title: The large GTPase dynamin regulates actin comet formation and movement in living cells.
abstract: The large GTPase dynamin (Dyn2) has been demonstrated by us and others to interact with several different actin-binding proteins. To define how Dyn2 might participate in actin dynamics in livings cells we have expressed green fluorescent protein (GFP)-tagged Dyn2 in cultured cells and observed labeling of comet-like vesicles and macropinosomes. The comet structures progressed with a constant velocity and were reminiscent of actin comets associated with motile vesicles in cells expressing type I phosphatidylinositol phosphate 5-kinases. Based on these observations we sought to determine whether Dyn2 is an integral component of actin comets. Cells expressing type I phosphatidylinositol phosphate 5-kinase and Dyn2-GFP revealed a prominent colocalization of Dyn2 and actin in comet structures. Interestingly, comet formation and motility were normal in cells expressing wild-type Dyn2-GFP but altered markedly in Dyn2 mutant-expressing cells. Dyn2K44A-GFP mutant cells displayed a significant reduction in comet number, length, velocity, and efficiency of movement. In contrast, comets in cells expressing Dyn2DeltaPRD-GFP appeared dark and did not incorporate the mutant Dyn2 protein, indicating that the proline-rich domain (PRD) is required for Dyn2 recruitment. Further, these comets were significantly longer and slower than those in control cells. These findings demonstrate a role for Dyn2 in actin-based vesicle motility.
authors.affiliation: Department of Biochemistry and Molecular Biology, 1721 Guggenheim Building, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: James D
authors.initials: JD
authors.lastname: Orth
authors.forename: E W
authors.initials: EW
authors.lastname: Krueger
authors.forename: H
authors.initials: H
authors.lastname: Cao
authors.forename: Mark A
authors.initials: MA
authors.lastname: McNiven
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002465:Cell Movement; D034281:Dynamins; D005347:Fibroblasts / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D049452:Green Fluorescent Proteins; D022781:Hepatocytes / Q000378:metabolism; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D008954:Models, Biological; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D000199:Actins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011392:Proline; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D020558:GTP Phosphohydrolases; D034281:Dynamins