Interactions between CLIP-170 and tubulin

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DESCRIPTION (provided by applicant): The process of cell organization underlies fundamental biological processes ranging from polarized growth to multicellular development. The long-term goal of the Goodson laboratory is to understand the origins of this organization. Because many aspects of cell organization depend on the microtubule cytoskeleton, a major part of this initally diffuse problem can be reduced to two questions:: a) How is the microtubule cytoskeleton itself morphologically defined? b) How do other cellular components interact with microtubules? To answer these questions requires knowledge of the proteins that control microtubule (MT) dynamics and mediate cargo-MT interactions. This proposal focuses on CLIP-170, an evolutionarily conserved MT-binding protein that is involved in both processes. CLIP-170 also has the intriguing property of localizing dynamically to growing MT plus ends. The goal of this proposal is to define interactions between CLIP-170 and MTs how CLIP-170 associates with MTs, the mechanism and significance of its dynamic plus-end tracking behavior, and the precise effects it has on MT dynamics. This knowledge will establish CLIP-170 function and mechanism, aid in understanding other MT plus-end tracking proteins, and help define the regulation of MT growth. Our Specific Aims are to: 1. Determine the affinity of CLIP-170 for different conformational states of tubulin/MTs by biophysical techniques including MT cosedimentation, fluorescence anisotropy, and surface plasmon resonance. This work will establish which interactions are physiologically relevant and test plus-end tracking mechanisms. 2. Define the effect of CLIP-170 on MT dynamics by performing in vitro MT dynamic instability assays in purified systems and in Xenopus extracts. This will define CLIP-170 activity and establish a system for further study of CLIP-170 regulation and protein partners. 3. Define the minimal plus-end tracking domain by transiently transfecting tagged CLIP-170 fragments into tissue culture cells and assaying their ability to track MT plus ends in vivo. This will allow us to establish the relationships between CLIP-170 structure, plus end tracking activity, and effects on MT dynamics, and will provide a simplified system for dissecting regulation involved in plus end tracking behavior.