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
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).
Sequence comparison has revealed two closely related transcripts having several exons in common, but differing at the N termini, suggesting two independent promoters. In addition, the larger transcript has a unique intermediate exon (exon 6). The two alternatively spliced transcripts were named rols7 and rols6 on the basis of their respective sizes. Conceptual translation indicates that rols7 encodes a 1900 amino acid protein, whereas rols6 reveals an open reading frame of 1670 amino acids. Rols6 harbors 79 specific amino acids at the N terminus with a high level of acetic amino acids, while Rols7 is characterized by 309 specific amino acids at the N terminus (Menon, 2001; Rau, 2001).
Conceptual translation of rols7 cDNA revealed a single open reading frame (ORF) that encodes a molecule with several interesting features. (1) Rols7 carries the signature sequence of a subfamily of lipolytic enzymes. This sequence, which contains the invariant nucleophilic serine essential for enzymatic catalysis, is also conserved in its relative position within the molecule, being always located near the N terminus. (2) The other regions that are conserved among many subfamily members are missing in Rols7, which raises the possibility that Rols7 may represent a divergent member of this lipolytic enzyme subfamily. (3) There is also a highly conserved and specialized zinc finger, known as the RING finger. At its C terminus, there are nine contiguous ankyrin repeats followed closely by three tetratricopeptide repeats (TPR) and a coiled-coil domain that overlaps with the last TPR repeat. All three motifs are known to mediate protein-protein interactions. With the exception of the lipolytic motif, all motifs are encoded for within the predicted ORF of the rols6 cDNA (Menon, 2001).
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).
date revised: 10 February 2002
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