Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape

The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is belie ved that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering mem brane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes, We have observed that the cytoplas mic distribution of dynamin changes dramatically in fibroblasts that have b een stimulated to undergo migration with a motagen/hormone, In quiescent ce lls, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicl es at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membran e ruffles and lamellipodia. Biochemical and morphological studies using ant ibodies and GFP-tagged dynamin demonstrate an interaction with cortactin, C ortactin is an actin-binding protein that contains a well defined SH3 domai n. Using a variety of biochemical methods we demonstrate that the cortactin -SH3 domain associates with the proline-rich domain (PRD) of dynamin. Funct ional studies that express wild-type and mutant forms of dynamin and/or cor tactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitmen t of endogenous or expressed dynamin into the cell ruffle. Further, express ion of a cortactin protein lacking the interactive SH3 domain (Cort Delta S H3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)Delta PRD) seq uester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morpholo gy. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first dem onstration that dynamin can interact with the actin cytoskeleton to regulat e actin reorganization and subsequently cell shape.