Browsing by Subject "Mitosis"
Now showing 1 - 13 of 13
- Results Per Page
- Sort Options
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 CDK5RAP2 Regulates Centriole Licensing to Restrict Centriole Duplication in Mice(2009-09-04) Barrera, Jose Anselmo; Megraw, TimothyCells division is a highly coordinated series of events that must occur with extreme precision. Defects during segregation of genetic material (DNA) can have adverse effects on the health of the cell, surrounding tissue, organ, and the organism as a whole. Accurate assembly of the bipolar mitotic spindle apparatus is crucial for precise chromosome segregation. Centrosomes play a crucial role in establishment of the mitotic spindle and therefore are vital to the maintenance of genetic stability. Centrosomes are composed of two centrioles that arrange a specialized conglomerate of proteins into a pericentriolar matrix. Centrosomes are highly regulated throughout the cell cycle, and duplicate only once per cell cycle ensuring that each cell inherits one centrosome after mitotic exit, and contains only two centrosomes at the following mitosis. Truncating mutations in the Cyclin-Dependent Kinase 5 Regulatory Associated Protein 2 gene (CDK5RAP2), which encodes a centrosomal protein, result in autosomal recessive primary microcephaly (MCPH, [MIM 251200]) in humans. The major phenotypic manifestation of this rare genetic disorder is a small head. Affected individuals have head circumferences at least 4 standard deviations below sex- and age-matched individuals and suffer mental retardation. In order to investigate how mutations in CDK5RAP2 affect centrosome structure and regulation, and how this leads to MCPH, we derived two distinct mouse mutant lines with truncating mutations within the CDK5RAP2 locus similar to those found in affected humans. We show that centriole engagement and cohesion, two distinct centriole-binding processes, are disrupted in CDK5RAP2 mutant cells. Partial disruption of CDK5RAP2 affected centriole cohesion, whereas complete CDK5RAP2 disruption deregulated the centriole duplication cycle leading to centriole/centrosome amplification. During mitosis amplified centrosomes in CDK5RAP2 mutant cells were potent microtubule organizing centers that drove formation of multipolar spindles. Furthermore, cells formed multiple primary cilia from multiple centrioles inherited from the previous cell cycle. Together these results define a role for CDK5RAP2 in the regulation of centriole duplication and also provide a basis for the development of MCPH.Item Centrosomin Self-Assembly and Centrosomal Protein Recruitment(2005-08-11) Bauer, Ruth Anne; Megraw, TimothyCentrosomes, the major microtubule organizing centers in animal cells, are important for mitotic spindle formation. Normally, each cell has two centrosomes which migrate to opposite sides of the nuclear envelope prior to entry into mitosis. Centrosomin (Cnn) is a major centrosomal protein that is important for nucleation and organization of bipolar spindle microtubules at mitosis. Cnn protein localizes to the pericentriolar matrix and from there other centrosomal proteins 'load' onto the centrosome, including gamma-tubulin. Centrosomes are non-functional without the addition of Cnn since it is responsible for recruitment of other centrosomal proteins. There are two conserved motifs in Cnn protein, currently of unknown function. One of these motifs is most likely responsible for interaction with gamma-tubulin and other centrosomal proteins which make the centrosome capable for microtubule nucleation. Cnn full-length and half proteins were expressed in E. coli and purified in vitro. The properties of these Cnn proteins show self-assembly and recruitment of centrosomal proteins. These activities of Cnn in vitro are novel and will help further the investigation of Cnn protein function in the context of biological systems. Cnn fusion proteins show characteristics similar to centrosomal 'satellite' or 'flare' particles described in animal cells. It is likely that these satellites communicate with the actin cytoskeleton in syncytial Drosophila embryos.Item Dissecting the Mitotic Golgi Membranes Mediated Microtubule Polymerization(2019-11-25) Guo, Haijing; Henne, W. Mike; Goodman, Joel M.; Yu, Hongtao; Seemann, JoachimA properly assembled astral microtubule network is required for correct mitotic spindle orientation, which is important in multiple development processes as it determines cell fate and function. The initiation and growth of astral microtubules was previously attributed to centrosomes and microtubule stabilizing proteins. Here in my dissertation research, I demonstrate that microtubules initiated by mitotic Golgi membranes contribute to the growth of astral microtubules and the proper orientation of the spindle. In turn, the microtubule initiation activity of mitotic Golgi membranes facilitates the proper inheritance of the single copy Golgi apparatus, which is essential in polarized cellular functions, including directional cell migration and secretion. Microtubule assembly is initiated by the Golgi resident protein GM130, which locally activates the spindle assembly factor TPX2 at the mitotic Golgi membranes. GM130 relieves TPX2 from inhibition by competing for importin α binding. The mitotic phosphorylation of importin α on Serine 62 by Cdk1 switches its substrate preference towards GM130 and enables the competition-based activation. The importin α S62A mutant impedes the local TPX2 activation and compromises the astral microtubules, which ultimately leads to misoriented spindles. Blocking of the GM130-importin α-TPX2 activation pathway reduces the astral microtubule growth rate. I also identified that the human GM130 homolog GLP harbors a domain that is highly similar to the TPX2 activating domain of GM130, which could potentially initiate microtubule assembly. My research reveals the novel role of mitotic Golgi membranes in astral spindle organization and the underlying mechanism that regulates this process in a spatio-temporal manner.Item Function And Recruitment Of Centromeric Heterochromatin Protein 1(2011-02-01) Chaudhary, Jaideep; Yu, HongtaoDuring early mitosis, the sister chromatids are held together by Cohesin, a protein complex composed of Smc3, Smc1, Scc1/Rad21 and Scc3. Cohesin is first released from the arms of chromosomes, leaving it intact at the centromere. At the metaphase – anaphase transition, centromeric cohesin is cleaved, allowing the chromatids to segregate to two daughter cells. Shugoshin (Sgo-1) is a known protector of cohesin at the centromere. It prevents phosphorylation of cohesin complex by Plk1 before the metaphase – anaphase transition, which would otherwise lead to cohesin release, causing the two chromatids to separate untimely. In this study we show that Sgo1 localizes on centromeres through HP1 during interphase in human cells. Also, Sgo1 binds all three forms of HP1 (i.e. alpha, beta, gamma) through its chromoshadow domain. We have determined the dissociation constant of this interaction to be in the sub-micromolar range. We have shown conclusively that Sgo1 binds to HP1 chromoshadow domain via one PxVxL motif. We have further shown that, in mitosis, HP1 is recruited to centromeres by Incenp, a subunit of the chromosomal passenger complex via the chromoshadow domain of HP1. This interaction is most likely at the HP1 CSD dimer interface, where PxVxL motifsbind. Hence, it seems that Incenp may provide competition to Sgo1 for HP1 binding.Item Generating the Spindle Assembly Checkpoint Signal at the Kinetochore(2004-08-19) Bharadwaj, Rajnish; Yu, HongtaoTo avoid missegregation of chromosomes during mitosis cells employ a surveillance mechanism termed Spindle assembly checkpoint that senses the lack of tension/attachment on the kinetochores and consequently blocks anaphase onset by inhibiting an E3 ubiquitin ligase called anaphase-promoting complex. The roles of two kinases- BubR1 and Mps1, implicated in spindle assembly checkpoint were investigated. A checkpoint complex containing BubR1 and Bub3 has been purified from mitotic human cells. BubR1 directly interacts with Cdc20 and inhibits the activity of APC in vitro,much more efficiently than Mad2. Surprisingly, the kinase activity of BubR1 or association with Bub3 is not required for the inhibition of APCCdc20. Furthermore, BubR1 restores the mitotic arrest in Cdc20-overexpressing cells treated with nocodazole. Mps1 is a dual specificity kinase that localizes to kinetochores in mitosis. Depletion of Mps1 by RNAi leads to the abrogation of spindle assembly checkpoint. The kinetochore proteins involved in the recruitment of checkpoint proteins and the generation of wait-anaphase signal have not been identified. Kinetochores also provide the attachment sites for spindle microtubules and are required for the alignment of chromosomes at the metaphase plate (chromosome congression). Components of the conserved Ndc80 complex have been implicated in both these function. To better understand the function of the Ndc80 complex, we have identified two novel subunits of the human Ndc80 complex, termed human Spc25 (hSpc25) and human Spc24 (hSpc24), using an immuno-affinity approach. Human Spc25 interacts with Hec1 (human Ndc80) throughout the cell cycle and localizes to kinetochores during mitosis. RNAi-mediated depletion of hSpc25 in HeLa cells causes aberrant mitosis followed by cell death, a phenotype similar to that of cells depleted for Hec1. Loss of hSpc25 also causes multiple spindle aberrations, including elongated, multipolar, and fractured spindles. In the absence of hSpc25, Mad1 and Hec1 fail to localize to kinetochores during mitosis whereas the kinetochore localization of Bub1 and BubR1 is largely unaffected. Interestingly, the kinetochore localization of Mad1 in cells with a compromised Ndc80 function is restored upon microtubule depolymerization. Thus, hSpc25 is an essential kinetochore component that plays a significant role in proper execution of mitotic events.Item Insights into the Metabolic Regulation of Growth and Proliferation in Saccharomyces Cerevisiae(2013-10-04) Cai, Ling; Liu, Yi; DeBerardinis, Ralph J.; Li, Bing; Ross, Elliott M.Cells needs to gauge their capacity to grow based on nutrient availability, and adopt different metabolic strategies for optimal growth and survival. We have investigated the molecular mechanism of how growth decisions are made based on metabolic status and how metabolic enzymes are regulated by nutrient availability. In the first part of this study, we report that acetyl-CoA is the downstream metabolite of carbon sources that represents a critical metabolic signal for growth and proliferation. Upon entry into growth, intracellular acetyl-CoA levels increase substantially and consequently induce the Gcn5p/SAGA-catalyzed acetylation of histones at genes important vi for growth, thereby enabling their rapid transcription and commitment to growth. Acetyl-CoA functions as a carbon-source rheostat that signals the initiation of the cellular growth program by promoting the acetylation of histones specifically at growth genes. In the second part of the study, we report the dynamic modification of ribosome biogenesis transcription factor Ifh1p regulated by different metabolic cues. Ribosome biogenesis requires an enormous commitment of energy and resources in growing cells. We show that Ifh1p is dynamically acetylated and phosphorylated as a function of the growth state of cells. Ifh1p is acetylated at numerous sites in its N-terminal region by Gcn5p and deacetylated by NAD+-dependent deacetylases of the sirtuin family. Acetylation of Ifh1p is responsive to intracellular acetyl-CoA levels and serves to regulate the stability of Ifh1p. The phosphorylation of Ifh1p is mediated by Protein Kinase A and is dependent on TORC1 signaling. Instead of modulating overall rates of RP gene transcription or growth, these nutrient-responsive modifications of Ifh1p play a more prominent role in the regulation of cellular replicative lifespan. Finally, we report the different roles of acetyl-CoA synthetases Acs1p and Acs2p in yeast metabolism. While Acs2p is important for rapid growth in glucose medium, Acs1p has unique roles in more challenging growth conditions. It is important for yeast metabolic cycling and is recruited to foci structure near mitochondria that might be involved in shuttling acetyl-CoA into the mitochondria during hypoxia.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.Item The Mitotic Spindle Mediates Inheritance of the Golgi Ribbon Structures(2010-05-14) Wei, Jen-Hsuan; Seemann, JoachimThe mammalian Golgi ribbon disassembles during mitosis and reforms in both daughter cells after division. Mitotic Golgi membranes concentrate around the spindle poles, suggesting that the spindle may control Golgi partitioning. To test this, cells were induced to divide asymmetrically with the entire spindle segregated into only one daughter cell. A ribbon reforms in the nucleated karyoplasts, whereas the Golgi stacks in the cytoplasts are scattered. However, the scattered Golgi stacks are polarized and transport cargo. Microinjection of Golgi extract together with tubulin or incorporation of spindle materials rescues Golgi ribbon formation. Therefore, the factors required for postmitotic Golgi ribbon assembly are transferred by the spindle, but the constituents of functional stacks are partitioned independently, suggesting that Golgi inheritance is regulated by two distinct mechanisms.Item Regulation of Sister Chromatid by the Acetyltransferase Naa50(2016-07-25) Rong, Ziye; Seemann, Joachim; Yu, Hongtao; Li, Bing; Olson, Eric N.During the cell cycle, sister-chromatid cohesion tethers sister chromatids together from S phase to the metaphase-anaphase transition and ensures accurate chromosome segregation of chromatids into daughter cells. N-terminal acetylation is one of the most prevalent protein covalent modifications in eukaryotes and is mediated by a family of N-terminal acetyltransferases (NAT). Naa50 (also called San or NatE) has previously been shown to play a role in sister-chromatid cohesion in metazoans. The mechanism by which Naa50 contributes to cohesion is not understood, however. Here, I show that depletion of Naa50 in HeLa cells weakens the interaction between cohesin and its positive regulator sororin and causes cohesion defects in interphase, consistent with a role of Naa50 in cohesion establishment or maintenance. Strikingly, co-depletion of NatA, a heterodimeric NAT complex that physically interacts with Naa50, rescues the sister-chromatid cohesion defects and the resulting mitotic arrest caused by Naa50 depletion, indicating that NatA and Naa50 play antagonistic roles in cohesion. Purified recombinant NatA and Naa50 do not affect each other's NAT activity in vitro. Because NatA and Naa50 exhibit distinct substrate specificity, I propose that they modify different effectors and regulate sister-chromatid cohesion in opposing ways.Item Regulation of Sister-Chromatid Cohesion(2017-10-17) Zheng, Ge; Burma, Sandeep; Yu, Hongtao; Mendell, Joshua T.; Tu, BenjaminOrderly execution of two critical events during the cell cycle--DNA replication and chromosome segregation--ensures the stable transmission of genetic materials. The cohesin complex physically connects sister chromatids during DNA replication in a process termed sister-chromatid cohesion. Timely establishment and dissolution of sister-chromatid cohesion is a prerequisite for accurate chromosome segregation, and is tight regulated by the cell cycle machinery and cohesin-associated proteins. Errors in this process can lead to aneuploidy and promote tumorigenesis. Research in this dissertation has provided several key insights into the regulation of sister-chromatid cohesion during the mitotic cell cycle. First, we report the crystal structure and functional characterization of human Wapl, a key negative regulator of cohesin that promotes cohesin release from chromatin. Our results indicate that Wapl-mediated cohesin release from chromatin requires extensive physical contacts between Wapl and multiple cohesin subunits. Second, we have determined the crystal structure of human SA2-Scc1 cohesin subcomplex, which is the interaction hub for cohesin regulators. Further biochemical and functional analyses reveal the direct competition between Wapl and the cohesion protector Sgo1 for binding to a conserved site on SA2-Scc1. Our results implicate a role for this direct antagonism in centromeric cohesion protection. Third, we report the crystal structure of human Pds5B bound to a conserved peptide motif found in both Wapl and Sororin. Further biochemical and functional studies suggest that Pds5 has both positive and negative roles in cohesion regulation and establish the molecular basis for how Wapl and the cohesin-stabilizing factor Sororin antagonistically influence cohesin dynamics on chromosomes. The structure reveals inositol hexakisphosphate (IP6) as an unexpected cofactor of Pds5. The IP6-binding segment of Pds5B engages the N-terminal region of Scc1 and inhibits the binding of Scc1 to Smc3. Our results suggest a direct role of Pds5 in cohesin release from chromosomes by stabilizing a transient, open state of cohesin during its ATPase cycle. Finally, we show that cohesin loading onto chromosomes requires the phosphorylation of MCM2-7 by Cdc7-Dbf4 kinase (DDK) during early S phase, when a mega-complex composed of MCM2-7, Scc2/4 and cohesin is formed. At active replication forks, inactivation of multiple replisome components impairs cohesin loading, weakens MCM-Scc2/4-cohesin interaction and leads to cohesion defects. By contrast, interfering Okazaki fragment processing and nucleosome assembly during DNA replication do not impact interphase cohesion, suggesting that cohesion establishment occurs before Okazaki fragment maturation and histone deposition. Our results demonstrate that DNA replication-coupled cohesin loading is required for the establishment of sister-chromatid cohesion. In conclusion, combining structural, biochemical and cellular approaches, our studies advance the molecular understanding of spatial and temporal regulation of the establishment and dissolution of sister-chromatid cohesion.Item Regulation of the Cytoskeleton by Kinesins(2013-11-20) Weil, Lauren Melissa; Albanesi, Joseph P.; Cobb, Melanie H.; Conrad, Nicholas; Scherer, PhilippKinesins are motor proteins that associate with microtubules. The position of the motor domain has been linked to kinesin function. While amino-terminal and carboxy-terminal localization of the motor domain is linked to cargo transport, kinesins with the motor domain in the middle (M-kinesins) have a role in microtubule depolymerization. The kinesin-13 family consists of four M-kinesins, KIF2A, KIF2B, KIF2C, and KIF24. These proteins regulate the cytoskeleton through their microtubule depolymerizing activity. All four kinesins have reported functions in mitosis, while little is known about their roles in interphase. KIF2A and KIF2C are upregulated in cancer cells and the increased protein expression influences cell migration and invasiveness. In order to understand how KIF2A and KIF2C influence migration, we analyzed the microtubule and actin cytoskeleton in cells manipulated for kinesin expression. We found that depletion of KIF2A increases the number of focal adhesions and stress fibers and results in defects in cell spreading. KIF2A does not influence the dynamics of focal adhesion assembly or disassembly. In contrast, depletion of KIF2C prevents re-formation of focal adhesions. has little or no effect on the actin cytoskeleton. Here we uncovered a functional divergence in regulation of the cytoskeleton between KIF2A and KIF2C. Furthermore, this is the first time that an M-kinesin, which does not transport cargo, has been shown to influence focal adhesion dynamics.Item Roles of HDACs and MEF2 in Adult Hippocampal Neurogenesis(2014-11-17) Jiang, Yindi; Zhang, Chun-Li; Olson, Eric N.; Johnson, Jane E.; Hsieh, JennyThe maintenance of the resident adult neural stem/progenitor cell (NSPC) pool depends on the precise balance of proliferation, differentiation, and maintenance of the undifferentiated state. Identifying the mechanisms that regulate this balance in adult hippocampal NSPCs can provide insight into basic neurogenesis principles important for tissue homeostasis and preventing tumor formation. Pharmacological inhibition of histone deacetylases (HDACs), a class of histone-modifying enzymes, have promising effects in cancer cells, yet the specific roles of individual HDACs in adult NSPCs are unclear. In this dissertation, I focus on dissecting the roles of two different HDACs in adult hippocampal neurogenesis: the Class I HDAC, HDAC3 and the Class IIa HDAC, HDAC5 as well as the Class IIa HDAC binding partner, myocyte enhancer factor 2 (MEF2). Using conditional knockout (cKO) mice and in vitro cell culture, I show that histone deacetylase 3 (HDAC3) is required for the proliferation of adult NSPCs. Detailed cell cycle analysis of NSPCs from Hdac3 cKO mice reveals a defect in cell cycle progression through G2/M phase, but not S phase. Moreover, HDAC3 controls G2/M phase progression mainly through post-translational stabilization of the G2/M cyclin- dependent kinase-1 (CDK1). These results demonstrate that HDAC3 plays a critical role in NSPC proliferation. HDAC5 is the most abundant Class IIa HDAC in adult dentate gyrus. HDAC5 is only expressed in immature and mature neurons. Using Hdac5 knockout mice and in vitro cell culture, I show that HDAC5 is necessary and sufficient to restrict the neuronal differentiation of NSPCs. However, the detailed mechanisms are yet to be determined. Class IIa HDACs bind to myocyte enhancer factor 2 (MEF2) in the nucleus to repress transcription of pro-neuronal genes. Thus, we also examined the function of Mef2 genes in adult hippocampal neurogenesis. In adult hippocampus, the three most highly expressed MEF2 proteins are MEF2A, 2C, and 2D, which are expressed in immature and mature neurons similar to HDAC5. We have shown that one synthetic small molecule, Isoxazole-9 (Isx-9) could trigger neuronal differentiation robustly in vitro and in vivo. Inducible knockout of all three Mef2 genes specifically in NSPCs and their progeny revealed their critical roles in mediating Isx-9 induced neurogenesis and baseline neurogenesis. In summary, these results demonstrate that HDACs and MEF2 control different stages of adult hippocampal neurogenesis and suggest that strategies aimed at pharmacological modulation of these proteins may be beneficial for tissue regeneration and controlling tumor cell growth in mammalian brain.