Pre-replicative complex (pre-RC) components the origin recognition complex (ORC), Cdc6, and Cdt1, play key roles in the recruitment, and loading of the replicative helicase, the minichromosome maintenance complex (Mcm2-7), onto DNA to license origins for replication. Until recently, the prevailing model for pre-RC assembly predicted that once MCMs are loaded at origins, ORC, Cdc6, and Cdt1 are dispensible for replication. Contrary to this model, previous work has shown that Orc6 is required following origin licensing, for the continued association of the MCM complex in late G1 phase. In this study, a similar role in pre-RC maintenance has been demonstrated for Cdc6, and Cdt1. Chromatin immunoprecipitation (ChIP) analysis has shown that late G1 phase depletion of either Cdc6, or Cdt1 leads to the destabilization of MCMs from origins, although this destabilization is more pronounced for Cdc6 depletion than for Cdt1. Furthermore, the resynthesis of Cdc6 following its depletion, allows for the reassembly of pre-RCs in late G1 phase, and restores competence for DNA replication. In this study, a potential role for Orc6 in mitosis/cytokinesis in budding yeast has also been characterized, as research with both Drosophila and human cell lines has pointed to a role for Orc6 in these processes. Deleting HOF1 and CYK3 (two proteins involved in cytokinesis in budding yeast) leads to a synthetic lethal phenotype, suggesting that the resulting gene products function in redundant cytokinetic pathways. Indeed, Hof1 has been shown to be primarily involved in actin ring contraction, while Cyk3 functions in septum formation, both pathways of which are important for budding yeast cytokinesis. Interestingly, previous work has identified an Orc6-Hof1 interaction in budding yeast. In this study, it has been demonstrated that following Orc6 depletion in a GAL1-ORC6/Δcyk3 strain, fluorescence activated cell sorting (FACS) analysis is consistent with a stronger cytokinetic defect phenotype than observed for Δcyk3 cells. Preliminary cell counts indicate that following Orc6 depletion, a higher percentage of GAL1-ORC6/Δcyk3 cells display misshapen mother bud necks than in an isogenic Δcyk3 strain. Cell synchronization experiments have demonstrated that Orc6 depletion during a G2/M phase arrest, leads to a block in cell cycle progression following release.

Pre-replicative complex (pre-RC) components the origin recognition complex (ORC), Cdc6, and Cdt1, play key roles in the recruitment, and loading of the replicative helicase, the minichromosome maintenance complex (Mcm2-7), onto DNA to license origins for replication. Until recently, the prevailing model for pre-RC assembly predicted that once MCMs are loaded at origins, ORC, Cdc6, and Cdt1 are dispensible for replication. Contrary to this model, previous work has shown that Orc6 is required following origin licensing, for the continued association of the MCM complex in late G1 phase. In this study, a similar role in pre-RC maintenance has been demonstrated for Cdc6, and Cdt1. Chromatin immunoprecipitation (ChIP) analysis has shown that late G1 phase depletion of either Cdc6, or Cdt1 leads to the destabilization of MCMs from origins, although this destabilization is more pronounced for Cdc6 depletion than for Cdt1. Furthermore, the resynthesis of Cdc6 following its depletion, allows for the reassembly of pre-RCs in late G1 phase, and restores competence for DNA replication. In this study, a potential role for Orc6 in mitosis/cytokinesis in budding yeast has also been characterized, as research with both Drosophila and human cell lines has pointed to a role for Orc6 in these processes. Deleting HOF1 and CYK3 (two proteins involved in cytokinesis in budding yeast) leads to a synthetic lethal phenotype, suggesting that the resulting gene products function in redundant cytokinetic pathways. Indeed, Hof1 has been shown to be primarily involved in actin ring contraction, while Cyk3 functions in septum formation, both pathways of which are important for budding yeast cytokinesis. Interestingly, previous work has identified an Orc6-Hof1 interaction in budding yeast. In this study, it has been demonstrated that following Orc6 depletion in a GAL1-ORC6/Δcyk3 strain, fluorescence activated cell sorting (FACS) analysis is consistent with a stronger cytokinetic defect phenotype than observed for Δcyk3 cells. Preliminary cell counts indicate that following Orc6 depletion, a higher percentage of GAL1-ORC6/Δcyk3 cells display misshapen mother bud necks than in an isogenic Δcyk3 strain. Cell synchronization experiments have demonstrated that Orc6 depletion during a G2/M phase arrest, leads to a block in cell cycle progression following release.

The objective of this work is to investigate the structures of two nucleotide binding proteins: mevalonate kinase (MVK) and FtsZ. MVK is the key enzyme involved in terpenoid biosynthesis. In this study, we solved the crystal structures of the M. jannaschii MVK apoprotein, as well as the protein in complex with ligands. Its fold was analyzed and firmly established within the GHMP kinase family, in which homoserine kinase (HSK), phosphomevalonate kinase and galactokinase also belong. Structural analysis in combination with enzyme kinetics studies revealed the mechanism of this enzyme upon substrate binding, catalysis and inhibition. In particular, the phosphate-binding loop was found to be critically involved in the binding of nucleotides and terpenoids, via the interaction with a di-phosphate moiety from the ligand. An enzymatic reaction mechanism was constructed based on our structural data and it is consistent with kinetics studies from the literature. In this mechanism, the invariant residue Asp 155 functions as a general base that increases the nucleophilicity of the phosphoryl acceptor. Finally, a virtual screening study has been performed to explore the ligand binding potential of MVK. Compounds predicted to bind MVK were tested and analyzed. FtsZ is a prokaryotic homologue of tubulin that forms the apparatus for bacterial cell division. The structure of a crystal filament of the M. tuberculosis FtsZ complexed with GDP was described in this study. It shows an anti-parallel, left-handed double helical architecture. Compared with the straight crystal filament revealed earlier by other groups, the catalytic T7 loop in our structure is found to be outside the nucleotide binding site, indicating the GTPase is inactive. Furthermore, the buried surface area in our crystal filament is less, probably suggesting the helical FtsZ filament is less stable. We therefore proposed that the hydrolysis of GTP and the releasing of the γ-phosphate group will trigger the rearrangement of the FtsZ fibler, characterized by the exclusion of the T7 loop, which might lead to a less stable helical filament and would be the first step for the disassembly of FtsZ polymer.

The Aurora kinases are highly conserved serine/threonine kinases that play essential roles throughout mitosis. In metazoans, these functions are mediated by Aurora A and B at the spindle poles and the equatorial region respectively. I show here that Dictyostelium contains a single Aurora kinase, DdAurora that displays characteristics of both Aurora A and B. Like Aurora A, DdAurora has an extended N-terminal domain with an A-box and localizes to the spindle poles during early mitosis. Like Aurora B, DdAurora localizes to centromeres in metaphase, the central spindle during anaphase and the cleavage furrow at the end of cytokinesis. In addition to these known features of Aurora A and B, I found that DdAurora remains associated with centromeres during anaphase and telophase which has not been shown in any other organisms. INCENP is known to be an important binding partner of Aurora B. In Dictyostelium the conserved C-terminal IN-box domain of DdINCENP is essential for its interaction with DdAurora and for the localization of DdAurora to the central spindle. In contrast, the centromeric and spindle pole localization of DdAurora does not require an interaction with DdINCENP. Surprisingly, a truncated DdINCENP protein lacking the IN-box domain can still localize on centromeres and the central spindle even though it does not bind to DdAurora. I also found that the localization of DdAurora to the central spindle requires Kif12, a protein similar to mitotic kinesin like protein 2 (MKLP2). However, this requirement is suppressed by the overexpression of GFP-DdINCENP. GFP-DdINCENP can localize to the central spindle in the absence of Kif12 and it probably recruits DdAurora to the same location through their strong interaction. Finally, I demonstrated that Myosin II heavy chain is important for the proper localization of the DdAurora/DdINCENP complex to the cleavage furrow during late cytokinesis. With the exception of DdINCENP, no other binding partner or substrate of DdAurora has been identified in Dictyostelium. By performing large-scale immunoprecipitation in wild-type cells, I identified several potential binding partners/substrates of DdAurora, including topoisomerase B and HspA. Future esearch on these proteins may help to elucidate DdAurora function in different stages of M phase.

Rho GTPases are highly conserved regulators of cell polarity and the actin cytoskeleton. The role of the Rho GTPase Cdc42 and its regulation during cell division is not well understood. Using biochemical and imaging approaches in budding yeast, I demonstrate that Cdc42 activation peaks during the G1/S transition and during anaphase, but drops during mitotic exit and cytokinesis. Cdc5/Polo kinase is an important upstream cell cycle regulator that suppresses Cdc42 activity. Failure to downregulate Cdc42 during mitotic exit prevents the normal localization of key cytokinesis regulators - Iqg1 and Inn1- at the division site, and results in an abnormal septum. The effects of Cdc42 hyperactivation are largely mediated by the Cdc42 effector p21-activated kinase (PAK) kinase, Ste20. Inhibition of Cdc42 and related Rho GTPases may be a general feature of cytokinesis in eukaryotes.

The contractile vacuole (CV) is an osmoregulatory organelle found exclusively in algae and protists. In addition to expelling excessive water out of the cell, it also expels ions and other metabolites and thereby contributes to the cell's metabolic homeostasis. The interest in the CV reaches beyond its immediate cellular roles. The CV's function is tightly related to basic cellular processes such as membrane dynamics and vesicle budding and fusion; several physiological processes in animals, such as synaptic neurotransmission and blood filtration in the kidney, are related to the CV's function; and several pathogens, such as the causative agents of sleeping sickness, possess CVs, which may serve as pharmacological targets. The green alga Chlamydomonas reinhardtii has two CVs. They are the smallest known CVs in nature, and they remain relatively untouched in the CV-related literature. Many genes that have been shown to be related to the CV in other organisms have close homologues in C. reinhardtii. We attempted to silence some of these genes and observe the effect on the CV. One of our genes, VMP1, caused striking, severe phenotypes when silenced. Cells exhibited defective cytokinesis and aberrant morphologies. The CV, incidentally, remained unscathed. In addition, mutant cells showed some evidence of disrupted autophagy. Several important regulators of the cell cycle as well as autophagy were found to be underexpressed in the mutant. Lipidomic analysis revealed many meaningful changes between wild-type and mutant cells, reinforcing the compromised-autophagy observation. VMP1 is a singular protein, with homologues in numerous eukaryotic organisms (aside from fungi), but usually with no relatives in each particular genome. Since its first characterization in 2002 it has been associated with several cellular processes and functions, namely autophagy, programmed cell-death, secretion, cell adhesion, and organelle biogenesis. It has been implicated in several human diseases: pancreatitis, diabetes, and several types of cancer. Our results reiterate some of the observations in VMP1's six reported homologues, but, importantly, show for the first time an involvement of this protein in cell division. The mechanisms underlying this involvement in Chlamydomonas, as well as other key aspects, such as VMP1's subcellular localization and interaction partners, still await elucidation. / Die kontraktile Vakuole ist ein osmoregulatorisches Organell, das ausschließlich in Algen und Protisten vorkommt. Zusätzlich zu ihrer Rolle als Ausstoßer überflüßigen Wassers aus der Zelle heraus, stößt sie auch Ionen und andere Metaboliten aus, und trägt dabei zur metabolischen Homöostase der Zelle bei. Das Interesse an der kontraktilen Vakuole erstreckt sich über seine unmittelbare zelluläre Rolle hinaus. Die Funktion der kontraktilen Vakuole ist mit einigen grundsätzlichen zellulären Verfahren, wie Membrandynamik und Vesikelknospung und -fusion, verwandt; einige physiologische Verfahren in Tieren, zum Beispiel synaptische Neurotransmission und das Filtrieren des Blutes in den Nieren, sind mit der Funktion der Vakuole eng verwandt; und einige Pathogene—der Ursacher der Schlafkrankheit als Beispiel—besitzen kontraktile Vakuolen, die als Ziele von Medikamenten dienen könnten. Die grüne Alge Chlamydomonas reinhardtii verfügt über zwei Vakuolen. Sie sind die kleinsten bekannten in der Natur, und bleiben bisher verhältnismäßig unerforscht. Viele Gene, die in anderen Organismen als kontraktile-Vakuole-bezogen erwiesen wurden, haben Homologe in C. reinhardtii. Wir versuchten, diese Gene auszuschalten und den Einfluss auf die Vakuole zu beobachten. Die Ausschaltung eines unserer Gene, VMP1, verursachte starke, beachtliche Phänotype. Die Zellen zeigten gestörte Zytokinese und aberrante Zellformen. Die kontraktile Vakuole blieb jedoch verschont. Des Weiteren zeigten Mutantzellen einige Hinweise auf gestörte Autophagie. Einige wichtige Gene des Zellzyklus und der Autophagie waren unterexprimiert in Mutantzellen. Lipidomische Analyse zeigte mehrere bedeutsame Unterschiede zwischen Wildtyp und Mutant, die die Beobachtungen der gestörten Autophagie verstärkten. VMP1 ist ein singularisches Protein, mit Homologen in zähligen eukaryotischen Organismen (jedoch nicht in Pilzen), aber üblicherweise ohne Verwandte in den jeweiligen Genomen. Seit seiner Erstcharakterisierung 2002 wurde es mit etlichen zellulären Verfahren, wie Autophagie, programmiertem Zelltod, Sekretion, Zelladhäsion, und Biogenese der Organellen, assoziiert. Es wurde auch mit einigen menschlichen Krankheiten wie Diabetes, Pankreatitis, und einigen Arten von Krebs in Verbindung gebracht. Unsere Ergebnisse wiederholen einige Beobachtungen in anderen Organismen, zeigen dennoch zum ersten Mal eine Beteiligung von VMP1 an der Zellteilung. Die unterliegenden Mechanismen dieser Beteiligung in Chlamydomonas, sowie andere wichtige Aspekte, etwa die subzelluläre Lokalisierung von VMP1 und dessen Interaktionspartner, warten noch auf Aufklärung.

No description available.

The scaffolding protein anillin plays a crucial role during cytokinesis – the physical separation of daughter cells following chromosome segregation. Anillin binds filamentous F-actin, non-muscle myosin II and septins, and in cell culture models has been shown to restrict actomyosin contractility to the cleavage furrow. Whether anillin also serves this function during the incomplete cytokinesis that occurs in developing germ cells has remained unclear. Localization of anillin to several actin-rich structures in developing male germ cells also suggests potential roles for anillin outside of cytokinesis. In this study, I demonstrate that anillin is required for cytokinesis in dividing Drosophila spermatocytes. In addition, spermatid individualization is defective in anillin-depleted cells, although similarities to another cytokinesis mutant, four wheel drive, suggest this may be a secondary effect of failed cytokinesis. Anillin, septins and myosin II stably associate with the cleavage furrow in wild-type dividing spermatocytes. Anillin is necessary for recruitment of septins to the cleavage furrow, and for maintenance of Rho, F-actin and myosin II at the equator in late stages of cytokinesis. Membrane trafficking appears unaffected in anillin-depleted cells, although, unexpectedly, ectopic expression of one membrane trafficking marker, DE-cadherin-GFP, suppresses the cytokinesis defect. DE-cadherin-GFP recruits β-catenin (armadillo) and α-catenin to the cleavage furrow and stabilizes F-actin at the equator. Taken together, my results suggest that the anillin-septin and cadherin-catenin complexes can serve as alternative means to promote tight physical coupling of F-actin and myosin II to the cleavage furrow and successful completion of cytokinesis.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Cytokinesis is the final chapter of cell division and its last page is abscission, the physical separation of two daughter cells. During cytokinesis of vertebrate cells, two future daughter cells are connected by an intercellular bridge within which the midbody (MB) is positioned. Since becoming the focus of intense investigations on cytokinesis completion, MB is now perceived as a complicated organelle where multiple pathways for abscission are targeted and coordinated. However, whether post-mitotic midbodies, the midbody derivatives (MBds), would be retained by either daughter cell post-abscission remains unexplored. In addition, how cells manage the fate of inherited MBds is also unclear or only sketchily proposed. Finally, whether the inherited MBds in cells may play non-cytokinetic roles is also unaddressed. In the first chapter, I review the historical and current understanding of MBs, with emphasis on their roles in cytokinesis whereas potential non-cytokinetic roles are also covered. In the second chapter, the aforementioned three questions are sequentially addressed. First, the newly-formed MBd appears to be inherited by the daughter cell with the older centrosome. Second, MBds are not only inherited but also accumulated in cancer and pluripotent stem cells, but not in normal somatic (differentiated) cells. In normal somatic cells, MBds are within membrane-bound compartments for lysosomemediated degradation via autophagosome engulfment. This partially explains why MBd-accumulation is rarely observed in these cells. In contrast to the previous model, colleagues and I showed that MBd-accumulation correlates well with the autophagosomal-lysosomal activity, but not with the proliferation rate. Finally, the experimental increase of MBd levels appears to enhance the anchorage-independent growth in cancer cells and the efficiency of reprogramming in fibroblasts. In the last chapter, I conclude our findings and discuss future directions in two aspects.