The role of the actin-binding protein Arp2/3 in growth cone actin dynamics and guidance

Abstract

Neuronal growth cones are responsible for wiring the immature nervous system. They rely on guidance cues present in the extracellular environment to guide axons to their targets. Guidance cues achieve effective changes in directionality by modulating the growth cone cytoskeleton. Particularly important for this process are the changes in actin dynamics at the leading edge of the growth cone. However, which and how actin-binding protein (ABP) regulate actin dynamics to achieve correct guidance is not clear. This thesis shows evidence on the role of two ABP's during growth cone guidance.The first chapter, The role of the Arp2/3 complex in actin dynamics and growth cone guidance is substrate dependent, characterizes the role of the actin nucleator Arp2/3 in growth cone actin dynamics and guidance. Arp2/3 has been shown to be important in leading edge actin dynamics in non-neuronal cells, but little was known about its role in neuronal growth cones. Because during development growth cones migrate in association with diverse adhesive substrates during development, we probed the hypothesis that the functional significance of Arp2/3 is substrate dependent. We report that Arp2/3 inhibition led to a reduction in the number of filopodia and F-actin content on laminin and L1. However, we found substrate-dependent differences in growth cone motility, actin retrograde flow, and guidance after Arp2/3 inhibition, suggesting that its role, and perhaps that of other ABP's, in growth cone motility is substrate-dependent. The next chapter, Ezrin/radixin/moesin family proteins mediate actin filament dynamics in attractive growth cone guidance to nerve growth factor, describes the role that the ezrin/radixin/moesin (ERM) family of membrane-cytoskeletal linker proteins have during attractive growth cone guidance. This family of proteins have been shown to link actin filaments to the cell membrane when activated. We found that endogenous guidance cues for dorsal root ganglion and retinal ganglion cell neurons can activate ERM proteins. Moreover, the increase in filamentous actin normally triggered by the stimulation of these attractive guidance cues was abolished when ERM protein function was disrupted. Additionally, we found that ERM activity is closely associated with correct placement of substrate adhesions in growth cones. Finally, we found that disruption of ERM protein function abolishes attractive growth cone guidance and causes mislocalization of ADF/cofilin, an active binding protein previously shown by our group to be required for correct guidance. These results suggest that the correct organization of filamentous actin and adhesions in growth cones by ERM proteins are necessary for correct growth cone guidance.The final chapter, The role of Arp2/3 during in-vivo axon guidance in the chick embryo, deals with the role of the Arp2/3 complex during growth cone guidance in-vivo. For this purpose studied the role of Arp2/3 during the development of chick retinotectal projections and that of the sensory-motor innervation of the hindlimb. We found that Arp2/3-inhibited neurons had no deficiencies during the development of the retinotectal projections. However, we did find that Arp2/3 was required for normal sensory-motor innervation of the hindlimb during development. Thus, suggesting that Arp2/3 is required during developmental axon guidance in some tissues.