The Large GTPase Dynamin Regulates Actin Comet Formation and Movement in Living Cells

• Published in: Proceedings of the National Academy of Sciences - PNAS 2002-01, Vol.99 (1), p.167-172
• Main Authors: Orth, James D., Krueger, E. W., Cao, H., McNiven, Mark A.
• Format: Article
• Language: English
• Subjects: 3T3 cells; Actins; Actins - chemistry; Actins - metabolism; Animals; Biological Sciences; Cell membranes; Cell Movement; Cellular biology; Comets; Dynamins; Fibroblasts; Fibroblasts - metabolism; Green Fluorescent Proteins; GTP Phosphohydrolases - chemistry; GTP Phosphohydrolases - genetics; GTP Phosphohydrolases - physiology; Hepatocytes; Hepatocytes - metabolism; Imaging; Immunohistochemistry; Luminescent Proteins - metabolism; Mice; Microscopy, Fluorescence; Microscopy, Video; Models, Biological; Mutation; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Physiological regulation; Plasmids - metabolism; Proline - chemistry; Protein Structure, Tertiary; Proteins; Rats; Recombinant Fusion Proteins - metabolism; Ruffles; Studies; Time Factors; Transfection
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.012607899

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 DynΔPRD-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.