The molecular mechanisms underlying directed motility of growth cones have not been determined. The role of myosin-V, an unconventional myosin, in growth cone dynamics was examined by chromophore-assisted laser inactivation (CALI). CALI of purified chick brain myosin-V absorbed onto nitrocellulose-coated cover slips inhibited the ability of myosin-V to translocate actin filaments. CALI of myosin-V in growth cones of chick dorsal root ganglion neurons resulted in rapid filopodial retraction. The rate of filopodial extension was significantly decreased, whereas the rate of filopodial retraction was not affected, which suggests a specific role for myosin-V in filopodial extension.

A molecular understanding of biology requires that we establish the in situ functions of the proteins in cellular processes. To address this, we developed chromophore-assisted laser inactivation (CALl) for probing the in vivo function of proteins. CALl inactivates specific proteins in living cells by using non-blocking antibodies conjugated with malachite green (MG) dye. MG absorbs 620 nm laser light (which is not absorbed by cells) to generate short lived free radicals with limited range of oxidative damage (15 A) around the dye. This inactivates the bound protein without significantly affecting its neighbors. CALl has been applied to 40 proteins and achieved specific inactivation in almost all those tested. We have developed micro-CALl which uses a focused laser beam (10 irn) to acutely inactivate specific proteins within cells. We have used this to address the molecular mechanisms of neuronal growth cone motility and has implicated a diversity of proteins, ( e.g. molecular motors, cytoskeletal, and signaling molecules) in discrete steps of growth cone motility. We hope that micro-CALl will be a useful research tool for addressing dynamic processes in biology and medicine.