Regulation of axon collateral branch morphogenesis by septin GTPases

Development of a functional nervous system requires the formation of correct innervation patterns, which are largely shaped by the axon collateral branching. Axon collateral branches are formed from actin-rich protrusions termed filopodia. Maturation of axon collateral branches requires the unbundling, fragmentation and entry of axonal microtubules into filopodia. During this process, the actin and microtubule cytoskeleton undergo dramatic remodeling and require coordination between actin and microtubules. However, the mechanisms involved remain elusive. Septin GTPases are a family of multifunctional proteins that associate with actin, microtubules and cell membranes. In this thesis, I show that septin 6 (SEPT6) and 7 (SEPT7) are essential for axon collateral branching. In embryonic sensory neurons (chicken dorsal root ganglia), SEPT6 localizes to axonal F-actin patches that serve as precursors to filopodia. In contrast, SEPT7 localizes to the base of axonal shaft filopodia. Both SEPT6 and SEPT7 accumulate at sites of incipient filopodia, but only SEPT6 affects the rate of filopodia formation. Quantitative analysis of actin dynamics revealed that SEPT6 increases the frequency with which filopodia emerge from F-actin patches, but does not affect the formation of F-actin patches and their lifespan. Although SEPT7 has no role in the protrusive activity of F-actin patches, it is essential for microtubule presence in nascent filopodia. Absence of the microtubule-bundling and stabilizing protein MAP1 B from SEPT7 sites and the splaying of microtubules in SEPT7 over-expressing axons indicate that SEPT7 might mediate the unbundling and targeting of microtubules into the nascent filopodia and thus, their maturation to axon collateral branches. Based on these data, I propose a two-step mechanism for the morphogenesis of axon collateral branches. First, SEPT6 triggers the conversion of F-actin patches to axonal filopodia, and second, SEPT7 facilitates the entry of axonal microtubules into the nascent filopodia, and thus the maturation of axon collateral branches. Because septin stabilization with the small molecule compound forchlorfenuron (FCF) triggers the proliferation of axon collateral branches, septins might be an ideal therapeutic target for the regeneration of neuronal branches after spinal cord injury.