Browsing by Subject "Cytokinesis"
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Item Analysis of Aurora B Regulation and Signaling(2006-05-16) Öncel, Dilhan; Yu, HongtaoAurora B is a serine/threonine kinase that functions in a complex with two other chromosomal passenger proteins called INCENP and Survivin. Its function is implicated in a variety of processes related to mitosis, such as chromosome condensation, regulation of arm cohesion, spindle assembly, chromosome bi-orientation and cytokinesis. During the cell cycle, the level of this protein is tightly controlled and its deregulated abundance is suspected to contribute to aneuploidy. The cell cycle profile for Aurora B is reminiscent of those for substrates of the anaphase-promoting complex/cyclosome (APC/C), an ubiquitin ligase essential for mitotic progression. Here, we showed that Aurora B is a substrate of APC/C both in vitro and in vivo. Aurora B is efficiently ubiquitinated iv in an in vitro reconstituted system by APC/C that had been activated by Cdh1. The recognition of Aurora B by APC/CCdh1 is specific as it requires the presence of a conserved KEN-box motif at the amino terminus of Aurora B. Degradation of Aurora B at the end of mitosis requires Cdh1 in vivo as the reduction of Cdh1 level by RNA interference stabilizes Aurora B protein. We conclude that, as a key mitotic regulator, Aurora B is degraded by APC/CCdh1 in late mitosis. Aurora B lies at the heart of the cellular mechanism that resolves synthelic and merotelic attachments. A failure to eliminate such events results in gain or loss of chromosomes. Therefore, identifying the physiological substrates of Aurora B is of pivotal importance for research. We screened Aurora B substrates using an in vitro expression cloning system. However, the methodology we employed didn't lead to candidate substrates to be further validated by more rigorous in vivo approaches. The use of high concentrations of misfolded recombinant Aurora B was partially responsible for the loss of specificity. Therefore, purifying active recombinant Aurora B has become a primary goal for future biochemical and structural work. Two molecular chaperones Hsp90 and Cdc37 assist the folding of a variety of kinases in vivo, among which Aurora B is also a candidate. This gave us the final idea of expressing Aurora B-INCENP complexes in bacteria via the coexpression of Hsp90-Cdc37 molecular chaperones.Item CDC14 Coordinates Cyclin Destruction with the Onset of Cytokinesis(2004-08-19) Bembenek, Joshua Nathaniel; Yu, HongtaoThe Cdc14 family of protein phosphatases operate during the final stages of mitosis in various organisms. The Cdc14 phosphatases are downstream components of two homologous signaling pathways: the mitotic exit network (MEN) of S. cerevisiae and septation initiation network (SIN) of S. pombe. Studies of these pathways have revealed divergent roles of Cdc14. In the MEN pathway, Cdc14 is required for cyclin degradation by dephosphorylating Cdh1. The dephosphorylated form of Cdh1 binds to and activates a ubiquitin ligase known as the anaphase-promoting complex (APC/C), which then ubiquitinates mitotic cyclins, targeting them for degradation by the 26S proteosome. In contrast, Cdc14 of the SIN is dispensable for cyclin degradation, but plays an important role during cytokinesis. Two Cdc14 homologues are found in vertebrates, hCdc14A and hCdc14B. I have investigated the regulation of Cdc14 phosphatases to obtain insights into the mechanisms of mitotic exit in higher eukaryotes. Biochemical studies demonstrate that recombinant hCdc14A and hCdc14B can dephosphorylate human Cdh1 and stimulate APC/CCdh1 ligase activity in vitro. Since both the MEN and SIN pathways control Cdc14 localization, I have examined the regulation of the subcellular localization of hCdc14A, hCdc14B and the budding yeast Cdc14. In HeLa cells, hCdc14A localizes to the centrosome whereas hCdc14B is nucleolar during interphase. Both hCdc14 homologues localize to the centrosome and midbody during mitosis. In budding yeast, Cdc14p localizes to the nucleolus during most of the cell cycle and is released in late anaphase when it localizes to the centrosome and the bud neck. The subcellular localization the Cdc14 homologues in HeLa cells is regulated by a nuclear export signal. S. cerevisiae strains carrying only NES mutant CDC14 alleles are capable of degrading mitotic cyclins and escaping mitosis. However, they exhibit a temperature-sensitive phenotype at 37°C because they fail to complete cytokinesis and lack centrosome and bud neck localization of Cdc14. This demonstrates that the Cdc14 phosphatases are regulated by nucleocytoplasmic shuttling. Collectively, my work strongly suggests that the Cdc14 phosphatases play a conserved role in coordinating the destruction of mitotic cyclins with the execution of cytokinesis.Item Investigating the Biological Functions of the Protein Kinase WNK1 in the Regulation of Cytoskeletal Structures and Membrane Trafficking(2013-06-19) Tu, Szu-Wei; Luby-Phelps, Katherine; Cobb, Melanie H.; Albanesi, Joseph P.; Rice, Luke M.With No Lysine (WNK) 1, a serine/threonine kinase, is a unique kinase to its catalytic lysine residue at a non-canonical position relative to all other kinases. Characterization of endogenous WNK1 distribution by immunofluorescence reveals a perinuclear punctate pattern. I have investigated this perinuclear distribution and how it might relate to the biological functions of WNK1 from two aspects. First, I investigated cytoskeletal structures mainly focused on the microtubules. WNK1 localized on mitotic spindles during mitosis as well as interphase microtubules. Depletion of WNK1 caused aberrant mitotic spindles, chromosomes and defect of abscission. In interphase cells, disruption of radiating microtubules from microtubule organization center was observed. Centrosomal structure was impaired. Cells showed a migratory defect. Clues from a former student and my mass spectrometry data suggested that dynein and its associated protein-dynactin, centrosomal protein of 70 and 170 kDa might be potential interactors mediating microtubule related phenotypes. Second, I examined WNK1 and membrane trafficking events. Depletion of WNK1 caused higher amount of epidermal growth factor receptors remained at the later step of endocytosis. Lysosomes and lysosome-related organelles were disrupted. Biochemical assay suggested that WNK1 could associate with active Rab 6 or 7 effector complexes. I have identified that the homotypic fusion and vacuole protein sorting (HOPS) complex, one of Rab7 effector complexes could interact with WNK1. Mass spectrometry results showed that WNK1 could pull down clathrin heavy chain and adaptor protein complex-3 (AP-3) β subunit. AP-3 vesicles are also HOPS complex-mediated vesicular trafficking between the Trans-Golgi network and late endosomes. Co-localization analysis suggested that WNK1 co-localized with AP-3 in a high pearson correlation coefficient (0.53). Depletion of WNK1 showed defect of the maturation of autophagosomes. Taken together, WNK1 might affect membrane trafficking through HOPS complex-mediated homotypic (the assembly of phagophores) and heterotypic (late endosomes and lysosomes) membrane fusion.