Micro-scale chromophore-assisted laser inactivation of nerve growth cone proteins

Abstract

Directed growth cone movement is crucial for the correct wiring of the nervous system. This movement is governed by the concerted actions of cell surface receptors, signaling proteins, cytoskeleton‐associated molecules, and molecular motors. In order to investigate the molecular basis of growth cone motility, we applied a new technique to functionally inactivate proteins: micro‐scale Chromophore‐Assisted Laser Inactivation [Diamond et al. (1993) Neuron 11:409–421]. Micro‐CALI uses laser light of 620 nm, focused through microscope optics into a 10‐μm spot. The laser energy is targeted via specific Malachite green‐labeled, non‐function‐blocking antibodies, that generate short‐lived protein‐damaging hydroxyl radicals [Liao et al. (1994) Proc Natl Acad Sci USA 91:2659–2663]. Micro‐CALI mediates specific loss of protein function with unachieved spatial and temporal resolution. Combined with time‐lapse video microscopy, it offers the possibility to induce and observe changes in growth cone dynamics on a real time base. We present here the effects of the acute and localized inactivation of selected growth cone molecules on growth cone behavior and morphology. Based on our observations, we propose specific roles for these proteins in growth cone motility and neurite outgrowth. Microsc. Res. Tech. 41:97–106, 2000.