Chemotactic Factors Regulate Focal Adhesion Formation And F-actin Organization In Neural Stem Cells Of Varying Differentiation States During Directional Migration

Directional migration of neural stem cells (NSCs) is crucial for the development ofthe nervous system and repair after injury. Cell migration is a complex, highly regulatedprocess that involves the continuous assembly and disassembly of focal adhesions(turnover of focal adhesions). The formation of focal adhesion initiates from a smallfocal complex (FX) and thenmatures to larger focal adhesion (FA) via a Rho-mediatedmyosin-based contractility in a spatial and timporal manner. Compared with focaladhesion, smaller focal complex displays more rapid turnover to promote cell migration.Our previous study showed that NSCs at varying differentiation states possess differentchemotactic responses, however, the cellular and molecular mechanisms remain largelyelusive. In this study, using a neural stem cell line, C17.2cells, we examine eventsduring the directional migration of NSCs at varying differentiation states, focusing onFA formation and F-actin assembly. We provide evidence that NSCs spreading needremodeling of FAs and arrangement of F-actin, and NSCs at varying differentiationstates possess different formation and of FAs and reorganization of F-actin.Furthermore, cells of0.5d and1d differentiation exhibit a prominent asymmetricdistribution of FAs, displaying a stronger directional migration. During this process,Y31-paxillin is firstly activated at cell periphery to form new focal complexes followedby the phosphorylation of Y118-paxillin in NSCs. VEGF and SDF-1α induce theremodeling of FAs and promote F-actin to form stress fibers depending on celldifferentiation, and cells of3d differentiation is not sensitive to the stimulation ofchemotactic factors. VEGF temporally and spatially regulates FAs formation in NSCsof0d,0.5d and1d differentiation while SDF-1α induces turnover of FAs by thestimulated time in those cells. Molecular mechanism analysis reveals that both VEGF and SDF-1α activate different patterns of phosphorylation of FAK at Y397and paxillinat Y31/118. In consistent with the number of FAs, higher activation of FAK at Y397accelerates turnover of FAs and decreases the number of FAs, while paxillin at Y118isphosphorylated to form stable FAs with an increase of their number or to disassembleFAs with a decrease of their number. VEGF leads to a prominent directional migrationof NSCs with generation of board lamellipodium and formation of focal complexes atleading edge compared to SDF-1α, and cells of1d differentiation display a strongestchemotaxis to VEGF. Time-lapse video analysis suggests that VEGF inducesasymmetric distribution of FAs between lamella and cell rear to provide those cells apersistent direction, with persistent formation of focal at the leading edge anddisassembly of FAs at the rear. Compared to cells at other differentiation states, cells of1d differentiation detach more rapidly at the rear to facilitate forward movement of cellbody with a concurrent enlarged size of FAs at the leading edge, while cells of3ddifferentiation display the slowest turnover of FAs and are not sensitive to thestimulation of VEGF. Collectively, our results suggest that differentiation of neuralstem cells influences their directional migration by remodeling of FAs andreorganization of F-actin，shedding light on optimization of the therapeutic potential ofNSCs to be employed for cure of neural degenerative diseases.