We address cellular function and have developed methodologies to address directly how proteins act in situ. In order to address this, we developed a novel technique called Chromophore-Assisted Laser Inactivation (CALI) to inactivate specific proteins in living cells and embryos at precise times and locations. We have ascertained the spatial specificity of CALI and established the physical basis of its mechanism. We have also developed micro-CALI which focuses the laser through microscope optics to inactivate specific protein functions in single cells with a spatial resolution of a few microns. These approaches are complementary to existing knockout strategies and offer several advantages including an unprecedented degree of spatial and temporal resolution, lack of genetic compensation and the ability to target proteins in cells and tissues that are not amenable to genetic approaches.
Figure 1. The diagram illustrates the versatility of light-based inactivation approaches to study gene function.
One major question addressed by the lab is what are the molecular mechanisms that determine how the nervous system is formed during embryonic development? One long-term goal of our lab is to elucidate the pathways of molecular interactions of axon guidance from the external environment to the cytoskeleton. These pathways are made up of membrane receptors, signal transduction molecules and cytoskeletal proteins that must act together to translate extracellular cues into directed motility. These techniques have been applied to a large variety of proteins inside growth cones and we have ascertained the functional roles of many of these proteins in growth cone motility and guidance. These include: calcineurin; myosin I b and V; talin; vinculin; ezrin; radixin; tau; NCAM-180; L1; ephrin-A5, GAP-43; protein kinase C and zyxin.
Our most recent work is directed at discovering proteins involved in cancer cell invasion using CALI and functional genomics. Our previous work has addressed the molecular mechanisms growth cone motility. Motility is also critical for how cancer cells invade. We have now applied CALI to cancer relevant proteins to address their cellular roles and have studied ezrin, pp60-c-src; pp59-fyn, and TSC I (hamartin). We have begun to use CALI with antibody libraries in a high throughput and automated fashion to discover new proteins that act in cancer cell invasiveness. We have initiated this high-throughput screen and begun to identify and validate targets. This will be the first proteome-wide screen for target validation that directly addresses cellular function. It has clinical importance as CALI provides a means of identifying and validating such proteins as targets for drug discovery and novel anti-cancer therapeutics.
Our research has shown that the molecular chaperone Heat Shock Protein 90 (Hsp90) is released by breast cancer cells that activate pro-invasive proteins in the tumor microenvironment. These studies have elucidated a novel cell biological process in which extracellular Hsp90 serves as a hub for the invasive niche and inhibitors that cannot cross the cell membrane could reduce invasion and hence metastasis without affect all the intracellular functions that Hsp90 does in all cells.