Mechanisms underlying Formin-2 function in the motility of neuronal growth cones

Abstract

Stereotyped wiring of the nervous system during development is accomplished by guidance cues which tightly control and shape neuronal trajectories and instruct the growth cone to make appropriate synaptic contacts. Tight regulation of growth cone steering is achieved by remodelling the underlying growth cone cytoskeleton that regulates polarity, protrusion, substrate adhesion and generation of coordinated traction forces. However, exact mechanisms of this regulation still remain less characterized in growth cones. In the following thesis, we focus on Formin-2 (Fmn2), a member of the formin family of actin-binding proteins. Earlier work from our group has shown that perturbation of Fmn2 in the developing chick spinal cord results in defective trajectories of spinal commissural neurons in vivo. In this study we show that Fmn2 transcript is not only enriched in the spinal cord but also higher expression coincides with the developmental window of commissural interneuron pathfinding in the spinal cord. Depletion of Fmn2 affects growth cone motility in vitro and results in a reduced lifetime of growth cone filopodia. On the other hand, reduction in Fmn2 does not affect the filopodial elongation and initiation rates suggesting a role in substrate adhesion based stability of filopodia. To understand the slow growth cone movement, in this study we have investigated growth cone substrate-dependent interactions in neurons that suggested the importance of Fmn2 in regulating growth cone point contacts. Earlier work has suggested that force-based maturation of point contacts is compromised upon Fmn2 depletion, implicating Fmn2 in the molecular clutch mechanism. Supporting this hypothesis, the current study reveals a regulatory role for Fmn2 in the generation of traction forces by growth cones. Additionally, retrograde flow of F-actin was found to be elevated upon knockdown of Fmn2, indicating slipping of the molecular clutch and further underscoring Fmn2 function as a component of the molecular clutch. Supporting studies in mouse fibroblasts show Fmn2 localization to ventral stress fibres and juxta focal adhesions suggesting its role in mediating their interaction. This crosstalk is further highlighted by knockdown studies that suggest a role in mechanotransduction-dependent stability of adhesions. This interaction is further exemplified by our observations of leading-edge stabilization defects during cell spreading of Fmn2 depleted cells. Taken together this study identifies Fmn-2 as a regulator of the molecular clutch in neuronal growth cones. It is likely that this function of Fmn-2 underlies its role in growth cone motility and consequent development of neural circuits.