Multi-parametric analysis of CLASP-interacting protein functions during interphase microtubule dynamics. Jennifer B Long¹, Maria Bagonis¹, Laura Anne Lowery¹, Haeryun Lee^1,2, Gaudenz Danuser¹, David Van Vactor¹. 1) Cell Biology, Harvard Medical School, Boston, MA; 2) Pohang University of Science and Technology, Pohang, Gyungbuk, KOREA.

   Regulation of microtubule (MT) dynamics is critical to many aspects of development, from control of cell proliferation to morphogenesis. MTs are key effectors downstream of various signaling pathways and are subject to tight regulation in response to intrinsic and extrinsic cues, often through transient interactions with a variety of microtubule-associated proteins. The MT plus-end tracking protein (+TIP) Orbit/MAST/CLASP, known to be involved in mitotic spindle formation, cell motility and axon guidance, mediates multiple dynamic cellular behaviors and interacts with numerous cytoplasmic proteins. While the action of some CLASP interactors on MT dynamics have been examined, a comprehensive survey of the proteins in the CLASP interactome as MT dynamic regulators has been missing. Ultimately, we are interested in understanding how CLASP collaborates with functionally linked proteins to regulate MT dynamics. Through an additional genetic screen of nearly 12,000 transposon insertion strains, we expanded the previously identified CLASP interactome from 24 to 118 interactors. We then utilize multi-parametric analysis of time-lapse MT+TIP imaging data acquired in Drosophila S2R+ cells to assess the effects on individual microtubule dynamics for RNAi-mediated depletion of 48 gene products previously identified as in vivo genetic CLASP interactors. While our analysis corroborates previously described functions of known CLASP-interactors, its multi-parametric resolution reveals more detailed functional profiles (fingerprints) that allow us to precisely classify the roles CLASP-interacting genes play in MT regulation. Using this data, we identify subnetworks of proteins with novel yet overlapping MT regulatory roles, and also uncover subtle distinctions between the functions of proteins previously thought to act via similar mechanisms.