Luini, Alberto

The size and integrity of the Golgi apparatus is maintained via a tightly controlled regulation of membrane traffic using a variety of different signaling and cytoskeletal proteins. We have recently observed that activation of c-Src has profound effects on Golgi structure, leading to dramatically vesiculated cisternae in a variety of cell types. As the large GTPase dynamin (Dyn2) has been implicated in Golgi vesiculation during secretion, we tested whether inhibiting Dyn2 activity by expression of a Dyn2K44A mutant or siRNA knockdown could attenuate active Src-induced Golgi fragmentation. Indeed, these perturbations attenuated fragmentation, and expression of a Dyn2Y(231/597)F mutant protein that cannot be phosphorylated by Src kinase had a similar effect . Finally, we find that Dyn2 is markedly phosphorylated during the transit of VSV-G protein through the TGN whereas expression of the Dyn2Y(231/597)F mutant significantly reduces exit of the nascent protein from this compartment. These findings demonstrate that activation of Dyn2 by Src kinase regulates Golgi integrity and vesiculation during the secretory process.

The degradation of extracellular matrix (ECM) by matrix metalloproteases is crucial in physiological and pathological cell invasion alike. Degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Herein, we show that the dynamin 2 (Dyn2), a GTPase implicated in the control of actin-driven cytoskeletal remodeling events and membrane transport, is necessary for focalized matrix degradation at invadopodia. Dynamin was inhibited by using two approaches: 1) expression of dominant negative GTPase-impaired or proline-rich domain-deleted Dyn2 mutants; and 2) inhibition of the dynamin regulator calcineurin by cyclosporin A. In both cases, the number and extension of ECM degradation foci were drastically reduced. To understand the site and mechanism of dynamin action, the cellular structures devoted to ECM degradation were analyzed by correlative confocal light-electron microscopy. Invadopodia were found to be organized into a previously undescribed ECM-degradation structure consisting of a large invagination of the ventral plasma membrane surface in close spatial relationship with the Golgi complex. Dyn2 seemed to be concentrated at invadopodia.