The cohesin complex is responsible for the fidelity of chromosomal segregation during mitosis. It consists of four core subunits namely Rad21/Mcd1/Sccl, Smc1, Smc3 and one of the yeast Scc3 orthologs SA1 or SA2. Sister chromatid cohesion is formed by the cohesin complex during DNA replication and maintained until the onset of anaphase. Among the many proposed models of how cohesin holds sister chromatids together, the 'core' cohesin subunits Smc1, Smc3 and Rad21/Mcd1/Scc1 are almost universally displayed as forming a contiguous ring. However, other than its supportive role in the cohesin ring, little is known about the fourth core protein SA1/SA2 - despite its physical association to the cohesin ring. To gain deeper insight into how physically and physiologically SA2 interacts with the cohesin complex, we performed structural characterization of SA2 and Rad21 and mapped the interaction region of the two proteins in vitro and ex vivo . We found SA2 interacts with Rad21 at multiple domains while Rad21 only interacts with SA2 through a 10 amino acid α-helical motif from 383-392aa. Deletion of these 10 amino acids or mutation of three conserved amino acids (L385, F389, and T390) in this α-helical motif prevents Rad21 from physically interacting with SA2/SA1 and causes premature sister chromatid separation in mitotic cells that often leads to aneuploidy. Our studies provide a model for how SA2 structurally strengthens the cohesin ring through its interaction with Rad21. Results from our structural characterization of these two proteins also provided directions for further investigation of the structural basis of protein-protein interaction in the cohesin complex.

In this study, the contributions of learning motivation, reasoning ability, learning orientation and gender to International Baccalaureate and National Program students&rsquo / mitosis and meiosis achievement was investigated. Participants of the study were 472 ninth grade students from a private high school in Ankara. Two hundred nineteen students (46%) were in International Baccalaureate Program and two hundred fifty three (54%) were in National Program. The study was conducted during the 2006-2007 Spring semester. Prior to the introduction of mitosis and meiosis topics, students&rsquo / motivations toward biology learning (self efficacy, active learning strategies, science learning value, performance goals, achievement goals, learning environment stimulation), formal reasoning abilities and learning approaches were measured by Students&rsquo / Motivation Towards Biology Learning Questionnaire, Test of Logical Thinking Ability scale and Learning Approach Questionnaire respectively. After the topics have been covered, a 20 item Mitosis and Meiosis Achievement Test was used to measure achievement in mitosis and meiosis topics. Multiple regression analysis revealed that achievement was explained in positive direction by formal reasoning ability and in negative direction by active learning strategies and rote learning in National Program classes. Self-efficacy and formal reasoning ability had significant contributions to achievement for International Baccalaureate students. The main predictor of achievement was formal reasoning ability for both International Baccalaureate and National Program students, explaining 4.7% and 10.9% variance respectively. Moreover, while 2.9% of the variance in achievement was explained by self efficacy in International Baccalaureate classes, rote learning explained 2.2% of the variance in achievement in negative direction in National Program classes.

Les centrosomes dont le rôle principal est d’organiser le cytosquelette de microtubules et le fuseau mitotique servent aussi de sites d’interaction pour plusieurs protéines régulatrices du cycle cellulaire et de la réponse aux dommages à l’ADN. Une de ces protéines est la kinase CHK2 et plusieurs publications montrent une sous-population de CHK2 localisée aux centrosomes dans les cellules en interphase et en mitose. Toutefois, la localisation de CHK2 aux centrosomes demeure controversée, car des doutes subsistent en ce qui concerne la spécificité des anticorps utilisés en immunocytochimie. En utilisant des lignées cellulaires du cancer du côlon, les cellules HCT116 sauvages et HCT116 CHK2-/- ainsi que différentes lignées d’ostéosarcome humain dans lesquelles l’expression de CHK2 a été inhibée par ARN interférence, nous montrons que les anticorps anti-CHK2 qui donnent un signal centrosomal sont non spécifiques et reconnaissent un antigène inconnu sur les centrosomes. Cependant, par des expériences d’immunofluorescence réalisées avec des cellules U2OS qui expriment les protéines de fusion GFP-CHK2 ou FLAG-CHK2, nous révélons une localisation centrosomale de CHK2 dans les cellules en mitose, mais pas en interphase. Ce résultat a été confirmé par vidéomicroscopie dans les cellules vivantes exprimant GFP-CHK2. Pour déterminer le ou les rôles potentiels de CHK2 en mitose nous avons réalisé des expériences pour explorer le rôle de CHK2 dans la progression de la mitose, la nucléation des microtubules aux centrosomes et la progression de la mitose en présence de problèmes d’attachement des chromosomes où de lésions génotoxiques. Nos données suggèrent que CHK2 n’est pas impliquée dans la régulation de la mitose dans les cellules U2OS. / Centrosomes function primarily as microtubule-organizing centres and play a crucial role during mitosis by organizing the bipolar spindle. In addition to this function, centrosomes act as reaction centers where numerous key regulators meet to control cell cycle progression. One of these factors involved in genome stability, the checkpoint kinase CHK2, was shown to localize at centrosomes throughout the cell cycle. Here, we clarify that CHK2 only localized at centrosomes during mitosis. Using wild-type and CHK2-/- HCT116 human colon cancer cells, or human osteosarcoma U2OS cells depleted for CHK2 with small hairpin RNAs, we show that several CHK2 antibodies are non-specific for immunofluorescence and cross-react with an unknown centrosomal protein(s). To analyse further CHK2 localization, we established cells expressing inducible GFP-CHK2 and Flag-CHK2 fusion proteins. We show that CHK2 localizes to the nucleus in interphase cells but that a fraction of CHK2 associates with centrosomes in mitotic cells, from early mitotic stages until cytokinesis. In contrast to previous data obtained by A. Stolz and colleagues with the human colon carcinoma HCT116 cell line, our experiments exploring the possible functions for CHK2 during mitosis did not support a role for CHK2 in the bipolar spindle formation and the timely progression of mitosis in human osteosarcoma U2OS cells.

No description available.

No description available.

Protein trafficking between the endoplasmic reticulum (ER) and Golgi complex is regulated by the activity of ADP-ribosylation factors (Arfs). Arf activation by guanine nucleotide exchange factors (GEFs) leads to the recruitment of the coatomer protein COPI and vesicle formation. By using fluorescently-tagged proteins in live cells, we have been able to identify novel functions for Arfs and the Arf-GEF GBF1 at the ER-Golgi intermediate compartment (ERGIC) and mitotic Golgi clusters. We first focused on Arf function at the ERGIC after observing both class I (Arf1) and class II (Arfs 4 and 5) Arfs at this structure. We discovered that class II Arfs remain bound to ERGIC membranes independently of GBF1 activity following treatment with brefeldin A (BFA). Further characterization of the class II Arfs using additional pharmacological agents such as Exo1 and inactive mutant forms of Arf4 demonstrated that the class II Arfs associate with the ERGIC membrane via receptors distinct from GBF1. Our work suggests that GBF1 accumulation on membranes in the presence of BFA is due to loss of Arfs from the membrane rather than the formation of an abortive complex with Arf and GBF1. Next, while studying GBF1 in live cells, we unexpectedly observed GBF1 localizing to large fragmented structures during mitosis. We identified these structures as mitotic Golgi fragments that are positive for GBF1 and COPI throughout mitosis. Again using live cells treated with BFA and Exo1, we demonstrated that GBF1 concentrates on these mitotic fragments suggesting that they are derived from Golgi membranes. By colocalization studies and fluorescence recovery after photobleaching, we demonstrated that these mitotic fragments maintain a cis-to-trans subcompartmental Golgi polarization and membrane dynamics of GBF1 similar to interphase cells. Interestingly, inactivation of GBF1 and loss of COPI from the membranes of the mitotic Golgi fragments did not delay progressing through mitosis. Our results from our second project indicate for the first time that the mitotic Golgi clusters are bona fide Golgi structures that exist throughout mitosis with a functional COPI machinery.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available in electronic form as viewed 2/16/2009.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 173-176.

In eukaryotes, cell proliferation and cell cycle transitions are strictly controlled by the anaphase-promoting complex/cyclosome (APC/C). The APC/C is an E3 ubiquitin ligase that regulates chromatid segregation at the metaphase to anaphase transition, exit from mitosis and the establishment and maintenance of G1. The APC/C’s catalytic activity and substrate specificity are controlled by its interactions with two coactivators, Cdc20 and Cdh1. In contrast to Cdh1, APC/C activation by Cdc20 during mitosis requires hyper-phosphorylation of APC/C subunits by cyclin-dependent kinase (Cdk) and polo kinase. The aim of the first part of this thesis was to understand how mitotic phosphorylation regulates APC/C activity. Using cryo-electron microscopy and biochemical analysis, we found that an auto-inhibitory segment of the Apc1 subunit acts as a molecular switch that in apo unphosphorylated APC/C interacts with a coactivator-binding site (C-box binding site), thereby obstructing engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box binding site to relieve APC/C auto-inhibition. Efficient phosphorylation of the auto-inhibitory segment requires the recruitment of the kinase Cdk-cyclin-Cks to a hyper-phosphorylated loop of Apc3. In addition to regulation of APC/C activity by phosphorylation, ordered cell progression is ensured by the ability of the APC/C to target substrate degradation in a defined order. At mitosis onset, degradation of securin and cyclin B1 is inhibited by the spindle assembly checkpoint, exerted by the mitotic checkpoint complex (MCC), whereas both cyclin A2 and Nek2A are not subject to MCC inhibition. The aim of the second part of the thesis was to elucidate the mechanism of how the APC/C achieves its substrate specificity. Our biochemical analysis showed that the resistance of cyclin A2 to MCC inhibition is due to its ABBA motif and the Cdk-associated Cks2 subunit. Furthermore, we found that it is the Cdc20 molecule of the MCC that binds to the ABBA motif to allow for cyclin A2 ubiquitination. Strikingly, mutating all three known degrons (KEN box, D box and ABBA motif) of cyclin A did not affect its ubiquitination by APC/CCdc20. Deletion of a potential novel degron found within residues 60-80 of cyclin A2 impaired cyclin A2 ubiquitination.

Les cellules multiciliées jouent un rôle essentiel dans la propulsion des fluides physiologiques. Leur dysfonctionnement provoque des maladies chroniques. Contrairement à la plupart des cellules de mammifères qui possèdent un centrosome composé de deux centrioles, les cellules multiciliées possèdent une centaine de centrioles qui servent de base à la nucléation des cils motiles. Les mécanismes d'amplification de centrioles ou de régulation du nombre de centrioles dans ce type cellulaire étaient jusque-là inconnus. Les centrioles nouvellement formés étaient considérés comme apparaissant " de novo ". Une approche de vidéomicroscopie et de microscopie de super-résolution corrélative nous a d'abord permis de déterminer que tous les procentrioles sont générés à partir du centrosome préexistant. Nous démontrons que le centriole fils du centrosome est le site principal de nucléation de 95% de centrioles nouvellement formés dans les cellules multiciliées. Ces résultats réfutent par conséquent l'origine " de novo " des centrioles dans ce type cellulaire. Puis, nous montrons que la machinerie mitotique orchestre la progression spatio-temporelle de la dynamique centriolaire dans ces cellules post-mitotiques et en phase terminale de différentiation. L'amortissement de l'activité de Cdk1 empêche la rentrée en mitose tout en permettant la coordination du nombre de centrioles, leur croissance, et leur désengagement par des transitions phasiques nécessaires à la nucléation de cils motiles. Cette thèse aide à mieux comprendre la différentiation des cellules multiciliées, les ciliopathies, ainsi que l'amplification centriolaire pathologique associée avec le cancer et la microcéphalie. / Multiciliated mammalian cells play a crucial role in the propulsion of physiological fluids. Their dysfunction causes severe chronic diseases. In contrast to the strict centriole number control in cycling cells, multiciliated cell differentiation is marked by the production of up to several hundred centrioles, each nucleating a motile cilium. The mechanisms of centriole amplification or centriole number control in these cells were unknown and new centrioles were thought to appear de novo in the cytoplasm. First, videomicroscopy combined with correlative super-resolution and electron microscopy has enabled us to determine that all procentrioles are generated via runs of nucleation from the pre-existing progenitor cell centrosome. We show that the daughter centriole of the centrosome is the primary nucleation site for 95% of the new centrioles in multiciliated cells and thus refute the de novo hypothesis. Then, we provide evidence of an activation of the mitosis regulatory network during the centriole dynamic. With single cell live imaging and pharmacological modulation of mitosis regulators, we show that the mitosis machinery orchestrates the spatiotemporal progression of centriole amplification in terminally differentiating multiciliated cell progenitors. The fine-tuning of Cdk1 activity prevents mitosis while allowing the timely coordination of centriole number, growth, and disengagement through checkpoint-like phase transitions necessary for subsequent functional motile ciliation. This PhD provides a new paradigm for studying multiciliated cell differentiation, cilia-related diseases and pathological centriole amplification associated with cancer and microcephaly.