Modulation of Rac localization and function by dynamin.

The GTPase dynamin controls a variety of endocytic pathways, participates in the formation of phagosomes, podosomal adhesions, and invadopodia, and in regulation of the cytoskeleton and apoptosis. Rac, a member of the Rho family of small GTPases, controls formation of lamellipodia and focal complexes, which are critical in cell migration and phagocytosis. We now show that disruption of dynamin(-2) function alters Rac localization and inhibits cell spreading and lamellipodia formation even though Rac is activated. Dominant-negative K44A dynamin(-2) inhibited cell spreading and lamellipodia formation on fibronectin without blocking cell adhesion; dynamin(-2) depletion by specific small interfering RNA inhibited lamellipodia in a similar manner. Dyn2(K44A) induced Rac mislocalization away from cell edges, into abnormal dorsal ruffles, and led to increased total Rac activity. Fluorescence resonance energy transfer imaging of Rac activity confirmed its predominant localization to aberrant dorsal ruffles in the presence of dominant-negative dyn2(K44A). Dyn2(K44A) induced the accumulation of tubulated structures bearing membrane-bound Rac-GFP. Constitutively active but not wild-type GFP-Rac was found on macropinosomes and Rac-dependent, platelet-derived growth factor-induced macropinocytosis was abolished by Dyn2(K44A) expression. These data suggest an indispensable role of dynamin in Rac trafficking to allow for lamellipodia formation and cell spreading.

Domain requirements for an endocytosis-independent, isoform-specific function of dynamin-2.

Endocytosis is inhibited by overexpression of either dynamin-1 or dynamin-2 mutants because both isoforms form heterotetramers with endogenous dynamin-2 and interfere with its function. By contrast, other phenotypes, which are specifically triggered by overexpression of dynamin-2, but not dynamin-1 are likely to reflect endocytosis-independent, dynamin-2-specific functions and/or interactions. Using Dyn2/Dyn1 chimeras, we explored the structural requirements for a readily quantifiable, isoform-specific function of dynamin-2, the activation of caspase-3 to trigger apoptosis. Strikingly, swapping the highly homologous GTPase domain of dynamin-2 into dynamin-1 was sufficient to confer caspase-3 activation. Moreover, assembly-defective mutations in GED, dynamin's GAP/assembly domain, that inhibit endocytosis enhance caspase-3 activation. Thus, this dynamin-2-specific function is mechanistically distinct from and independent of its role in endocytosis. These findings have important implications for interpreting dynamin-2 dependent phenotypes in overexpression studies.