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Novel small GTPase M-Ras participates in reorganization of actin cytoskeleton
1Department of Biology, Faculty of Science, Chiba University, Chiba, Japan.
Abstract:
During an attempt to elucidate regulatory mechanisms of skeletal muscle cell differentiation, we cloned cDNAs encoding a novel small GTPase from cDNA libraries of the mouse skeletal muscle cell line C2 and rat brain. It was designated as M-Ras due to the structural similarity to Ras family proteins. M-Ras contained conserved motifs for GDP/GTP-binding and GTPase activities, whereas it varied from the other Ras family proteins at several amino acids within the extended effector domain. From the C-terminal sequence, M-Ras is presumed to be anchored to the cell membrane with a geranylgeranyl group in combination with a polybasic region. Bacterially expressed recombinant M-Ras exerted the GTP-binding and GTPase activities. A mutant M-RasG22V was unable to hydrolyze bound GTP, indicating that it serves as a constitutively active form. Epitope-tagging experiments showed that M-Ras was concentrated on certain plasma membrane-associated structures. Transfection of M-Ras cDNA and microinjection of the M-RasG22V protein into fibroblasts induced formation of the peripheral microspikes. In addition, the actin stress fibers disappeared and instead numerous actin foci were formed in the injected cells. The transfected cells eventually exhibited dendritic appearances with microspikes. Consequently, M-Ras is likely to participate in reorganization of the actin cytoskeleton.
Insights
Researchers identified a novel small GTPase, M-Ras, involved in regulating skeletal muscle cell differentiation. M-Ras reorganizes the actin cytoskeleton, influencing cell shape and potentially cell differentiation processes.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Skeletal muscle cell differentiation involves complex regulatory mechanisms.
- Small GTPases are key regulators of cellular processes, including cytoskeleton dynamics.
- Ras family proteins share conserved motifs but exhibit functional diversity.
Purpose of the Study:
- To identify novel proteins involved in skeletal muscle cell differentiation.
- To characterize the function and localization of a newly discovered small GTPase, M-Ras.
- To investigate the role of M-Ras in actin cytoskeleton reorganization.
Main Methods:
- Cloning of cDNAs encoding M-Ras from mouse and rat cell lines.
- Bacterial expression and purification of recombinant M-Ras.
- Site-directed mutagenesis to create a constitutively active M-Ras mutant (M-RasG22V).
- Epitope-tagging for M-Ras localization studies.
- Cell transfection and microinjection experiments in fibroblasts.
Main Results:
- A novel small GTPase, M-Ras, was identified and characterized.
- M-Ras exhibits GTP-binding and GTPase activities, with a constitutively active mutant (M-RasG22V) identified.
- M-Ras localizes to plasma membrane-associated structures.
- Expression of M-Ras induces peripheral microspike formation and actin stress fiber disappearance.
- M-Ras promotes dendritic cell morphology with microspikes.
Conclusions:
- M-Ras is a novel Ras family GTPase implicated in actin cytoskeleton remodeling.
- M-Ras plays a role in cellular morphological changes, potentially influencing cell differentiation.
- Further research is warranted to fully elucidate M-Ras's regulatory mechanisms in skeletal muscle and other cell types.