Dynamin1 is a novel target for IRSp53 and works with Mena and Eps8 to regulate filopodial dynamics [Cell Biology]

July 16th, 2014 by Chou, A. M., Sem, K. P., Wright, G. D., Sudhaharan, T., Ahmed, S.

Filopodia are dynamic actin-based structures that play roles in processes such as cell migration, wound healing and axonal guidance. Cdc42 induces filopodia formation through IRSp53, an Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain protein. Previous work from a number of laboratories has shown that IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics through its SH3 domain binding partners. Here we show that Dynamin1 (Dyn1), the large guanosine triphosphatase, is an interacting partner of IRSp53 through pulldown and Forster resonance energy transfer (FRET) analysis, and explore its role in filopodia formation. In neuroblastoma cells, Dyn1 localizes to filopodia, associated tip complexes and the leading edge just behind the anti-capping protein Mena. Dyn1 knockdown reduces filopodia formation, which can be rescued by over-expressing wild-type Dyn1 but not GTPase mutant Dyn1-K44A and the loss-of-function ABD mutant Dyn1-K/E. Interestingly, dynasore, an inhibitor of Dyn GTPase, also reduced filopodia number and increased their lifetime. Using rapid time-lapse TIRF microscopy we show that Dyn1 and Mena localize to filopodia only during initiation and assembly. Dyn1 actin binding domain mutant inhibits filopodia formation, suggesting a role in actin elongation. In contrast, Eps8, an actin capping protein, is seen most strongly at filopodia tips during disassembly. Taken together, the results suggest IRSp53 partners with Dyn1, Mena and Eps8 to regulate filopodial dynamics.