The fusion of myoblasts leading to the formation of myotubes is an integral part of skeletal myogenesis in many organisms. In Drosophila, specialized founder myoblasts initiate fusion through expression of the receptor-like attractant Dumbfounded, which brings them into close contact with other myoblasts. Rols7, a gene expressed in founders, has been identified as an essential component for fusion during myotube formation. Rols7 is a 7kb alternative splice of rolling pebbles. During fusion, Rols7 localizes in a Duf-dependent manner at membrane sites that contact other myoblasts. These sites are also enriched with D-Titin, which functions to maintain myotube structure and morphology. When Rols7 is absent or its localization is perturbed, the enrichment of D-Titin fails to occur. Rols7 integrates the initial event of myoblast attraction with the downstream event of myotube structural organization by linking Duf to D-Titin (Menon, 2001).
dumbfounded is the only other fusion gene that is known to be restricted in expression to the founders and is absent in fusion competant myoblasts (FCM) (Ruiz-Gomez, 2000). In addition to the overlap in spatial expression, the temporal expression profiles of rols7 and duf in these tissues appear identical. These observations, the receptor-like nature of the molecule encoded by duf, and the loss of membrane-enriched Rols7 in the duf mutant led the authors to test whether Duf expression is sufficient to promote Rols7 membrane localization. This was done by overexpressing Rols7 early throughout the mesoderm and later in all muscles, either by itself or together with Duf, using the 24B-GAL4 driver. Under either of these conditions, no change was observed in the patterning of the somatic muscles. Rols7 localization was then examined late at stage 16, a time at which the expression of endogenous Rols7 and probably that of Duf is lost in wt muscles. When Rols7 is overexpressed alone, the protein appears as speckles throughout the cytosol of mature muscles. Despite its abundance, no membrane patches could be detected. In contrast, co-overexpression with Duf results in Rols7 becoming membrane enriched, with little or no protein remaining in the cytoplasm of muscles. In addition, higher levels of Rols7 are found along membranes that come in direct contact with other muscles. Muscles such as VL1, VO4, and VO6 that abut other muscles only on one side show significantly higher levels of Rols7 on the side in contact with its neighbor, whereas muscles such as VL2 and VO5, which lie between muscles, show equally high levels of Rols7 expression on either side of the membrane. This and observations in wt embryos, where Rols7 accumulates at discrete sites along the myotube membrane suggest that Duf and Rols7 may be present at specialized sites along the founder (or myotube) membrane that contact the FCM (Menon, 2001).
The protein encoded by the rols7 transcript has several distinct domains that can potentially participate in protein-protein interactions. At its N terminus, Rols7 carries a C3HC4 zinc finger, called the RING finger. While studies on several RING finger-containing proteins such as Cbl suggest that this domain is essential for E2-dependent ubiquitin protein ligase activity leading to protein destruction, the RING fingers in other proteins have been implicated in different modes of protein-protein interactions. Of note, the RING finger in the vertebrate muscle-specific proteins termed MURFs is essential to establish stable interaction between a specific MURF and Titin or the cytoskeletal network of microtubules (Centner, 2001; Spencer, 2000). At its C terminus, Rols7 encodes three different protein interaction motifs: a tandem array of nine ankyrin repeats followed closely by three TPR repeats and a coiled-coiled domain. Based on its overall structure, it is plausible that Rols7 could act as a focal point for the assembly of a multiprotein complex at the membrane where the Duf receptor is located, bringing the fusion machinery and directing changes in the cytoskeleton to sites where fusion would take place. In support of this, it has been shown that Rols7 is required for the enrichment of D-Titin to fusion sites in founders (or myotubes). However, this appears to be only one of the roles served by Rols7, since founders in the rols mutant either remain unfused or develop into small precursors, whereas fusion is arrested at a later stage in the D-Titin null allele (Menon, 2001).
Somatic muscle formation in Drosophila requires fusion of muscle founder cells with fusion-competent myoblasts. In a genetic screen for genes that control muscle development, antisocial (ants; alternative name for rolling pebbles), a gene that encodes an ankyrin repeat-, TPR repeat-, and RING finger-containing protein, was shown to be required for myoblast fusion. In ants mutant embryos, founder cells and fusion-competent myoblasts are properly specified and patterned, but they are unable to form myotubes. Ants, which is expressed specifically in founder cells, interacts with the cytoplasmic domain of Dumbfounded, a founder cell transmembrane receptor, and with Myoblast city, a cytoskeletal protein, both of which are also required for myoblast fusion. These findings suggest that Ants functions as an intracellular adaptor protein that relays signals from Dumbfounded to the cytoskeleton during myoblast fusion (Chen, 2001).
In order to gain insights into the function of rols during myoblast fusion, tests were conducted to determine whether Rols is present in founder cells or fusion-competent myoblasts. An antibody double-labeling experiment was performed with anti-Rols and anti-ß-galactosidase (ß-gal) antibodies using the rp298 enhancer trap line, which carries a P element insertion in the 5' promoter of the duf gene. Confocal microscopy has demonstrated that Rols is localized to the lacZ-expressing founder cells. Another founder cell-specific marker, even-skipped (eve), is also localized to the same cells as Rols. Interestingly, Rols is a cytoplasmic protein that aggregates to discrete foci. The aggregated appearance of Rols staining is reminiscent of that of Sns, the transmembrane receptor of fusion-competent myoblasts, which is localized to discrete sites associated with the cell membrane as fusion progresses (Chen, 2001).
Two transmembrane receptors, Duf and Sns, are implicated in cell recognition during myoblast fusion in Drosophila, whereas the cytoplasmic protein Mbc has been implicated in mediating changes in the cytoskeleton. It is not clear whether or how the known fusion molecules interact with each other during the fusion process. In addition, given the multistep nature of the fusion process, it is likely that additional components of the pathway(s) remain to be identified. Rols physically interacts with both Duf and Mbc. Thus, Rols could serve as a linker molecule that relays essential signals from a membrane receptor to changes in the cytoskeleton of founder cells (Chen, 2001).
Ankyrin proteins contain three domains, including a membrane binding domain at the amino terminus, a central spectrin binding domain, and a carboxy-terminal regulatory domain. The membrane binding domain, which contains multiple ankyrin repeats, binds to the cytoplasmic domains of specific integral membrane proteins, including adhesion molecules. Rols is not a conventional ankyrin protein, since its ankyrin repeats are located at the carboxy-terminal region and it lacks the central spectrin binding domain. Nevertheless, Rols can associate with the founder cell receptor Duf and the cytoplasmic protein Mbc. The conserved regions between Rols and its vertebrate orthologs, including the ankyrin repeats, are required for Rols' interaction with Duf, since a deletion construct lacking the conserved domains does not associate with Duf. The fact that a rols allele (antsT321) that deletes the conserved region behaves as a null mutation is consistent with this region being important for the function of Rols in vivo. Preliminary results indicate that Mbc maintains the ability to interact with an Rols protein lacking the conserved carboxy-terminal region, suggesting that the amino-terminal domain of Rols is likely to interact with Mbc (Chen, 2001).
Antibody staining has shown that Rols is a cytoplasmic protein. Two other fusion molecules, Mbc and Blow, are also expressed in the cytoplasm. However, the localization of Rols is distinct from that of Mbc and Blow. While Mbc and Blow are expressed in both founder cells and fusion-competent myoblasts, Rols is only expressed in founder cells. In addition, while Mbc and Blow are expressed throughout the cytoplasm of myoblasts, Rols is localized in discrete domains in the cytoplasm. These results, together with the protein-protein interaction between Rols and Duf, raise the possibility that the Rols localization domains might correlate with the sites of cell recognition and adhesion between founder cells and fusion-competent myoblasts. The subcellular structures in which Rols is localized and how these domains might be related to the expression of Duf on the founder cell membrane remain to be determined. While the lack of Duf antibody prevents the examination of the Duf protein expression pattern on the founder cell membrane and the relative localization of Duf and Rols, the Sns protein has been shown to be clustered in discrete regions on the membrane of fusion-competent cells (Bour, 2000). It is conceivable that Duf may also be localized to specific membrane regions in founder cells during the fusion process. However, the possibility that there is an excessive amount of Duf on the founder cell membrane such that no localization of Duf is necessary during cell recognition and cell adhesion cannot be ruled out. Nevertheless, the altered Rols localization in duf mutant embryos supports the hypothesis that Duf is required to localize Rols to specific subcellular foci, presumably through the physical interaction between the two proteins (Chen, 2001).
Myoblast fusion requires not only the recognition and adhesion between founder cells and fusion-competent cells, but also subsequent cytoskeletal rearragements that lead to the proper alignment of the two populations of cells. Previous studies on the founder cell-specific receptor Duf have shown that it acts as an attractant for fusion-competent cells (Ruiz-Gómez, 2000). Although duf is necessary for myoblast fusion, it is not sufficient, since ectopic expression of duf in fusion-competent cells did not result in fusion among this population of myoblasts (Ruiz-Gómez, 2000). Based on this observation, it was suggested that besides duf, there must exist at least one additional protein that is present in founder cells but absent from fusion-competent myoblasts. This protein could interact with the intracellular domain of Duf to initiate fusion (Ruiz-Gómez, 2000). Rols may represent such a molecule: (1) Rols is expressed in founder cells just before and during the fusion process; (2) Rols physically interacts with the cytoplamic domain of Duf; (3) the Rols protein is localized in discrete regions in the cytoplasm of founder cells during the fusion process, and the specific localization of Rols is altered in duf mutant embryos, consistent with the possible interaction with a localized membrane receptor during the fusion process (Chen, 2001).
Given the conservation of numerous signaling pathways between Drosophila and vertebrates, it is possible that vertebrate homologs of genes required for Drosophila myoblast fusion might play similar roles in skeletal muscle development. However, none of the myoblast fusion genes identified in Drosophila so far have been implicated in a similar role in vertebrate skeletal muscle development. For example, the closest vertebrate homolog of Duf and Sns is the human Nephrin protein, which is essential for kidney development. The vertebrate homolog of Mbc, DOCK180, interacts with focal adhesion molecules and seems to be a general factor that regulates cytoskeletal events. Studies of two mouse orthologs of rols suggest that one of them, mants1, could be involved in skeletal muscle development in vertebrates. The temporal expression pattern of mants1 in the developing mouse embryo is reminiscent of rols expression in the Drosophila embryo. mants1 expression coincides with the early stages of mesodermal development, and its expression is dramatically reduced after skeletal muscle formation. The transient expression of mants1 in the mesoderm is consistent with a potential role in early skeletal muscle development, including myoblast fusion. Interestingly, mants1 is also expressed at the time of fusion in the C2 myoblast cell line. However, it should be pointed out that the expression of mants1 in the mouse embryo is not solely restricted to skeletal muscle precursors but rather is more broadly distributed throughout the mesoderm at E11.5. Obviously, further studies will be required to confirm if mants1 indeed plays a role in myoblast fusion in vertebrates as does rols in Drosophila (Chen, 2001).
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).
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).
The rolling pebbles gene of Drosophila encodes two proteins, one of which, Rols7, is essential for myoblast fusion. In addition, Rols 7 is expressed during myofibrillogenesis and in the mature muscles. Here it overlaps with alpha- Actinin (a-Actn) and the N-terminus of D-Titin/Kettin/Zormin in the Z-line of the sarcomeres. In the attachment sites of the somatic muscles, Rols7 and the immunoglobulin superfamily protein Dumbfounded/Kin of irreC (Duf/Kirre) colocalise. As Duf/Kirre is detectable only transiently, it may be involved in establishing the first contact of the outgrowing muscle fiber to the epidermal attachment site. It is proposed that Rols7 and Duf/Kirre link the terminal Z-disc to the cell membrane by direct interaction. This is supported by the fact that in yeast two hybrid assays the tetratricopeptide repeat E (TPR E) of Rols7 shows interaction with the intracellular domain of Duf/Kirre. The colocalisation of Rols7 with a-Actn and with D-Titin/Kettin/Zormin in the Z-dics is reflected in interactions with different domains of Rols7 in this assay. In summary, these data show that besides the role in myoblast fusion, Rols7 is a scaffold protein during myofibrillogenesis and in the Z-line of the sarcomere as well as in the terminal Z-disc linking the muscle to the epidermal attachment sites (Kreiskother, 2006).
The scaffold protein Rols7 has been shown to be essential for myoblast fusion in the somatic mesoderm during Drosophila embryogenesis where it might interact with several components of the fusion machinery. Evidence is presented that Rols7 has an additional function in the establishment of the muscle attachment and the formation of the Z-discs, as well as in the Z-discs of the mature muscles (Kreiskother, 2006).
During myoblast fusion, Rols7 mRNA decays at stage 15. Antibody staining of stage 17 embryos reveal a concentration of Rols7 at the muscle ends next to the epidermal attachment sites, which are caused by new transcription in RT-PCR experiments. Later on in the mature larval muscles Rols7 is detected in the sarcomeric Z-discs (Kreiskother, 2006).
During the early stages of myogenesis, the interaction of the founder cell specific protein Duf/Kirre and the fusion competent myoblasts (fcm) specific Sns leads to the adhesion of the two cell types, which is a prerequisite for further steps of the fusion process. Besides this, Duf/Kirre transiently are concentrated at the end of the developing muscles at stage 15 and 16, while it disappears again at stage 17. This led to a hypothesis that Duf/Kirre might participate in the first contact of the outgrowing muscle to the attachment site, as does Vein. This would require an interaction partner in the extracellular matrix or at the epidermal site. Since the sns transcript is present in the muscle attachment sites at a low level at stage 17, antibody staining for Sns was performed, but a distinct signal in the attachment sites could not be detected. As well as the transcript of sns, its paralog, Hibris (Hbs), is also found in the muscle attachment sites, and, more exactly, localised to the contact site between the cells at the epidermal attachments. Thus, it could function as an interaction partner for Duf/Kirre (Kreiskother, 2006).
As a further possible interaction partner Rst/IrreC was considered, since Rst/IrreC, the paralogue of Duf/Kirre, shows expression in the epidermal tendon cells during embryonic stages. Due to the fact that Duf/Kirre and Rst/IrreC are indeed able to undergo heterophilic interaction in cell culture experiments, the conclusion is drawn that Rst/IrreC might be the candidate for an interaction partner of Duf/Kirre on the epidermal site, thus enabling an early contact of the muscle to the epidermal attachment site (Kreiskother, 2006).
Rols7, which interacts with the intracellular domain of Duf/Kirre, is also localised at the muscle ends from late stage 16 onwards shortly before Duf/Kirre disappears. It is speculated that Rols7 is brought to the membrane where it interacts with Duf/Kirre (Kreiskother, 2006).
alpha actinin (α-Actn) and D-Titin/Kettin, both found at the muscle attachment site in a similar pattern, also interact with Rols7 (at least in the yeast two hybrid assay) and participate in the establishment of the terminal Z-disc. For the flight muscle it was shown that α-Actn is essential for the formation of this structure and for obtaining a correct insertion of the myofibril to the epidermal tendon cell. Furthermore the yeast assay showed an interaction of α-Actn with Duf/Kirreintra (Kreiskother, 2006).
kettin mutants show strong defects in terminal Z-disc function. This study proposes that, in addition to Kettin, Rols7 and α-Actn are important for the formation of this structure. The process might be connected to Muscleblind (Mbl), since in mutants for mbl, Z-discs are not assembled correctly. Unfortunately, a mutant analysis of Rols7 function in terminal Z-disc formation is difficult due to its essential function during myoblast fusion (Kreiskother, 2006).
Apart from the myoblasts and attachment sites, Rols7 is expressed in the developing sarcomeres of larval and adult muscles and localises to the Z-discs, as was shown using antibodies for α-Actn and D-Titin/Kettin as markers. Yeast interaction assays revealed that Rols7 might directly interact with α-Actn and Zormin, which, like Kettin, is an isoform derived from the sallimus (sls) gene and also localises to the Z-discs. Therefore, it is postulated that Rols7 serves as a scaffold protein that links α-Actn and Zormin in the Z-disc. Furthermore, the analyses of alpha actinin mutants showed that the presence of α-Actn is not necessary for Rols7 localisation to the Z-discs. In addition, Rols7, as well as α-Actn and D-Titin/Kettin, is present during the assembly of the sarcomere. In vertebrates it has been shown that in spreading edges of rat cardiomyocytes, dense bodies that contain Z-disc proteins assemble at the spreading membrane and align to premyofibrils in cooperation with newly formed actin filaments and small myosin filaments (Kreiskother, 2006).
Antibody staining showed protein aggregates that aligned to form kinds of premyofibrils and demonstrated that in Drosophila, the assembly of the Z-discs seems to be similar to that of vertebrates. So, Rols7 is the first protein that is essential for myoblast fusion and plays an additional role in the sarcomere assembly as well as in the Z-discs of mature muscles, where it is proposed that it links α-Actn and D-Titin/Kettin/Zormin. Δ-titin/kettin-mutants have a weaker fusion phenotype than rols7-mutants, however, D-Titin/Kettin is clearly expressed during myoblast fusion as a component of the adhesion complex between founder cell and fcm. Individual Rols7 domains serve different function in distinct processes of myogenesis (Kreiskother, 2006).
From coimmunoprecipitation experiments it was already supposed that the intracellular domain of Duf interacts with Rols. Furthermore, cell culture cotransfection assays showed colocalisation of Duf, Rols and D-Titin. In yeast interaction assays the individual domains of Rols7 were tested for interaction with potential partners that included components of the fusion machinery which might be relevant for muscle attachment or sarcomere assembly as well. Indeed, the different domains interact with different partners in different developmental contexts, and it is concluded that Rols7 is a multifunctional protein (Kreiskother, 2006).
The interaction of Rols7 with the intracellular domain of Duf/Kirre was confirmed and it was found that the interaction probably is mediated by the TPR repeats of Rols7, respectively, by the most C-terminal TPR E repeat and the R1 fragment that contains the RING finger and an additional part of 321 amino acids downstream. In contrast α-Actn interacts with the R1 domain and with both TPR repeats, the TPR E and the TPR X, whereas the N-terminal part of Zormin interacts only with the R1 domain in this assay. No interaction was detected for the N-terminal part of Kettin and the Rols7 domains. These results, together with the rescue capability of truncated Rols7 versions, led to the proposal of certain functions to individual Rols7 domains. Either the RING finger domain, the TPR repeats or the ankyrin repeats and the TPR repeats have been deleated and the remaining parts of Rols7 were examined for their competence to rescue the rols fusion defect. A deletion of the RING finger domain does not affect the rescue of the rols fusion phenotype, whereas a deletion of the TPR repeats leads to a partial rescue and a deletion of ankyrin repeats and TPR repeats together does not rescue fusion at all. The Rols7 version without the RING finger rescues the fusion phenotype. This RING finger is included in the R1 fragment which interacts with Duf/Kirreintra, Blow, Zormin and α-Actn. Thus, it is proposed that the R1 domain is a candidate to mediate the transient interaction of Duf/Kirreinttra at the muscle attachment sites. The R1 domain of Rols7 could then mediate the interaction with Zormin in the Z-discs in all larval muscles. R1 is the only Rols7 fragment that interacts with Zormin. R1 and TPR E as well as TPR X have the capability to interact with α-Actn. It cannot be decide whether both domains of Rols7 interact with α-Actn in the Z-discs. The interaction of Duf/Kirreintra with the TPR E repeat indicates a function of the TPR E repeat during myoblast fusion, since its deletion only leads to a partial rescue of the rols fusion phenotype. The ankyrin repeats did not interact with any of the proteins which have been tested in the yeast assay and which are characteristic for sarcomere assembly and muscle attachment. Taking this together with the fact that a deletion of this domain, in addition to a deletion of the TPR repeats, prevents the rescue of the fusion defect, indicates that the ankyrin repeats predominantly function during myoblast fusion (Kreiskother, 2006).
Rols7 is a scaffold protein which contains distinct domains characteristic of protein-protein interaction. It is proposed that the interaction of the appropriate domain with certain proteins is specific for the process of myogenesis, myoblast fusion, muscle attachment or sarcomere assembly (Kreiskother, 2006).
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).
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