Role of Formin-2 in actin-microtubule coordination during axonal pathfinding and it's characterization in axonal branching

Abstract

Neural circuits are formed by directed translocation of axonal growth cones to their synaptic targets and specific patterns of branching. The axon, with the growth cone at the tip, moves in a directed fashion by sensing the environmental cues through structures like the filopodia. As the neuron reaches its target tissue, it innervates the tissue to form multiple connections. This is achieved by the arborization of the terminal end of the axon or by collateral branching of the axon. These processes of outgrowth, guidance and branching are driven by active and coordinated remodelling of the underlying cytoskeletal components. One such cytoskeletal regulator is Formin-2 (Fmn2), an actin nucleator, which is highly expressed in the developing and adult central nervous system (Leader and Leder, 2000) and has been implicated in cognition (Law et al., 2014; Agís-Balboa et al., 2017). Recent studies from our lab have provided glimpses into the mechanism of Fmn2 function during the development of the nervous system. Fmn2 is involved in maintaining optimum outgrowth speed and directionality of migration. Moreover, deficiency of Fmn2 resulted in pathfinding defects of spinal commissural neurons (Sahasrabudhe et al., 2016). Since axonal pathfinding requires coordination between actin and microtubule cytoskeletons, this study investigated the role of Fmn2 in mediating actin-microtubule crosstalk. We find that Fmn2 facilitates the exploration of microtubules into the peripheral domain of the growth cone.In the filopodia, Fmn2 stabilizes the microtubules, most likely, by physically coupling themto the F-actin bundles. This coupling appears to occur through the tail region of Fmn2, which binds to both actin and microtubule. In the absence of Fmn2, disruption of actin-microtubule crosstalk in filopodia results in deficits in sensing and/or turning that underlie the axon guidance defects. Along with the requirement of Fmn2 in the growth cone, Fmn2 was found to be involved in axonal collateral branching. Axonal branching is an important component of connectivity patterns in neural circuits. A collateral is initiated by the seeding of F-actin bundles from actin accumulation in the axonal shaft which result in the formation of a filopodium on the axonal shaft. This protrusion is stabilized by microtubule innervation and ultimately matures into a stable branch (Gallo,2015). Fmn2 was found to localize at the base of axonal filopodia and its deficiency reduced branching by half. On the other hand, Fmn2 lead to increased branching when over-expressed. Moreover, the density of these protrusions is dependent on the actin nucleation activity of Fmn2. Preliminary evidence suggests that Fmn2 may facilitate microtubule presence in axonal filopodia, in a manner analogous to that uncovered in the growth cone filopodia, thereby ultimately aiding their maturation into branches. In conclusion, this study provides a mechanistic understanding of Fmn2 in processes critical to the development of neuronal circuits and its mediation of actin-MT cross-talk in the developing neuron.