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Axonal growth is mediated by coordinated changes of the actin and microtubule (MT) cytoskeleton. Ample evidence suggests that members of the formin protein family are involved in the coordination of these cytoskeletal rearrangements, but the molecular mechanisms of the formin-dependent actin–microtubule crosstalk remains largely elusive. Of the six Drosophila formins, DAAM was shown to play a pivotal role during axonal growth in all stages of nervous system development, while FRL was implicated in axonal development in the adult brain. Here, we aimed to investigate the potentially redundant function of these two formins, and we attempted to clarify which molecular activities are important for axonal growth. We used a combination of genetic analyses, cellular assays and biochemical approaches to demonstrate that the actin-processing activity of DAAM is indispensable for axonal growth in every developmental condition. In addition, we identified a novel MT-binding motif within the FH2 domain of DAAM, which is required for proper growth and guidance of the mushroom body axons, while being dispensable during embryonic axon development. Together, these data suggest that DAAM is the predominant formin during axonal growth in Drosophila, and highlight the contribution of multiple formin-mediated mechanisms in cytoskeleton coordination during axonal growth.

Flightless-I is a unique member of the gelsolin superfamily alloying six gelsolin homology domains and leucine-rich repeats. Flightless-I is an established regulator of the actin cytoskeleton, however, its biochemical activities in actin dynamics are still largely elusive. To better understand the biological functioning of Flightless-I we studied the actin activities of Drosophila Flightless-I by in vitro bulk fluorescence spectroscopy and single filament fluorescence microscopy, as well as in vivo genetic approaches. Flightless-I was found to interact with actin and affects actin dynamics in a calcium-independent fashion in vitro. Our work identifies the first three gelsolin homology domains (1–3) of Flightless-I as the main actin-binding site; neither the other three gelsolin homology domains (4–6) nor the leucine-rich repeats bind actin. Flightless-I inhibits polymerization by high-affinity (∼nM) filament barbed end capping, moderately facilitates nucleation by low-affinity (∼μM) monomer binding, and does not sever actin filaments. Our work reveals that in the presence of profilin Flightless-I is only able to cap actin filament barbed ends but fails to promote actin assembly. In line with the in vitro data, while gelsolin homology domains 4–6 have no effect on in vivo actin polymerization, overexpression of gelsolin homology domains 1–3 prevents the formation of various types of actin cables in the developing Drosophila egg chambers. We also show that the gelsolin homology domains 4–6 of Flightless-I interact with the C-terminus of Drosophila Disheveled-associated activator of morphogenesis formin and negatively regulates its actin assembly activity.