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).
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).
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).
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).
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).
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