Filopodia formation induced by active mDia2/Drf3

Filopodia Eire rod-shaped cell surface protrusions composed of a parallel bundle of actin filaments. Since filopodia frequently emanate from lamellipodia, it has been proposed that they form exclusively by the convergence and elongation of actin filaments generated in lamellipodia networks. However, filopodia form without Arp2/3-complex, which is essential for lamellipodia formation, indicating that actin filaments in filopodia may be generated by other nucleators. Here we analyzed the effects of ectopic expression of GFP-tagged full length or a constitutively active variant of the human formin mDia2/Drf3. By contrast to the full-length molecule, which did not affect cell behaviour and was entirely cytosolic, active Drf3 lacking the C-terminal regulatory region (Drf3 Delta DAD) induced the formation of filopodia and accumulated at their tips. Low expression of Drf3 Delta DAD induced rod-shaped or tapered filopodia, whereas over-expression resulted in multiple, club-shaped filopodia. The clubs were filled with densely bundled actin filaments, whose number but not packing density decreased further away from the tip. Interestingly, clubs frequently increased in width after protrusion beyond the cell periphery, which correlated with increased amounts of Drf3 Delta DAD at their tips. These data suggest Drf3-induced filopodia form and extend by de now nucleation of actin filaments instead of convergent elongation. Finally, Drf3 Delta DAD also induced the formation of unusual. lamellipodia-like structures, which contained both lamellipodial markers and the prominent filopodial protein fascin. Microarray analyses revealed highly variable Drf3 expression levels in different commonly used cell lines. reflecting the need for more detailed analyses of the functions of distinct formins in actin cytoskeleton turnover and different cell types.