| Nerve growth cones are sensory-motile structures that guide developing neurites to form of trillions of synapses in the human brain. Growth cones interact with the extracellular matrix (ECM), such as laminin (LN), through integrin receptors at adhesion sites. Adhesions link ECM proteins to the actin cytoskeleton through numerous adaptor and signaling proteins. One presumed function of growth cone adhesions is to restrain or "clutch" myosin-II-based filamentous actin (F-actin) retrograde flow (RF) to promote leading edge membrane protrusion. In motile non-neuronal cells, myosin-II binds and exerts force upon actin filaments at the leading edge, where clutching forces occur. However, in growth cones, it is unclear whether similar F-actin-clutching forces affect axon outgrowth and guidance. I found in Xenopus spinal neurons RF is reduced in rapidly migrating growth cones on laminin (LN) compared with non-integrin-binding poly-D-lysine (PDL). I further illustrate this relationship between adhesion and RF locally via two-channel imaging of RF and paxillin, as well as on micropatterns of PDL and LN. Finally, I showed that RF is significantly attenuated in vivo, suggesting that it is restrained by molecular clutching forces within the spinal cord. In the second part of my thesis I investigated how extracellular mechanical forces influence neuritogenesis. I examined the role of mechanical forces on neurite development of human forebrain (hFBs) neurons and human motor neurons (hMNs) derived from induced pluripotent stem cells (iPSCs). These two populations of neurons extend axons into distinct environments. Using polyacrylamide (PAA) and collagen substrata of varying elasticity I found that the extension of hFB neurites exhibit little differential outgrowth, while hMN neuritogenesis was strongly promoted on more rigid substrata. I found that the observed differential growth rates are in part due to greater adhesion of the growth cone leading edge to more rigid environments, suggesting hMN growth cones form more stable adhesions leading to greater motility. Furthermore, I found RhoA, an important adhesion regulating GTPase, activity was higher on stiffer substrata in hMNs. Taken together, these data illustrate the mechanistic role of intrinsic and extrinsic forces during neurite outgrowth. |
