The role of cytoskeletal proteins talin and vinculin in neuronal growth cone motility

One of the major questions of neurodevelopment is how the movement of the neuronal growth cone is guided so that axons reach the correct targets for the establishment of the nervous system. While several molecules that can guide axons have been identified, little is known about the mechanisms of growth cone motility that these molecules must affect. Talin and vinculin are both good candidates to be links between the actin cytoskeleton and the extracellular matrix that play a role in motility by generating tension on actin filaments that could be used as a motive force for growth cone motility. In this way, talin and vinculin are both proposed to act as a "molecular clutch" that transduces the force of actin retrograde flow into forward motility of the growth cone. Micro-scale chromophore assisted laser inactivation (micro-CALI), is a technique that inactivates proteins by directing the energy of a 620 nm laser beam to the protein via the chromophore malachite green. I have adapted micro-CALI to the study of intracellular proteins in order to generate a spontaneous and localized functional inactivation of talin and vinculin. Inactivation of talin in filopodia caused a transient loss of filopodial extension and retraction whereas vinculin inactivation increased filopodial bending and buckling. These results are consistent with talin but not vinculin acting as a "molecular clutch," and suggest that vinculin plays a role in maintaining filopodial structure. By varying the location of the laser spot during talin inactivation, I have also shown that the growth cone is sensitive to differences in talin function at the leading edge.