The objective of this application is to establish a state-of-the-art facility for the execution of cellular and subcellular """"""""knockouts"""""""" of protein function through the technique of Chromophore-Assisted Laser Inactivation (CALI). In this technique the chromophore Malachite green is covalently coupled to an antibody. The Malachite green-antibody conjugate is microinjected into living cells. Controlled laser pulses from a nitrogen pumped dye laser are applied. At the wavelengths used, only the chromophore absorbs the laser energy. Proteins bound by the Malachite green-labeled antibody are inactivated by free radical damage. Because antibodies are used as targeting probes, the CALI technique provides a high degree of molecular specificity. Because inactivation occurs only upon laser irradiation, the technique provides excellent temporal control and the ability to image cells at high resolution before and after inactivation. Finally, because the laser can be focused on the entire cell or onto a small subregion, the echnique provides excellent spatial resolution in disrupting cellular pathways. The members of the major user group intend to apply this approach in four areas of mammalian cell biology: 1) signaling components of the M phase checkpoint (Gorbsky), 2) the role of SCAMPS and other vesicle trafficking proteins (Castle), 3) signaling and assembly of focal adhesions, 4) activation of the Map kinase pathway in G1 and mitosis (Weber). Currentiy each laboratory has been microinjecting function-blocking antibodies into living cells as a means of studying cellular signaling and physiology. However, most antibodies are neutral; i.e they have no inherent function-blocking ability. The CALI technique uses these neutral antibodies as knockout reagents to examine the role of target proteins in vivo. The protein ablation microscope workstation is intended to complement other knockout strategies (anti- sense, mouse gene knockouts) and become a pivotal resource for the community of researchers seeking a simple, inexpensive, and effective way to study the effects of inactivating specific proteins in living mammalian cells.
