Multiparametric Analysis of CLASP-Interacting Protein Functions during Interphase Microtubule Dynamics

Abstract

The microtubule (MT) plus-end tracking protein (؉TIP) CLASP mediates dynamic cellular behaviors and interacts with numerous cytoplasmic proteins. While the influence of some CLASP interactors on MT behavior is known, a comprehensive survey of the proteins in the CLASP interactome as MT regulators is missing. Ultimately, we are interested in understanding how CLASP collaborates with functionally linked proteins to regulate MT dynamics. Here, we utilize multiparametric analysis of time-lapse MT ؉TIP imaging data acquired in Drosophila melanogaster S2R؉ cells to assess the effects on individual microtubule dynamics for RNA interference-mediated depletion of 48 gene products previously identified to be in vivo genetic CLASP interactors. While our analysis corroborates previously described functions of several known CLASP interactors, its multiparametric resolution reveals more detailed functional profiles (fingerprints) that allow us to precisely classify the roles that CLASP-interacting genes play in MT regulation. Using these 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. The orchestration of cytoskeletal dynamics is critical for a broad range of cellular behaviors, including mitosis, polarity, motility, morphogenesis, and cell-cell interaction (1-3). Microtubule (MT) polymer networks participate in numerous signaling pathways, often helping to assemble and/or deliver effector protein complexes and to define the spatial organization of cellular responses. Many classes of cytoskeletal binding proteins regulate the configuration of MT arrays and often interact with other protein networks. However, our understanding of how these extended effector networks function to control cytoskeletal dynamics is still limited. Large-scale screens for MT regulators have primarily relied on endpoint phenotypes that affect mitosis (4-6). The mitotic spindle is a unique apparatus whose gross architecture can be severely disturbed by accumulated effects of altered MT dynamics and thus offers a simple readout for such studies. However, these readouts report screening hits only on the basis of indirect MT phenotypes in a large complex system without pinpointing the actual role that they play in terms of bona fide MT regulation. Direct detection of altered MT dynamics has been much more challenging. For this reason, we adopted a quantitative live-imaging approach that allowed us to identify with single-MT resolution shifts in MT dynamics induced by RNA interference (RNAi)-mediated depletion of putative MT regulators.CLASP (cytoplasmic linker protein [CLIP]-associated protein) is a well-conserved MT plus-end interacting protein (ϩTIP), which modulates dynamic instability and facilitates the interaction of MTs with other cellular structures, including the cell cortex (7, 8) and kinetochores (9-11). CLASP functions as an MT-stabilizing factor, promoting MT rescue both in cultured ...

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