THE VANDERBILT UNIVERSITY
The purpose of this supplement is to fund equipment infrastructure upgrade to allow uninterrupted progress of proposed study in the parent grant. The goal of this study is to understand how the subcellular localization and activation of WASP is regulated during chemotaxis. We hypothesize that a complex interplay among PCH family proteins, RacC, and WASP leads to polarized trafficking of WASP and ACA, resulting in cell polarity establishment via the polarized organization of actin cytoskeleton and asymmetric distribution of ACA during Dictyostelium chemotaxis. Examinination and tracking of polarized trafficking of WASP- or ACAvesicles along the microtubule via live-cell imaging was proposed in the second aim. Spinning disk technology offers several advantages over conventional laser scanning confocal microscope: a wide dynamic range and high sensitivity; minimal phototoxicity and photobleaching which is critical for long-term observation of living cells; fast acquisition of images. Combining the spinning-disk module and an EMCCD camera would allow us to track vesicle movements with significantly higher fidelity and efficiency, which is critical for the completion of the second aim.