InteractiveFly: GeneBrief

rolling pebbles : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - rolling pebbles

Synonyms - antisocial

Cytological map position - 68F1-2

Function - signaling

Keywords - mesoderm, myoblast fusion

Symbol - rols

FlyBase ID: FBgn0041096

Genetic map position -

Classification - RING-finger motif, ankyrin repeat and a TPR repeat

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

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 (Duf: Kin of irre/Kirre), that brings founder myoblasts into close contact with other myoblasts. Rolling pebbles (Rols), a gene expressed in founders, is an essential component for fusion during myotube formation. During fusion, Rols 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 Rols is absent or its localization is perturbed, the enrichment of D-Titin fails to occur. Rols encodes an ankyrin repeat-, TPR repeat-, and RING finger-containing protein. Rols, 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. Thus Rols integrates the initial event of myoblast attraction with the downstream event of myotube structural organization by linking Duf to D-Titin (Chen, 2001; Menon, 2001; Rau, 2001).

The formation of skeletal muscle requires the commitment of multipotent mesodermal stem cells to a myogenic fate, followed by the fusion of mononucleated myoblasts to form multinucleated myotubes and the patterning, morphogenesis, and innervation of mature muscle fibers. The somatic musculature of Drosophila is composed of a stereotyped, segmentally repeated pattern of 30 muscle fibers per hemisegment. Larval body wall muscle development begins during embryogenesis and can be divided into two distinct stages -- myoblast fate determination and myoblast fusion. During mid-embryogenesis, a population of mesodermal cells, marked by the expression of the twist gene, acquires a myoblast cell fate. Subsequently, a subset of myoblasts, marked by the expression of lethal of scute, is selected via a lateral inhibition process to become muscle founder cells while the remaining twist-expressing cells become fusion competent. It is believed that the founder cells serve as sources of attractant for the surrounding fusion-competent cells to fuse with these fusion-competent cells and form myotubes that typically comprise between 4 and 25 myoblasts. Thus, the founder cells act as 'seeds' for the future muscle fibers to determine their position, orientation, size, and pattern of motorneuron innervation (Chen, 2001; Menon, 2001; Rau, 2001 and references therein).

Electron microscopic studies have revealed that myoblast fusion is a multistep process that involves similar ultrastructural changes in vertebrate and Drosophila muscle cells. Based on these studies, Drosophila myoblast fusion can be divided into four steps, including cell-cell recognition, adhesion, alignment, and membrane fusion. Initially, a myoblast recognizes an appropriate cellular target for fusion, for example, a founder cell or a forming myotube. Then, the myoblast adheres to the founder cell or the myotube. At this point, a prefusion complex forms along closely apposed plasma membranes. The prefusion complex consists of groups of paired vesicles with associated electron-dense material on each side of the membrane. Later, the prefusion complex resolves into electron-dense plaques along the plasma membranes of the apposed cells. The fusing cells align along their long axes, and pores form between the apposed plasma membranes. Finally, the plasma membranes vesiculate along their shared lengths, followed by vesiculation of the apposed membranes (Chen, 2001).

Recent genetic studies have identified several genes essential for myoblast fusion in Drosophila. dumbfounded (kirre) (duf) encodes a transmembrane protein with extracellular immunoglobulin (Ig) domains and is expressed in founder cells (Ruiz-Gómez, 2000); sticks and stones (sns), which also encodes a transmembrane protein with Ig domains, is expressed in fusion-competent cells (Bour, 2000). It has been suggested that Duf acts as an attractant for fusion-competent cells by interacting with the Sns protein (Frasch, 2000). Myoblast city (Mbc), a Drosophila homolog of human DOCK180, has been proposed to mediate changes in the cytoskeleton during myoblast fusion, since human DOCK180 has been implicated in signaling by the Rho/Rac family of GTPases to the cytoskeleton. Another gene required for myoblast fusion is Blown fuse (blow), which encodes a cytoplasmic protein with no significant sequence homology to known proteins (Doberstein, 1997). The structures and functions of these proteins suggest the existence of a signaling pathway for myoblast fusion in which transmembrane receptors are linked to components of the cytoskeleton. However, to date, there has been no biochemical evidence for direct interactions between these proteins, and the mechanism whereby they cooperate to control myoblast fusion remains a mystery (Chen, 2001).

rolling pebbles was identified independently in three labs in screens to identify genes involved in myoblast fusion. One study used a GFP reporter, driven by the muscle-specific myosin heavy chain promoter (MHC-tauGFP), which allowed the examination of muscle morphology in live embryos (Chen, 2001). Two other studies examined collections of P-element induced mutations (Menon, 2001; Rau, 2001).

Fusion is severely disturbed rols mutant alleles. Before dorsal closure, many unfused myoblasts per segment are often observed. In embryos at stage 16/17, only a small number of irregularly shaped myofibers are present, leading to a very rudimentary muscle pattern and a varying proportion of unfused myoblasts. The disappearance of many myoblasts might be explained in part by cell death of unfused myoblasts, which are cleared away by macrophages. Embryos homozygous for the mutant rols alleles develop until shortly before hatching since dorsal closure is evident. Many unfused myoblasts are present and some muscle-like fibers with only a few nuclei are found. It is proposed that these muscle-like fibers represent muscle precursor cells that stretched and tried to contact the epidermis, as originally observed for founder cells in mbc mutant embryos. The persisting myoblasts often adhere to the myofiber-like cells. Furthermore, these myoblasts often extend filopodia, which are directed towards muscle-like fibers. However, the extent of the phenotype is variable, mainly after stage 16; the number of unfused myoblasts, and the appearance and number of myofibers also differ significantly among embryos of the same stage. Cardioblast development is not obviously disturbed, as the typical repetitive pattern of four ß3 tubulin stained cardioblasts and two unstained cardioblasts is evident. Analysis of gut morphogenesis often reveals incomplete formation in at least a quarter of the mutants when compared with the wild type, which might be evidence for defects in the visceral muscles of the midgut. The visceral musculature also consists of small syncytia. In duf mutants and in sticks and stones mutants no fusion can be detected in the visceral mesoderm, implying that the founder cell hypothesis also holds true for the visceral musculature of the midgut (Rau, 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).

Based on these observations and the interaction between Rols and Mbc, the following sequence of events during myoblast fusion is proposed. Initially, Duf acts as an attractant for fusion-competent myoblasts. Through either direct or indirect interaction(s) between Duf and Sns, fusion-competent myoblasts recognize and adhere to founder cells. In this process, Sns is localized to discrete sites in the membrane of fusion-competent myoblasts, presumably sites of cell adhesion. It is possible that Duf is also localized to discrete domains in the membrane of the founder cells. Next, within the founder cells, through interaction(s) between the cytoplasmic domain of Duf and Rols, Rols is recruited to discrete cytoplasmic domains close to the membrane. Meanwhile, interaction between Rols and Mbc, and perhaps additional cytoskeleton-associated molecules, leads to changes in the cytoskeleton that are necessary for the proper alignment of founder cells with fusion-competent cells. This model predicts that in rols mutant embryos, despite a block of cell alignment, which requires the transmission of signals from Duf to the cytoskeleton, cell recognition and adhesion should take place normally. This is indeed what is observed. In rols mutant embryos, fusion-competent myoblasts extend filopodia toward their fusion targets. Such phenotypes are not observed in duf mutant embryos in which fusion is blocked at the cell recognition step (Ruiz-Gómez, 2000). Taken together, the model is favored that Rols acts as a linker molecule that relays signals from the membrane receptor Duf to changes in the cytoskeleton in the founder cells (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).


REGULATION

Protein Interactions

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 aggregation of Rols in distinctive cytoplasmic locations in founder cells, and the presence of multiple protein-protein interaction motifs in the Rols protein prompted an examination of whether Rols plays a role during myoblast fusion by mediating interactions between molecules in the myoblast fusion pathway(s). To test whether Rols interacts with other fusion molecules, coimmunoprecipitation assays were performed in Drosophila S2 cells using MYC-tagged Rols and other fusion proteins, including Blow, Duf, Mbc, and Sns, tagged with the V5-epitope at their carboxyl termini. Rols interacts with the founder cell receptor Duf but not the fusion-competent cell receptor Sns, despite the high homology shared by Duf and Sns. This specific interaction between Rols and Duf is consistent with the founder cell-specific expression of Rols. A cleaved form of is generated when full-length Duf is expressed in S2 cells. This form migrates slightly slower than the Duf cytoplasmic domain alone, suggesting that it is likely to contain both the transmembrane and the cytoplasmic domains. Interestingly, this cleaved form also associates with Rols. However, when the Duf cytoplasmic domain alone was tested, no interaction was detected. These results suggest that the transmembrane domain of Duf is required for its interaction with Rols. In addition, protein-protein interaction was detected between an amino-terminal fragment of Mbc and Rols, while no interaction was detected between Blow and Rols. The interactions between full-length Mbc and Rols could not be tested, since the full-length Mbc was not expressed at a detectable level (Chen, 2001).

To locate the specific domain(s) of Rols that are required for its interaction with Duf, a carboxy-terminal deletion (Rols-DeltaC) was created that truncates the conserved region between Drosophila Rols and its mouse orthologs. This deletion construct was tested for its ability to associate with Duf in coimmunoprecipitation experiments. No interaction between the truncated Rols protein and Duf was detected, suggesting that the conserved region of Rols is required for its interaction with Duf. This conclusion is consistent with the genetic mutants, since the antsT321 allele produces carboxy-terminal-truncated protein that deletes the entire conserved region (Chen, 2001).

The interaction between Rols and Duf, together with the subcellular aggregation of the Rols protein, suggests that Rols is likely to colocalize with Duf during myoblast fusion. Because of the lack of Duf antibody, this hypothesis could not be tested directly. However, if Duf is involved in recruiting Rols to specific subcellular locations during fusion, one would expect a change in the pattern of Rols localization in duf mutant embryos. Examination of Rols protein in duf mutant embryos has shown this to be the case. Instead of localizing to discrete sites in the cytoplasm, Rols protein is distributed throughout the cytoplasm at the peripheral membrane region and appears as rings that outline the founder cells in the duf mutant embryo (Chen, 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).

Drosophila rolling pebbles colocalises and putatively interacts with alpha-Actinin and the Sls isoform Zormin in the Z-discs of the sarcomere and with Dumbfounded/Kirre, alpha-Actinin and Zormin in the terminal Z-discs

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).

The intracellular domain of Dumbfounded affects myoblast fusion efficiency and interacts with Rolling pebbles and Loner

Drosophila body wall muscles are multinucleated syncytia formed by successive fusions between a founder myoblast and several fusion competent myoblasts. Initial fusion gives rise to a bi/trinucleate precursor followed by more fusion cycles forming a mature muscle. This process requires the functions of various molecules including the transmembrane myoblast attractants Dumbfounded (Duf) and its paralogue Roughest (Rst), a scaffold protein Rolling pebbles (Rols) and a guanine nucleotide exchange factor Loner (Schizo). Fusion completely fails in a duf, rst mutant, and is blocked at the bi/trinucleate stage in rols and loner single mutants. This study analysed the transmembrane and intracellular domains of Duf, by mutating conserved putative signaling sites and serially deleting the intracellular domain. These were tested for their ability to translocate and interact with Rols and Loner and to rescue the fusion defect in duf, rst mutant embryos. Studying combinations of double mutants, further tested the function of Rols, Loner and other fusion molecules. This study shows that serial truncations of the Duf intracellular domain successively compromise its function to translocate and interact with Rols and Loner in addition to affecting myoblast fusion efficiency in embryos. Putative phosphorylation sites function additively while the extreme C terminus including a PDZ binding domain is dispensable for its function. It was also shown that fusion is completely blocked in a rols, loner double mutant and is compromised in other double mutants. These results suggest an additive function of the intracellular domain of Duf and an early function of Rols and Loner which is independent of Duf (Bulchand, 2010).

This study has shown that in order to ensure successful fusion a large part of the intracellular region of Duf is required for its function. Serial truncations of the intracellular domain reveal that the efficiency of fusion is decreased as larger regions are removed. Also, conserved putative phosphorylation signalling sites function additively resulting in efficient myoblast fusion and the formation of a mature myotube. Several parallels can be drawn from this data and that published by Kocherlakota (2008), on the intracellular domain of the Duf ligand SNS. Similar to what has been found for SNS, the PDZ binding domain is not required for the function of Duf during myoblast fusion. This is contrary to the role of this domain in the function of Rst in the developing eye. While the intracellular domain of SNS is important for its function, the C terminal end of SNS is dispensable similar to that of Duf as shown by Duf ΔCT1-flag in the Rols/Loner translocation assay in S2 cells and rescue of the fusion defect in duf, rst embryos. The membrane proximal intracellular regions of SNS and Duf are more important for their functions. While SNS is phosphorylated on tyrosine residues, the ability of Duf 4 phos-flag to only partially rescue the duf, rst mutant, implies that phosphorylation of these sites also contributes to Duf function (Bulchand, 2010).

Membrane anchored forms of Duf irrespective of the sequence of the transmembrane domain, appear to be sufficient for successful fusion. This suggests that the transmembrane domain of Duf does not perform any essential role or contribute to downstream signalling activity and only serves to anchor Duf to the plasma membrane. The PADVI motif, though not essential for myoblast fusion, might have a function in the context of a different tissue type that has not been tested so far. That the functions of Duf cannot be attributed to particular motifs might be a strategy utilised to ensure that normal myotube development occurs in a robust manner and compromising the function of any of these motifs singly, does not drastically affect the overall process. As has been suggested for the downstream functions of SNS, Duf too might transduce signals to cytoskeletal elements via its intracellular domain, to ensure successful myoblast fusion (Bulchand, 2010).

Previous studies proposed that myoblast fusion molecules can be categorised into those that participate in the early versus later phases of fusion. More recently it has been proposed that all fusion molecules are required in all fusion events. Molecules like Rols and Loner have been individually shown to function in the second phase of fusion after the formation of the bi/trinucelate precursor. This study has shown that removal of both rols and loner completely blocks fusion similar to the duf, rst mutant. Analyses of other similar double mutants demonstrate that genes involved in myoblast fusion might interact with each other to affect fusion efficiency. It is possible that what this study has shown with a few myoblast genes is true for other genes that have thus far been characterised for their role in the later stages of fusion. Such interactions have been shown for Kette/Hem/Nap1/GEX-3 and Blow (Bulchand, 2010).

This study has shown that membrane anchored Duf without its intracellular domain and without any interaction with Rols and Loner, is sufficient to initiate fusion. It is possible that even in the absence of robust Duf dependent signal transduction, requirements for the formation of a bi/trinucleate precursor are met. It was also shown that Rols and Loner are required, albeit redundantly, for precursor formation or the initial phase of fusion suggesting that this 'early function' of these molecules appears to be independent of Duf. This fusion defect was observed in late stage 15-early stage 16 embryos to ensure that the observations and interpretation thereof are not due to a delay in fusion. Rols and Loner may perform different roles early versus later on during myoblast fusion. In the later phase of fusion, Rols and Loner appear to sustain fusion by interacting with and translocating Duf to the surface of the myotube. As has been suggested in the case of Rols, Loner too might serve to regulate Duf at the surface of the myotube through as yet unknown mechanisms. It is possible that these supposed distinct early versus late mechanisms are used in mutant conditions in an effort to overcome fusion blocks, thus leading to delayed fusion events (Bulchand, 2010).


DEVELOPMENTAL BIOLOGY

Protein Interactions

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 aggregation of Rols in distinctive cytoplasmic locations in founder cells, and the presence of multiple protein-protein interaction motifs in the Rols protein prompted an examination of whether Rols plays a role during myoblast fusion by mediating interactions between molecules in the myoblast fusion pathway(s). To test whether Rols interacts with other fusion molecules, coimmunoprecipitation assays were performed in Drosophila S2 cells using MYC-tagged Rols and other fusion proteins, including Blow, Duf, Mbc, and Sns, tagged with the V5-epitope at their carboxyl termini. Rols interacts with the founder cell receptor Duf but not the fusion-competent cell receptor Sns, despite the high homology shared by Duf and Sns. This specific interaction between Rols and Duf is consistent with the founder cell-specific expression of Rols. A cleaved form of is generated when full-length Duf is expressed in S2 cells. This form migrates slightly slower than the Duf cytoplasmic domain alone, suggesting that it is likely to contain both the transmembrane and the cytoplasmic domains. Interestingly, this cleaved form also associates with Rols. However, when the Duf cytoplasmic domain alone was tested, no interaction was detected. These results suggest that the transmembrane domain of Duf is required for its interaction with Rols. In addition, protein-protein interaction was detected between an amino-terminal fragment of Mbc and Rols, while no interaction was detected between Blow and Rols. The interactions between full-length Mbc and Rols could not be tested, since the full-length Mbc was not expressed at a detectable level (Chen, 2001).

To locate the specific domain(s) of Rols that are required for its interaction with Duf, a carboxy-terminal deletion (Rols-DeltaC) was created that truncates the conserved region between Drosophila Rols and its mouse orthologs. This deletion construct was tested for its ability to associate with Duf in coimmunoprecipitation experiments. No interaction between the truncated Rols protein and Duf was detected, suggesting that the conserved region of Rols is required for its interaction with Duf. This conclusion is consistent with the genetic mutants, since the antsT321 allele produces carboxy-terminal-truncated protein that deletes the entire conserved region (Chen, 2001).

The interaction between Rols and Duf, together with the subcellular aggregation of the Rols protein, suggests that Rols is likely to colocalize with Duf during myoblast fusion. Because of the lack of Duf antibody, this hypothesis could not be tested directly. However, if Duf is involved in recruiting Rols to specific subcellular locations during fusion, one would expect a change in the pattern of Rols localization in duf mutant embryos. Examination of Rols protein in duf mutant embryos has shown this to be the case. Instead of localizing to discrete sites in the cytoplasm, Rols protein is distributed throughout the cytoplasm at the peripheral membrane region and appears as rings that outline the founder cells in the duf mutant embryo (Chen, 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).

Drosophila rolling pebbles colocalises and putatively interacts with alpha-Actinin and the Sls isoform Zormin in the Z-discs of the sarcomere and with Dumbfounded/Kirre, alpha-Actinin and Zormin in the terminal Z-discs

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).

The intracellular domain of Dumbfounded affects myoblast fusion efficiency and interacts with Rolling pebbles and Loner

Drosophila body wall muscles are multinucleated syncytia formed by successive fusions between a founder myoblast and several fusion competent myoblasts. Initial fusion gives rise to a bi/trinucleate precursor followed by more fusion cycles forming a mature muscle. This process requires the functions of various molecules including the transmembrane myoblast attractants Dumbfounded (Duf) and its paralogue Roughest (Rst), a scaffold protein Rolling pebbles (Rols) and a guanine nucleotide exchange factor Loner (Schizo). Fusion completely fails in a duf, rst mutant, and is blocked at the bi/trinucleate stage in rols and loner single mutants. This study analysed the transmembrane and intracellular domains of Duf, by mutating conserved putative signaling sites and serially deleting the intracellular domain. These were tested for their ability to translocate and interact with Rols and Loner and to rescue the fusion defect in duf, rst mutant embryos. Studying combinations of double mutants, further tested the function of Rols, Loner and other fusion molecules. This study shows that serial truncations of the Duf intracellular domain successively compromise its function to translocate and interact with Rols and Loner in addition to affecting myoblast fusion efficiency in embryos. Putative phosphorylation sites function additively while the extreme C terminus including a PDZ binding domain is dispensable for its function. It was also shown that fusion is completely blocked in a rols, loner double mutant and is compromised in other double mutants. These results suggest an additive function of the intracellular domain of Duf and an early function of Rols and Loner which is independent of Duf (Bulchand, 2010).

This study has shown that in order to ensure successful fusion a large part of the intracellular region of Duf is required for its function. Serial truncations of the intracellular domain reveal that the efficiency of fusion is decreased as larger regions are removed. Also, conserved putative phosphorylation signalling sites function additively resulting in efficient myoblast fusion and the formation of a mature myotube. Several parallels can be drawn from this data and that published by Kocherlakota (2008), on the intracellular domain of the Duf ligand SNS. Similar to what has been found for SNS, the PDZ binding domain is not required for the function of Duf during myoblast fusion. This is contrary to the role of this domain in the function of Rst in the developing eye. While the intracellular domain of SNS is important for its function, the C terminal end of SNS is dispensable similar to that of Duf as shown by Duf ΔCT1-flag in the Rols/Loner translocation assay in S2 cells and rescue of the fusion defect in duf, rst embryos. The membrane proximal intracellular regions of SNS and Duf are more important for their functions. While SNS is phosphorylated on tyrosine residues, the ability of Duf 4 phos-flag to only partially rescue the duf, rst mutant, implies that phosphorylation of these sites also contributes to Duf function (Bulchand, 2010).

Membrane anchored forms of Duf irrespective of the sequence of the transmembrane domain, appear to be sufficient for successful fusion. This suggests that the transmembrane domain of Duf does not perform any essential role or contribute to downstream signalling activity and only serves to anchor Duf to the plasma membrane. The PADVI motif, though not essential for myoblast fusion, might have a function in the context of a different tissue type that has not been tested so far. That the functions of Duf cannot be attributed to particular motifs might be a strategy utilised to ensure that normal myotube development occurs in a robust manner and compromising the function of any of these motifs singly, does not drastically affect the overall process. As has been suggested for the downstream functions of SNS, Duf too might transduce signals to cytoskeletal elements via its intracellular domain, to ensure successful myoblast fusion (Bulchand, 2010).

Previous studies proposed that myoblast fusion molecules can be categorised into those that participate in the early versus later phases of fusion. More recently it has been proposed that all fusion molecules are required in all fusion events. Molecules like Rols and Loner have been individually shown to function in the second phase of fusion after the formation of the bi/trinucelate precursor. This study has shown that removal of both rols and loner completely blocks fusion similar to the duf, rst mutant. Analyses of other similar double mutants demonstrate that genes involved in myoblast fusion might interact with each other to affect fusion efficiency. It is possible that what this study has shown with a few myoblast genes is true for other genes that have thus far been characterised for their role in the later stages of fusion. Such interactions have been shown for Kette/Hem/Nap1/GEX-3 and Blow (Bulchand, 2010).

This study has shown that membrane anchored Duf without its intracellular domain and without any interaction with Rols and Loner, is sufficient to initiate fusion. It is possible that even in the absence of robust Duf dependent signal transduction, requirements for the formation of a bi/trinucleate precursor are met. It was also shown that Rols and Loner are required, albeit redundantly, for precursor formation or the initial phase of fusion suggesting that this 'early function' of these molecules appears to be independent of Duf. This fusion defect was observed in late stage 15-early stage 16 embryos to ensure that the observations and interpretation thereof are not due to a delay in fusion. Rols and Loner may perform different roles early versus later on during myoblast fusion. In the later phase of fusion, Rols and Loner appear to sustain fusion by interacting with and translocating Duf to the surface of the myotube. As has been suggested in the case of Rols, Loner too might serve to regulate Duf at the surface of the myotube through as yet unknown mechanisms. It is possible that these supposed distinct early versus late mechanisms are used in mutant conditions in an effort to overcome fusion blocks, thus leading to delayed fusion events (Bulchand, 2010).


EFFECTS OF MUTATION

The scaffold-like protein D-Titin (Sallimus) is expressed in prefusion myoblasts: during fusion, it accumulates along the membrane at sites of myoblast-myotube contact. Although fusion is initiated in the absence of D-Titin, it does not go to completion, and results in the formation of significantly smaller muscles that show aberrant myotube morphology with many unfused myoblasts. This suggests that proper myotube formation is achieved through the synchronization of fusion events occurring at the cell (or myotube) surface and changes in cytoskeletal architecture that take place within the cytoplasm. Whether Rols7 might serve to coordinate these two processes in the founder (or myotube) by linking Duf function to D-Titin expression and/or localization was tested (Menon, 2001).

In wt embryos, D-Titin is strongly expressed along the periphery of the FCM, whereas its expression around the founder cell periphery is relatively weak. Upon fusion, the myotube expresses D-Titin along its periphery, with enrichment at sites of myoblast-myotube contact that coincide with sites where Rols7 appears to be membrane associated. In D-mef2, sns, duf, and Df(3L)BK9 embryos, D-Titin expression in unfused founders is initially weak but increases with time. However, while the founders in D-mef2 and sns embryos continue to show peripheral D-Titin expression and enrichment at discrete sites that colocalize with membrane-associated Rols7, its localization in duf and Df(3L)BK9 founders is different. In duf, D-Titin no longer shows membrane enrichment and the protein appears cytosolic. In Df(3L)BK9 embryos, D-Titin can sometimes be seen around the membrane of some precursors without showing enrichment. More often, membrane-associated D-Titin is not detectable and the protein appears cytosolic. However, by reintroducing Rols7 into the founders of Df(3L)BK9 embryos using rP298-GAL4/UAS-rols7, D-Titin is seen to regain peripheral localization and becomes clearly enriched at discrete sites along the myotube membrane that colocalizes with Rols7 and contacts other myoblasts. Since Rols7 expression and membrane localization remain unaffected in the D-Titin mutant, Rols7 appears to function upstream of D-Titin. From these results, it is concluded that the enrichment of D-Titin at fusion sites within the founder or precursor requires Rols7 (Menon, 2001).

The expression profile of rols7 suggests that it may act at the level of muscle founder or precursor cells. The analysis of the mutant phenotype reveals the appearance of elongated mini-muscles containing more than one nucleus in several cases. In order to test the hypothesis that Rols acts on the precursor level, the fusion competence of rols mutant cells were examined in a wild-type background with a cell transplantation strategy. The wild-type host embryos contain a daGAL4 construct, which is expressed in all cells, while the transplanted cells of the rols mutant contained a UAS-lacZ gene. Thus rols mutant cells in a wild-type background express only the reporter ß-galactosidase after successful cell fusion with the wild-type host cells. One might expect that successful fusion is dependent on the transplanted cell type. Since ventral mesodermal cells were transplated at the cellular blastoderm stage, they develop either to founders or to fusion-competent cells, the latter being the far more abundant cell type. If Rols is indeed required in muscle precursor cells -- as suggested by its expression and mutant phenotype -- but not in FCMs, wild-type precursor cells of the host embryo should be able to recruit FCMs from rols mutants to form myofibers that express ß-galactosidase. However, transplanted donor cells that develop into precursors should not be able to recruit further host myoblasts for fusion (Rau, 2001).

The deficiency Df(3L)BK9 was chosen as a null allele for rols. Since mutant mesodermal cells were transplanted into wild-type embryos, the loss of rols6 in the endoderm and semaphorin 5c in the ectoderm as well as the loss of other genes localized in the deficiency should not influence this assay. After transplantation the recipient embryos were allowed to develop until 3rd instar larval stage and the muscle pattern was examined with respect to myotube development (Rau, 2001).

From a total of 191 transplantations, 119 embryos (62%) reached the third larval instar. Of these larvae, 53 showed a clone derived from the transplanted cell descendants, which had fused to host cells. In 11 cases, the clones derived from homozygous rols donors and were found in the musculature. These clones were compared at the morphological level with 42 clones derived from control donors with regard to size, shape and correct attachment. The 11 clones derived from homozygous rols embryos were found in the ventral, lateral and dorsal body wall muscles of third instar larvae, and they demonstrate that at least a population of rols mutant cells is able to fuse with host cells to multinucleate myotubes. These data show that rols mutant cells can participate in fusion. Since these large muscle clones are abundant, it is suggested that in these clones rols mutant FCMs fuse with wild-type founders. Moreover the clones were found at the same positions in the thoracic and the abdominal segments and approximately in the same size as the control clones. In the case of transplanted rols mutant cells, small muscle-like structures referred to as mini-muscles or compact, not elongated muscle-like cells were found in five larvae. These mini-muscles represent only a small part of the observed clones and contain two to five nuclei each. These mini-muscles are considered as precursor cells formed by a rols mutant founder and fusion competent cells of the donor. In one case, two of these mini-muscles were observed close to each other. The appearance of duplicated mini-muscles is interpreted as evidence for two precursor cells derived from asymmetric cell division of a progenitor. On the basis of these observations, it is concluded that the mini-muscles can be hybrid precursor cells derived from a rols mutant founder (with UAS-lacZ) and one to four cells of the wild-type host (carrying daGAL4). Therefore it is suggested that Rols acts in muscle precursors to recruit further FCMs. With rols-deficient cells, nearly 50% of the host embryos were found to contain mini-muscles. In four cases of the 43 control transplantations (in about 10% of the embryos) with hetero- or homo-zygous balancer embryos, similar defects were detected and it is assumed that this is probably a result of a dosage effect or may be due to incompatibility of homozygous balancer cells in the mosaic clones (Rau, 2001).

The Rolling pebbles isoform 6 (Rols6) is essential for proper Malpighian tubule morphology

During myoblast fusion, cell-cell recognition along with cell migration and adhesion are essential biological processes. The factors involved in these processes include members of the immunoglobulin superfamily like Sticks and stones (Sns), Dumbfounded (Duf) and Hibris (Hbs), SH3 domain-containing adaptor molecules like Myoblast city (Mbc) and multidomain proteins like Rolling pebbles (Rols). For rolling pebbles, two differentially expressed transcripts have been defined (rols7 and rols6). However, to date, only a muscle fusion phenotype has been described and assigned to the lack of the mesoderm-specific expressed rols7 transcript. This study shows that a loss of the second rolling pebbles transcript, rols6, which is expressed from the early bud to later embryonic stages during Malpighian tubule (MpT) development, leads to an abnormal MpT morphology that is not due to defects in cell determination or proliferation but to aberrant morphogenesis. In addition, when Myoblast city or Rac are knocked out, a similar phenotype is observed. Myoblast city and Rac are essentially involved in the development of the somatic muscles and are proposed to be interaction partners of Rols7. Because of the predicted structural similarities of the Rols7 and Rols6 proteins, it is argued that genetic interaction of rols6, mbc and rac might lead to proper MpT morphology. It is also proposed that these interactions result in stable cell connections due to rearrangement of the cytoskeleton (Putz, 2005).

The Malpighian tubules (MpTs) of Drosophila arise as four buds from the hindgut anlage close to its boundary with the posterior midgut primordium. The cells of the four buds are characterised by the expression of the transcription factor Cut (Ct) at stage 10 of embryogenesis. During germ band extension at stage 11, the cells of the four tubule primordia undergo cell proliferation, and the tubules begin to bud out. By stage 13, proliferation is complete and short tubules have formed. From stage 13 onwards, cells from the caudal mesoderm join the MpT primordia and later the stellate cells (SCs). From the end of germ band retraction, the tubules begin to elongate due to cell rearrangement. In stage 15 and 16 embryos, the characteristic stereotypic course of the four renal tubules through the embryonic body is clearly visible. The paired posterior tubules span the posterior abdominal and terminal segments of the embryo. The anterior tubules extend forwards into abdominal segments 2/3 where the tubule loops back on itself so that the tips of both anterior tubules lie more posteriorly within the abdomen (Putz, 2005).

Since Rols6 is expressed in the Malpighian tubules (MpTs) throughout their development, the role of Rols6 in the generation of this tissue was investigated. For this purpose, a rols6-specific mutant was generated, in which the majority of the putative promoter region of rols6 was deleted, and thereby rols6 transcription was knocked out, while rols7 expression persisted as in the wild type. In this rols6-specific mutant, the early phase of organogenesis is the same as in wild type, i.e. the MpTs consist of two cell types, the principal cells (PCs) and the SCs. As the SCs originate from the mesoderm, one might expect that they would be affected in rols mutants. However, in the specific rols6 mutation generated, the SCs are able to migrate and integrate between the PCs as observed in the wild type. However, the PCs and SCs do not arrange correctly, and therefore, the typical MpT arrangement as found in wild-type embryo is not observed for stage 15 embryos onwards. The anterior tubules often show abnormal curves and lasso-like structures and fail to extend through the abdominal cavity. These navigation defects might well result from incorrect cell rearrangements, indicated by thickened regions of the tubules, whereas other parts seem to have a typical wild-type organisation (Putz, 2005).

Evidence is presented that correct cell rearrangement is dependent on Rols6 and proteins such as Mbc and Rac. These factors have been proposed to act with Rols7 in a common signalling cascade during myoblast fusion. An additional defect is the disorientation of MpTs in the body cavity, which again is characteristic for rols6, mbc and rac mutants (Putz, 2005).

Homozygous rols6 mutants are viable, indicating that the physiological functions of principal cells and stellate cells are largely unaffected. Loss of rols6 expression only moderately affects embryonic viability. Furthermore, homozygous EP(3)3330*5a flies do not die prematurely in contrast to those lacking another gene essential for MpT formation, hibris. The strongest hibris allelic combination die early as adults. Also, in contrast to rols6 mutants, in hibris mutants, the number of SCs is strongly reduced. This might cause defects in excretory function of the tubules, and thus leads to the observed lethality (Putz, 2005).

rols6-specific mutants show no distortion in rols7 transcription and in muscle development indicating that rols6 is specific for MpT development, while rols7 is essential for myogenesis. This is consistent with the observatio that Rols6 is not able to rescue the myogenic defect in rols mutants (Putz, 2005).

myoblast city mutants, rolling pebbles mutants and rac1/rac2 double mutants show late defects in Malpighian tubule differentiation. mbc mutants exhibit a MpT phenotype and it is proposed that this might be due to a failure to complete cell rearrangement; a phenomenon which is more apparent in mbc mutants than for rolling pebbles ones. Mbc, the homologue of vertebrate DOCK180 in Drosophila, associates with the adapter protein Crk. This interaction regulates cell migration and cytoskeleton organisation in a Rac-dependent manner. This agrees with the finding that rac1/rac2 double mutants exhibit the characteristic MpT defects as rols6 and mbc mutants do. Rols7 and Duf have been shown to interact in myogenesis. The strong similarity between the Rols proteins and their proposed functions leads to the hypothesis that Rols6 interacts with a so far unknown partner in the PCs. It is proposed that Rols6 initiates a signalling cascade via Mbc and Rac that leads to the correct rearrangement of cells, presumedly by rearranging the cytoskeleton, as has been proposed for Rols7 in the myogenic precursor cells. In the development of the somatic musculature, rearrangement of cytoskeleton is mediated by Blown fuse (Blow) and Kette in the second fusion wave (Putz, 2005).

Individual factors and protein complexes involved in cell migration and cytoskeleton arrangement have been described from many model organisms as well as from cell culture experiments. DOCK180/CED-5, the homologues of Drosophila Myoblast city (Mbc) in vertebrates and in C. elegans, form a complex with ELMO1/CED-12 that functions as a guanine nucleotide exchange factor (GEF). This functional GEF promotes Rac activation, and thus facilitates cell migration and rearrangement of the cytoskeleton. In vertebrates, additional protein complexes are built via DOCK180/p130Cas/Crk interaction and regulate cell migration and cytoskeletal organisation in a Rac-dependent manner. From kidney cells of human and mouse, the signalling molecule NEPHRIN is known to be of major importance in the podocyte for slit-diaphragm formation. Mutations in the nephrin gene are the major cause of congenital nephrotic syndrome in humans. In Drosophila, the homologue of vertebrate Nephrin, Hibris (hbs), is expressed during MpT development specifically in SCs. Therefore, it is likely that during MpT differentiation, Hibris mediates cell adhesion and arrangement between the PCs and the SCs, a mechanism comparable to myogenesis. In vertebrates, CMS/CD2AP has been identified as an interaction partner for Nephrin. The CMS/CD2AP homologue in Drosophila can be detected in silico as CG11316. CD2AP knock-out mice die due to kidney failure. Moreover, the Nephrin/CD2AP complex is able to bind to actin and to p130Cas (corresponding to CG1212). In Drosophila, homologues have been identified for all the above-mentioned factors involved in these protein complexes. However, little is known about their role in the developmental processes taking place during MpT development (Putz, 2005).

In Drosophila, a group of immunoglobulin-like proteins act in cell-cell recognition and attraction during myogenesis. These processes are also of importance in MpT development. Rolling pebbles is a multidomain and adapter-like protein. It is proposed that Rols6 interacts in Malpighian tubule development with proteins also involved in myogenesis such as Mbc and Rac. It is assumed that Rolling pebbles interacts with Mbc, and thus activates Rac. This hypothesis is supported by the observations that mbc and rac mutants exhibit defects in MpT development which might be linked to cell organisation in this tissue (Putz, 2005).

The mechanisms underlying the stereotypic course of the MpTs through the body cavity are still unclear. However, studies of phenotypes of early determination mutants like numb show that the tip cell and its sibling might both play a critical role in controlling the spatial arrangement of the growing tubules. This is indicated by the MpT phenotypes of numb mutants and UAS-numb embryos, where numb is overexpressed. These embryos lack either the tip cell or the sibling cell but form elongated MpTs with normally rearranged PCs. Although the PCs rearrange normally in these numb alleles, the MpTs are misrouted through the body cavity, as has been observed for rols and rac mutants. This raises the question whether determination of the tip cells is affected in rols mutants (Putz, 2005).

Essential transcription factors for Tip cell determination and PC cell proliferation are the A-SC, Krüppel and Seven up. These factors could be required for rols6 expression in the MpTs. However, since rols6 is expressed in the rudimentary primordia of MpTs in Krüppel mutants and in seven up mutants, this is unlikely. It is assumed, therefore, that Rolling pebbles is not a signalling molecule involved in cell specification through direct regulation of early genes, but rather that it plays a role as an adapter molecule in a protein complex connecting the cells in the tissue as Rols7 does in myogenesis. Since rols6, mbc and rac mutant embryos exhibit the described MpT phenotype, it is likely that they belong to a group of genes that can be helpful in discovering the mechanisms in MpT development that lead to the typical thin tubule morphology through cell rearrangement (Putz, 2005).

Drosophila and mammalian models uncover a role for the myoblast fusion gene TANC1 in rhabdomyosarcoma

Rhabdomyosarcoma (RMS - see Drosophila as a Model for Human Diseases: Rhabdomyosarcoma) is a malignancy of muscle myoblasts, which fail to exit the cell cycle, resist terminal differentiation, and are blocked from fusing into syncytial skeletal muscle. In some patients, RMS is caused by a translocation that generates the fusion oncoprotein PAX-FOXO1, but the underlying RMS pathogenetic mechanisms that impede differentiation and promote neoplastic transformation remain unclear. Using a Drosophila model of PAX-FOXO1–mediated transformation, this study shows that mutation in the myoblast fusion gene rolling pebbles (rols) dominantly suppresses PAX-FOXO1 lethality. Further analysis indicates that PAX-FOXO1 expression causes upregulation of rols, which suggests that Rols acts downstream of PAX-FOXO1. In mammalian myoblasts, gene silencing of Tanc1, an ortholog of rols, reveals that it is essential for myoblast fusion, but is dispensable for terminal differentiation. Misexpression of PAX-FOXO1 in myoblasts upregulates Tanc1 and blocks differentiation, whereas subsequent reduction of Tanc1 expression to native levels by RNAi restorrs both fusion and differentiation. Furthermore, decreasing human TANC1 gene expression causes RMS cancer cells to lose their neoplastic state, undergo fusion, and form differentiated syncytial muscle. Taken together, these findings identify misregulated myoblast fusion caused by ectopic TANC1 expression as a RMS neoplasia mechanism and suggest fusion molecules as candidates for targeted RMS therapy (Avirneni-Vadlamudi, 2012).

This study uses a Drosophila chromosomal deletion, Df(3L)vin5, that dominantly suppresses PAX7-FOXO1–induced lethality. Human PAX7 demonstrates slightly higher sequence identity to Drosophila PAX3/7 than does human PAX3 and is therefore used in flies in this study. Df(3L)vin5 deletes segments 68A2–69A1 on chromosome 3, which includes the muscle-patterning gene rols, located at 68F1. rols encodes an essential adaptor molecule that links the Kirre transmembrane receptor with the machinery that drives myoblast cell-cell fusion and syncytial muscle formation; therefore, rols expression in the somatic mesoderm temporally coincides precisely with embryonic myoblast fusion. However, it was found by mRNA expression profiling that rols is misexpressed in PAX7-FOXO1 larval muscle. Thus, the study hypothesizes that heterozygous deletion of the rols locus might account for Df(3L)vin5-mediated PAX7-FOXO1 suppression and that rols might act as a PAX7-FOXO1 target gene (Avirneni-Vadlamudi, 2012).

Of the 2 alternative transcripts expressed from the rols locus, only one of which is expressed in myoblasts; expression of the second is restricted to endodermal/ectodermal precursors. In this study, 2 rols homozygous-lethal, P-element insertion loss-of-function alleles, P1027 and P1729, were tested for suppression of PAX7-FOXO1. Of these 2 alleles, only the P1729 insertion disrupts expression of the myoblast rols transcript (myoblast expression of rols is unperturbed in P1027); accordingly, only the rolsP1729 allele suppresses PAX7-FOXO1–induced lethality and muscle pathogenicity (Avirneni-Vadlamudi, 2012).

To investigate whether rols acts as a downstream PAX-FOXO1 target, the daughterless-Gal4 transgene was used to drive ubiquitous embryonic expression of UAS-PAX7-FOXO1 and probed for Rols misexpression. Since native Rols expression initiates at embryonic stage 11, the study focused only on embryos stage 10 or earlier. Diffuse expression of PAX7-FOXO1 and Rols is observed in blastoderm (stage 4–5) embryos, which consist of uncommitted precursor cells, and expression persists in all examined cells — including nonmyogenic ectodermal and endodermal cells — of gastrulated (stage 9–10) embryos. Taken together, these Drosophila studies reveal that rols acts as a PAX7-FOXO1 downstream target gene, direct or indirect, and as a bona fide genetic effector (Avirneni-Vadlamudi, 2012). 

RMS model systems conveniently promote insights into not only neoplasia, but also muscle development. Although ultrastructural studies suggest that myoblast fusion biology is conserved, few of the Drosophila fusigenic genes have been identified as essential in mammals, and none of these are from the founder subfamily. As the name implies, founder myoblasts are seminal to Drosophila myogenesis, uniquely dictating the location and physiology of each individual muscle. With rols and Tanc1, it was shown that founder gene function is conserved in mammals and, furthermore, participates in human disease. How founder gene activity influences other forms of neuromuscular disease now becomes an intriguing issue (Avirneni-Vadlamudi, 2012). 

Genetic screening in a Drosophila model and loss-of-function/gain-of-function studies in mammalian platforms have collaboratively uncovered a PAX-FOXO1-to-TANC1 neoplasia axis, a finding that the study suggests to be novel. Results from this study also argue that the relationship between myogenesis transcription factor (e.g., MyoD) signaling and myoblast fusion genes is intricate. In the presence of altered fusion potential, both Drosophila and mammalian myoblasts transition to differentiated myocytes, which suggests that later aspects of myogenesis signaling must uncouple from the TANC1 fusigenic pathway. Yet correcting PAX-FOXO1–mediated overexpression of rols/TANC1 rescues PAX-FOXO1–induced differentiation and arrest. These results intimate that correction of the TANC1 fusigenic axis feeds back to and rescues PAX-FOXO1–mediated misregulation of myogenic signaling, raising fascinating questions regarding the mechanisms by which this occurs. The observation in this study that PAX3-FOXO1 protein levels remain unchanged in TANC1-silenced cells argues that rescue does not originate from decreased expression of PAX3-FOXO1 from the PAX3 promoter. Thus, the study speculates that rescue occurs epistatically downstream of PAX3-FOXO1 (Avirneni-Vadlamudi, 2012).


EVOLUTIONARY HOMOLOGS

Database searches have identified two predicted mouse proteins, mCP20090 and mCP14686 (Celera mouse genome annotation), and two human ESTs, KIAA1728 and KIAA1636, that encode apparent orthologs of rols. The human EST KIAA1728 (1644 amino acids) is 591 amino acids longer at its carboxyl terminus than its mouse homolog, mCP20090 (1051 amino acids), suggesting that the predicted mouse protein is missing a portion of its carboxy-terminal sequence (Chen, 2001).

To investigate whether the mammalian orthologs could also be involved in skeletal muscle development, the expression of the mouse orthologs was examined in the developing embryonic mesoderm by in situ hybridization. For simplicity, the mouse orthologs are referred to as mants1 (mCP20090) and mants2 (mCP14686), referring to the alternative Drosophila name for Rolling pebbles, Antisocial. mants1 is expressed in a broad range of the embryonic mesodermal tissues, including the limb buds and the somites at embryonic day 11.5, coincident with the time period when myoblast fusion occurs. Mants1 expression dramatically decreases at E13.5, when muscle differentiation is almost completed. Northern blot of adult tissues has shown that mants1 is not detectable in adult skeletal muscle. Thus, mants1 is expressed during a short time window when myoblast fusion takes place. The expression pattern of mants2, in contrast, is completely different from that of mants1. While mants1 expression is absent from the neural tube and dorsal root ganglia in the E11.5 embryo, mants2 is expressed strongly in these neural tissues. The neural expression of mants2 persists into adult stages. The transient expression of mants1 in mouse embryonic tissue is consistent with the transient expression of rols during myoblast fusion in Drosophila embryos and suggests that mants1 could play a role in skeletal muscle differentiation (Chen, 2001).

A novel rat gene, tanc (GenBank Accession No. AB098072), has been cloned that encodes a protein containing three tetratricopeptide repeats (TPRs), ten ankyrin repeats and a coiled-coil region, and is possibly a rat homolog of Drosophila rolling pebbles. The tanc gene is expressed widely in the adult rat brain. Subcellular distribution, immunohistochemical study of the brain and immunocytochemical studies of cultured neuronal cells indicate the postsynaptic localization of TANC protein of 200 kDa. Pull-down experiments have shown that TANC protein binds PSD-95, SAP97, and Homer via its C-terminal PDZ-binding motif, -ESNV, and fodrin via both its ankyrin repeats and the TPRs together with the coiled-coil domain. TANC also binds the alpha subunit of Ca2+/calmodulin-dependent protein kinase II. An immunoprecipitation study shows TANC association with various postsynaptic proteins, including guanylate kinase-associated protein (GKAP), alpha-internexin, and N-methyl-D-aspartate (NMDA)-type glutamate receptor 2B and AMPA-type glutamate receptor (GluR1) subunits. These results suggest that TANC protein may work as a postsynaptic scaffold component by forming a multiprotein complex with various postsynaptic density proteins (Suzuki, 2004).


REFERENCES

Search PubMed for articles about Drosophila rolling pebbles

Avirneni-Vadlamudi, U., Galindo, K.A., Endicott, T.R., Paulson, V., Cameron, S. and Galindo, R.L. (2012). Drosophila and mammalian models uncover a role for the myoblast fusion gene TANC1 in rhabdomyosarcoma. J Clin Invest 122: 403-407. PubMed

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

Bulchand, S., Menon, S. D., George, S. E. and Chia, W. (2010). The intracellular domain of Dumbfounded affects myoblast fusion efficiency and interacts with Rolling pebbles and Loner. PLoS One 5(2): e9374. PubMed Citation: 20186342

Centner, T., et al. (2001). Identification of muscle specific ring finger proteins as potential regulators of the titin kinase domain. J. Mol. Biol. 306: 717-726. 11243782

Chen, E. H. and Olson, E. N. (2001). Antisocial, an intracellular adaptor protein, is required for myoblast fusion in Drosophila. Dev. Cell 1: 705-715. 11709190

Doberstein, S. K., Fetter, R. D., Mehta, A. Y. and Goodman, C. S. (1997). Genetic analysis of myoblast fusion: blown fuse is required for progression beyond the prefusion complex. J. Cell Biol. 136: 1249-1261. 9087441

Frasch, M. and Leptin, M. (2000). Mergers and acquisitions: unequal partnerships in Drosophila myoblast fusion. Cell 102: 127-129. 10943831

Kocherlakota, K. S., et al. (2008), Analysis of the cell adhesion molecule sticks-and-stones reveals multiple redundant functional domains, protein-interaction motifs and phosphorylated tyrosines that direct myoblast fusion in Drosophila melanogaster. Genetics 178: 1371-1383. PubMed Citation: 18245830

Kreiskother, N., Reichert, N., Buttgereit, D., Hertenstein, A., Fischbach, K. F. and Renkawitz-Pohl, R. (2006). Drosophila rolling pebbles colocalises and putatively interacts with alpha-Actinin and the Sls isoform Zormin in the Z-discs of the sarcomere and with Dumbfounded/Kirre, alpha-Actinin and Zormin in the terminal Z-discs. J. Muscle Res. Cell Motil. 27(1): 93-106. 16699917

Menon, S. D. and Chia, W. (2001). Drosophila Rolling pebbles: A multidomain protein required for myoblast fusion that recruits D-Titin in response to the myoblast attractant Dumbfounded. Dev. Cell 1: 691-703. 11709189

Putz, M., et al. (2005). In Drosophila melanogaster, the Rolling pebbles isoform 6 (Rols6) is essential for proper Malpighian tubule morphology. Mech. Dev. 122(11): 1206-17. 16169193

Rau, A., et al. (2001). rolling pebbles (rols) is required in Drosophila muscle precursors for recruitment of myoblasts for fusion. Development 128: 5061-5073. 11748142

Rúiz-Gomez, M., Coutts, N., Price, A., Taylor, M. V. and Bate, M. (2000) Drosophila dumbfounded: a myoblast attractant essential for fusion. Cell 102: 189-198. 10943839

Spencer, J. A., Eliazer, S., Ilaria, R. L., Richardson, J. A. and Olson E. N. (2000) Regulation of microtubule dynamics and myogenic differentiation by MURF, a striated muscle RING-finger protein. J. Cell Biol. 150: 771-784. 10953002

Suzuki, T., et al. (2004). A novel scaffold protein, TANC, possibly a rat homolog of Drosophila Rolling pebbles (Rols), forms a multiprotein complex with various postsynaptic density proteins. Eur. J. Neurosci. 21(2): 339-50. 15673434


Biological Overview

date revised: 20 July 2012

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.