Because here intracellular components of the fusion apparatus are affected, fusion-competent cells still aggregate around founders, but the tight membrane junctions are not formed and fusion does not occur. As is consistent with the somatic phenotype, in visceral myogenesis, fusion is totally blocked although founder and fusion-competent cells are in direct contact and still express Sns and Duf (Klapper, 2002).

Taken together, these results provide evidence that the founder-cell hypothesis also applies to visceral myogenesis employing the same genetic components as used in the somatic myoblast fusion processes. Thus, the specification of myoblasts as either founder or fusion-competent cells might be a fundamental step preceding syncytia formation (Klapper, 2002).

Previous analyses have provided evidence that the primordium for the longitudinal midgut musculature is located at the posteriormost tip of the mesoderm anlage. In byn mutant embryos the hindgut as well as the longitudinal musculature are absent. The longitudinal musculature can be rescued, if byn is ectopically expressed exclusively within the posterior tip of the mesoderm anlage, leading to the conclusion that the entire anlage of the longitudinal musculature is located within this posterior region. However, after transplantation within the central region of the trunk, mesoderm labelled longitudinal muscles were frequently obtained. Taking into account that the founder-cell hypothesis is also valid for this tissue, this seeming contradiction can now be solved. The founder cells of the longitudinal musculature that comprise the genetic information for the specific tissue identity arise from the posterior tip of the mesoderm anlage, while the fusion-competent cells originate from the entire trunk region as indicated by confocal analysis. During the fusion processes it is possible to distinguish between two different events of syncytia formation. (1) At stage 12 all the ventrally located founder cells simultaneously become extended dorsally. Each of them fuse with a single SNS-expressing fusion-competent cell, eventually differentiating into a binucleate circular muscle. (2) The remaining SNS-expressing cells migrate to both margins of the visceral band and successively fuse with the longitudinal founder cells that invade from the posterior tip of the mesoderm. It is thus reasonable that in byn mutant embryos the entire longitudinal musculature is missing, since the fusion-competent cells alone are not capable of differentiating longitudinal muscles (Klapper, 2002).

The immunoglobulin superfamily member Hbs functions redundantly with Sns in interactions between founder and fusion-competent myoblasts

The body wall muscle of a Drosophila larva is generated by fusion between founder cells and fusion-competent myoblasts (FCMs). Initially, a founder cell recognizes and fuses with one or two FCMs to form a muscle precursor, then the developing syncitia fuses with additional FCMs to form a muscle fiber. These interactions require members of the immunoglobulin superfamily (IgSF), with Kin-of-IrreC (Kirre) and Roughest (Rst) functioning redundantly in the founder cell and Sticks-and-stones (Sns) serving as their ligand in the FCMs. Previous studies have not resolved the role of Hibris (Hbs), a paralog of Sns, suggesting that it functions as a positive regulator of myoblast fusion and as a negative regulator that antagonizes the activity of Sns. The results reported in this study resolve this issue, demonstrating that sns and hbs function redundantly in the formation of several muscle precursors, and that loss of one copy of sns enhances the myoblast fusion phenotype of hbs mutants. It was further shown that excess Hbs rescues some fusion in sns mutant embryos beyond precursor formation, consistent with its ability to drive myoblast fusion, but show using chimeric molecules that Hbs functions less efficiently than Sns. In conjunction with a physical association between Hbs and SNS in cis, these data account for the previously observed UAS-hbs overexpression phenotypes. Lastly, it was demonstrated that either an Hbs or Sns cytodomain is essential for muscle precursor formation, and signaling from IgSF members found exclusively in the founder cells is not sufficient to direct precursor formation (Shelton, 2009).

Sns and Hbs function redundantly in the initial fusion event between founder cells and FCMs. As observed in other mutants, precursor formation in sns mutant embryos is delayed over that occurring in wild-type embryos, but is readily observed in stage 13 embryos in at least some segments. By contrast, no fusion was observed by late stage 15 in sns, hbs double mutant embryos. Although the possibility of a temporal delay of fusion in sns, hbs double mutants cannot be eliminated because reporter expression declines after this stage, a model is favored in which a crucial first step is not occurring in the absence of both Sns and Hbs. Using new FCM reporters that facilitate quantitation of unfused myoblasts, re-examination of the hbs loss-of-function phenotype reveals that the loss of one copy of sns actually worsens the hbs mutant phenotype, as expected if these proteins have some functional redundancy. Finally, both snsGal4 and mef2Gal4 directed Hbs can drive a significant amount of fusion in sns mutants, arguing that Hbs is capable of directing fusion beyond precursor formation (Shelton, 2009).

Although Hbs can rescue the sns mutant phenotype beyond precursor formation, replacing any domain of Hbs with the comparable domain of Sns improves the ability of the chimeric protein to rescue fusion over that achieved by Hbs alone. The activity of the Hbs cytodomain is most dramatically different from that of Sns, providing an explanation for the observation that intact Hbs or a membrane-anchored Hbs cytoplasmic domain both interfere with myoblast fusion in wild-type embryos. Rather than acting as an antagonist of Sns, these high levels of Hbs probably interfere competitively with endogenous Sns. First, an excess of Hbs may drive its interaction with a limiting component that is normally used more efficiently by Sns. Alternatively, given their ability to form hetero- and homodimers in vivo, excess Hbs may sequester Sns in a less functional form. Although the data do not fully resolve this issue, the co-localization of Hbs and Sns is consistent with the latter model. Of note, dimer formation between the related IgSF proteins Boc and Cdo can be directed by sequences in both the extracellular and intracellular domains (Kang, 2002), and both the extracellular and intracellular domains of Sns are capable of mediating its interaction with Hbs, raising the possibility that either full-length Hbs or a membrane-anchored cytodomain may sequester Sns under conditions of overexpression (Shelton, 2009).

The finding that Hbs functions positively but much less efficiently than Sns in directing later rounds of myoblast fusion provides an explanation for the previously observed behavior of Hbs in overexpression assays (Artero, 2001; Dworak, 2001). Additionally, the data appear to be inconsistent with a model in which excess Sns is deleterious, as inferred if a decrease in sns copy number compensates for the loss of hbs (Artero, 2001). The possibility that Sns activity is negatively regulated cannot be excluded. Possible mechanisms could include limitations in the machinery for tyrosine phosphorylation, such that unphosphorylated Sns even in excessive amounts would be unable to transduce a signal to downstream events. Downstream targets of Sns may also be limiting, such that no further activation of the pathway can be accomplished by Sns. It is also noted that Sns protein is transient, appearing just before fusion and being eliminated shortly thereafter. Despite the issue of whether Sns activity is regulated in some fashion, the data are not consistent with a model in which its activity is negatively regulated by endogenous Hbs (Shelton, 2009).

Current models for myoblast fusion suggest that it occurs in two steps that differ genetically and/or temporally. Consistent with the two genetically distinct steps, fusion does not occur in embryos mutant for genes encoding the guanine nucleotide exchange factors Schizo, Mbc or Duf and Rst. By contrast, precursor formation is observed in embryos lacking the Hem-2/Nap1 homolog Kette, the Kirre-associated protein Rols, the Arp14D/66B regulators WASp and Vrp1 or Sns. These data support a model in which the molecular requirements for precursor formation differ from those for subsequent myotube formation. An alternative model, using three dimensional analyses and quantitating fusing myoblasts over time, revealed that fusion occurs in two temporal phases, comprising an initial phase of limited fusion between cells that are in close proximity and a second phase when most myoblast fusion occurs. Moreover, precursor formation is temporally delayed in embryos lacking molecules such as Rols and Kette, suggesting that these molecules do influence the first step in fusion (Shelton, 2009).

The present study does not address whether the genetic requirements for precursor formation differ from those for subsequent rounds of fusion, or whether these steps utilize the same set of proteins. The data do not eliminate the possibility of two distinct genetic steps, with Sns and Hbs acting redundantly in precursor formation but not in later events. Hbs is capable of directing precursor formation in the absence of Sns. However, the ability of Hbs to drive fusion beyond precursor formation when in excess, and the observation that removal of one copy of sns enhances fusion defects in hbs mutants, suggests that Hbs can assist in later rounds of myoblast fusion. These data are consistent with models in which molecular interactions in precursor formation and subsequent fusion differ kinetically but not genetically. One possibility, independent of the process of fusion itself, is that Sns and Hbs differ in their ability to drive FCM cell migration. Although the role of cell migratory behavior in myoblast fusion is unclear, the ability to migrate may contribute to the rate of fusion. While these questions remain to be addressed, the present study advances the understanding of fusion by resolving the interaction of two proteins that function early in the process, thereby providing additional perspectives for sorting out the different mechanisms of myoblast fusion (Shelton, 2009).


For information on Sns homologs, inclucing mammalian Nephrin and Drosophila Hibris, see hibris.


Search PubMed for articles about Drosophila sticks and stones

Abmayr, S. M., Balagopalan, L. Galletta, B. J. and Hong, S. J. (2003). Cell and molecular biology of myoblast fusion. Int. Rev. Cytol. 225: 33-89. PubMed Citation: 12696590

Artero, R. D., Castanon, I. and Baylies, M. K. (2001). The immunoglobulin-like protein Hibris functions as a dose-dependent regulator of myoblast fusion and is differentially controlled by Ras and Notch signaling. Development 128: 4251-4264. 11684661

Bao, S. (2014). Notch controls cell adhesion in the Drosophila eye. PLoS Genet 10: e1004087. PubMed ID: 24415957

Benzing, T. (2004). Signaling at the slit diaphragm. J. Am. Soc. Nephrol. 15: 1382-1391. PubMed Citation: 15153549

Bour, B. A., Chakravarti, M., West, J. M. and Abmayr, S. M. (2000). Drosophila Sns, a member of the immunoglobulin superfamily that is essential for myoblast fusion. Genes Dev. 14: 1498-1511. 10859168

Donoviel, D. B., Freed, D. D., Vogel, H., Potter, D. G., Hawkins, E., Barrish, J. P., Mathur, B. N., Turner, C. A., Geske, R., Montgomery, C. A. et al. (2001). Proteinuria and perinatal lethality in mice lacking NEPH1, a novel protein with homology to NEPHRIN. Mol. Cell. Biol. 21: 4829-4836. PubMed Citation: 11416156

Dworak, H. A., Charles, M. A., Pellerano, L. B. and Sink, H. (2001). Characterization of Drosophila hibris, a gene related to human nephrin. Development 128: 4265-4276. 11684662

Galletta, B. J., Chakravarti, M., Banerjee, R. and Abmayr, S. M. (2004). Sns: Adhesive properties, localization requirements and ectodomain dependence in S2 cells and embryonic myoblasts. Mech. Dev. 121(12): 1455-68. 15511638

Garg, P., Verma, R., Nihalani, D., Johnstone, D. B. and Holzman, L. B. (2007). Neph1 cooperates with nephrin to transduce a signal that induces actin polymerization. Mol. Cell. Biol. 27: 8698-8712. PubMed Citation: 17923684

Hamano, Y., Grunkemeyer, J. A., Sudhakar, A., Zeisberg, M., Cosgrove, D., Morello, R., Lee, B., Sugimoto, H. and Kalluri, R. (2002). Determinants of vascular permeability in the kidney glomerulus. J. Biol. Chem. 277: 31154-31162. PubMed Citation: 12039968

Haralalka, S., Shelton, C., Cartwright, H. N., Guo, F., Trimble, R., Kumar, R. P. and Abmayr, S. M. (2014). Live imaging provides new insights on dynamic F-Actin filopodia and differential endocytosis during myoblast fusion in Drosophila. PLoS One 9: e114126. PubMed ID: 25474591

Jones, N., Blasutig, I. M., Eremina, V., Ruston, J. M., Bladt, F., Li, H., Huang, H., Larose, L., Li, S. S., Takano, T., et al. (2006). Nck adaptor proteins link nephrin to the actin cytoskeleton of kidney podocytes. Nature 440: 818-823. PubMed Citation: 16525419

Kesper, D. A., et al. (2007). Myoblast fusion in Drosophila melanogaster is mediated through a fusion-restricted myogenic-adhesive structure (FuRMAS). Dev. Dyn. 236: 404-415. PubMed Citation: 17146786

Kang, J. S., Mulieri, P. J., Hu, Y., Taliana, L. and Krauss, R. S. (2002). BOC, an Ig superfamily member, associates with CDO to positively regulate myogenic differentiation. EMBO J. 21: 114-124. PubMed Citation: 11782431

Kestilä, M., Lenkkeri, U., Männikkö, M., Lamerdin, J., McCready, P., Putaala, H., Ruotsalainen, V., Morita, T., Nissinen, M., Herva, R., et al. (1998). Positionally cloned gene for a novel glomerular protein -- nephrin -- is mutated in congenital nephrotic syndrome. Mol. Cell 1: 575-582. 9660941

Khoshnoodi, J., Sigmundsson, K., Ofverstedt, L. G., Skoglund, U., Obrink, B., Wartiovaara, J. and Tryggvason, K. (2003). Nephrin promotes cell-cell adhesion through homophilic interactions. Am. J. Pathol. 163(6): 2337-46. 14633607

Kim, S., et al. (2007). A critical function for the actin cytoskeleton in targeted exocytosis of prefusion vesicles during myoblast fusion. Dev. Cell 12: 571-586. PubMed Citation: 17419995

Klapper, R., (2002). The formation of syncytia within the visceral musculature of the Drosophila midgut is dependent on duf, sns and mbc. Mech Dev. 110(1-2): 85-96. 11744371

Kocherlakota, K. S., Wu, J. M., McDermott, J. and Abmayr, S. M. (2008). Analysis of the cell adhesion molecule sticks-and-stones reveals multiple redundant functional domains, protein-interaction motifs and phosphorylated tyrosines that direct myoblast fusion in Drosophila melanogaster. Genetics 178(3): 1371-83. PubMed Citation: 18245830

Lenkkeri, U., et al. (1999). Structure of the gene for congenital nephrotic syndrome of the Finnish type (NPHS1) and characterization of mutations. Am. J. Hum. Genet. 64: 51-61. 9915943

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Liu, G., Kaw, B., Kurfis, J., Rahmanuddin, S., Kanwar, Y. S. and Chugh, S. S. (2003). Neph1 and nephrin interaction in the slit diaphragm is an important determinant of glomerular permeability. J. Clin. Invest. 112: 209-221. PubMed Citation: 12865409

Massarwa, R., Carmon, S., Shilo, B.-Z. and Schejter, E. D. (2007). WIP/WASp-based actin-polymerization machinery is essential for myoblast fusion in Drosophila. Dev. Cell 12: 557-569. Medline abstract: 17419994

Paululat, A., Burchard, S., Renkawitz-Pohl, R. (1995). Fusion from myoblasts to myotubes is dependent on the rolling stone gene (rost) of Drosophila. Development 121(8): 2611-20. 7671823

Paululat, A., Goubeaud, A., Damm, C., Knirr, S., Burchard, S. and Renkawitz-Pohl, R. (1997). The mesodermal expression of rolling stone (rost) is essential for myoblast fusion in Drosophila and encodes a potential transmembrane protein. J. Cell Biol. 138: 337-348. Medline abstract: 923007

Paululat, A., Holz, A. and Renkawitz-Pohl, R. (1999). Essential genes for myoblast fusion in Drosophila embryogenesis. Mech. Dev. 83: 17-26. Medline abstract: 10507836

Putaala, H., Soininen, R., Kilpelainen, P., Wartiovaara, J. and Tryggvason, K. (2001). The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death. Hum. Mol. Genet. 10: 1-8. PubMed Citation: 11136707

Shelton, C., Kocherlakota, K. S., Zhuang, S. and Abmayr, S. M. (2009). The immunoglobulin superfamily member Hbs functions redundantly with Sns in interactions between founder and fusion-competent myoblasts. Development 136(7): 1159-68. PubMed Citation: 19270174

Verma, R., Kovari, I., Soofi, A., Nihalani, D., Patrie, K. and Holzman, L. B. (2006). Nephrin ectodomain engagement results in Src kinase activation, nephrin phosphorylation, Nck recruitment, and actin polymerization. J. Clin. Invest. 116: 1346-1359. PubMed Citation: 16543952

Weavers, H., Prieto-Sanchez, S., Grawe, F., Garcia-Lopez, A., Artero, R., Wilsch-Brauninger, M., Ruiz-Gomez, M., Skaer, H. and Denholm, B. (2009). The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm. Nature 457: 322-326. PubMed Citation: 18971929

Zhuang, S., Shao, H., Guo, F., Trimble, R., Pearce, E. and Abmayr, S. M. (2009). Sns and Kirre, the Drosophila orthologs of Nephrin and Neph1, direct adhesion, fusion and formation of a slit diaphragm-like structure in insect nephrocytes. Development 136(14):2335-44. PubMed Citation: 19515699

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