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).

GENE STRUCTURE

cDNA clone length - 8035 bp

Bases in 5' UTR - 548

Exons - 30

Bases in 3' UTR - 3038

PROTEIN STRUCTURE

Amino Acids - 1482

Structural Domains

The sns cDNA sequence, determined from overlapping clones, includes an apparent open reading frame (ORF) of 1483 amino acids, and untranslated regions of 549 and 3480 nucleotides on the 5' and 3' ends, respectively. The predicted size of the Sns protein without modification is 162 kD. The amino acid sequence strongly suggests that sns encodes a cell-adhesion molecule in the immunoglobulin superfamily. Sns contains eight putative Ig domains in the amino-terminal region, a single fibronectin type III domain and a putative transmembrane domain. A potential signal anchor sequence characteristic of membrane bound proteins is present in the amino terminus. This sequence, centered around a glutamine-rich stretch, is composed of 13 contiguous uncharged polar residues flanked by basic residues. A similar sequence of unknown function is present in the COOH terminal portion of Sns. BLAST database searches for proteins with structural homology to Sns have identified the human Nephrin protein (Kestila, 1998; Lenkkeri, 1999), which is expressed specifically in the kidney glomerulus and is associated with Congenital Nephrotic Syndrome. Sns has homology to Nephrin throughout the eight immunoglobulin domains and the fibronectin type III repeat. In Sns, this extracellular region includes 14 NXS and NXT consensus triplets for N-glycosylation. Like Nephrin, Sns also contains several SG doublets that are potential attachment sites for heparin sulfate. Homology to Nephrin continues through the putative transmembrane domain and into the cytosolic carboxy-terminal region. Although Sns has a longer cytoplasmic domain, the Nephrin-related region of Sns includes several tyrosine residues that may act as sites for phosphorylation. In addition to Nephrin, other genes with significant homology to Sns include the recently reported hibris gene of Drosophila (Bour, 2000).

Of note, the available Drosophila genomic sequence has revealed the presence of at least 20 introns, and a transcription unit that covers a minimum of 50 kb. These preliminary findings suggest that the genomic region that corresponds to the sns locus is quite large, not unlike that of the human Nephrin gene, which includes 29 introns spanning a total of 26 kb (Bour, 2000).

EVOLUTIONARY HOMOLOGS

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

date revised: 5 July 2005

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.