Molecular Mechanisms Underlying Regulations Of Cellular Dynamics By MCAK And DDA3-two Microtubule Plus-end Tracking Proteins

Cytoskeleton defines the architecture and internal organization of cells by positioning organelles and activities, as well as by supporting cell shape and mechanics, and thereby serves various vital cellular functions. Microtubule (MT), one of the three main types of cytoskeleton, is essential for a variety of cellular processes, including mitosis, cell polarity and migration, and vesicle transport. The roles of microtubule depend on its dynamics. In cells, microtubule-associated proteins (MAPs) regulate both the dynamics of microtubules as well as the interactions of microtubules with other cellular components.MT plus-end tracking proteins (+TIPs) are a subgroup of MAPs that specifically accumulate at the growing ends of microtubules.+TIPs hence, regulate microtubule plus-end dynamics and microtubule-based functions underlying various cellular activities. It has been indicated that deregulation of +TIPs is correlated with various pathological processes, including tumorigenesis, infection and cardiovascular diseases. Thus,+TIPs have attracted a lot of attentions these years. Studies on +TIPs together with microtubule dynamics will help to understand the molecular regulation of microtubule dynamics underlying different cellular activities but also to provide theoretical bases for relevant diseases treatments. Most +TIPs use either CAP-Gly domain or Ser-x-Ile-Pro (SxIP) motif to bind to EB proteins-the the core components of protein complexes at MT plus ends to localize to and track microtubule growing plus ends. By taking advantage of computing analyses combined with biochemical characterizations, we assessed many potential +TIPs with SxIP motif. To characterize and functionally dissect +TIPs in depth, we selected two representative +TIPs. One is the previously identified +TIP-MCAK, which is widely investigated during the last decade due to its function in ensuring faithful chromosome segregation. Another is DDA3, a potential +TIP.During cell division, dynamic interaction between kinetochores and dynamic spindle microtubules governs chromosome movements. As a microtubule depolymerase, microtubule plus-end tracking protein MCAK is a key regulator of mitotic spindle assembly and dynamics in cells. However, the regulatory mechanisms underlying its depolymerase activity during the cell cycle remain elusive. In the first part of my work, we show that PLK1 is a novel regulator of MCAK. Biochemical characterizations reveal that MCAK interacts with PLK1 both in vitro and in vivo. The neck and motor domain of MCAK associates with the kinase domain of PLK1. MCAK is a novel substrate of PLK1 and the phosphorylation in the C-terminus of MCAK stimulates its microtubule depolymerization activity in vivo. Interestingly, intra-molecular interaction exists in MCAK and it is regulated by PLK1-mediated phosphorylation. We speculate that PLK1 may regulate the depolymerase activity of MCAK via modulating its molecular conformation. Fluorescence and Time-lapse microscopic assays demonstrate that overexpression of a PLK1-phosphomimetic mutant MCAK causes a dramatic increase in misaligned chromosomes and in multi-polar spindles in mitotic cells, while overexpression of a non-phosphorylatable MCAK mutant results in aberrant anaphase with sister chromatid bridges. Based on our data, the former is probably due to the hyper-activity of MCAK which results in spindle assembly defects, while the latter may due to the hypo-activity of MCAK which can not timely correct the aberrant microtubule-kinetochore attachments before anaphase onset. These suggest that precise regulation of the MCAK activity by PLK1 phosphorylation is critical for proper microtubule dynamics and essential for the faithful chromosome segregation. We reason that dynamic regulation of MCAK phosphorylation by PLK1 is required to orchestrate faithful cell division whereas the high levels of PLK1 and MCAK activities seen in cancer cells may account for a mechanism underlying the pathogenesis of genomic instability.After elucidation of the new molecular mechanism underlying MCAK regulation during mitosis, we moved to investigate the function of +TIPs in interphase cells. Cell motility and adhesion involve orchestrated interaction of microtubules with their +TIPs. However, the mechanisms underlying regulations of microtubule dynamics and directional cell migration are still elusive. Therefore, the emphases of the second part of my work is to elucidate the +TIP properties of DDA3 and its roles in cell migration. We show that DDA3-EB1 interaction, potentially regulated by EB1 acetylation, orchestrates MT plus-end dynamics and facilitates directional cell migration. Biochemical characterizations reveal that DDA3 interacts with EB1 via its SxIP motif within the C-terminal Pro/Ser-rich region. Time-lapse and total internal reflection fluorescence (TIRF) microscopic assays demonstrate that DDA3 exhibits EB1-dependent, MT plus-end loading and tracking. The EB1-based loading of DDA3 is responsible for MT plus-ends stabilization at the cell cortex, which in turn orchestrates directional cell migration. Thus, the EB1-based function of DDA3 links MT dynamics to directional cell migration. More importantly, we found that EB1 acetylation regulates DDA3-EB1 interaction and such regulation might be a potential mechanism underlying DDA3 regulation in EGF-elicited cell migration. To our knowledge, this is the first time to show that acetylation is a significant regulatory mechanism underlying +TIPs-microtubules interaction in cell migration. It undoubtedly provides a unique view for investigation of signal transduction during cell migration.