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INCENP Translation during Oocyte Maturation Is a Maternal Factor of Xenopus Laevis DevelopmentLeblond, Geoffrey January 2011 (has links)
During vertebrate oocyte maturation, the chromosomes progress to and arrest at metaphase of meiosis II in preparation for fertilization. This process includes emission of the first polar body. The second polar body is emitted after fertilization. A number of proteins are accumulated during oocyte maturation. Inhibition of this de novo translation does not appear to affect the progression of meiosis during oocyte maturation. The role of these pools of proteins has yet to be elucidated. Curiously, several of the upregulated proteins are key players in mitosis, including INCENP, a subunit of the chromosome passenger complex implicated in chromosome segregation and cytokinesis. During early stages of development in Xenopus laevis, the embryo cycles through mitosis, also known as embryo cleavage, every 30min with little to no time for transcription/translation. Our goal is to determine if the de novo translation of these mitotic proteins during oocyte maturation has a role in early embryogenesis. We used morpholino oligonucleotides antisense to INCENP mRNA (INCENPmorpho) to inhibit de novo translation during oocyte maturation. Using confocal imaging and the host transfer technique, these injected oocytes were matured, fertilized and assessed for developmental competency. INCENPmorpho and a control morpholino (ctrlmorpho) had no discernable effect on 1st or 2nd polar body emission. Whereas ctrlmorpho embryos developed normally, INCENPmorpho embryos did not cleave. Thus, de novo translation of INCENP during oocyte maturation is necessary for embryogenesis. Specifically, accumulation of INCENP and other mitotic proteins during oocyte maturation may be a common strategy in this species to prepare for the rapid and synchronous mitoses during early embryogenesis.
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Calcium Signaling During Polar Body Emission in the Xenopus laevis OocyteLeblanc, Julie January 2014 (has links)
Polar body emission (PBE), a form of asymmetric division, occurs twice during vertebrate oocyte maturation and is required to produce a haploid egg for sexual reproduction. Our lab elucidated parts of the mechanism that regulates PBE in Xenopus laevis oocytes. Cdc42 and RhoA, two GTPases, were shown to mediate membrane protrusion and the contractile ring, respectively. It is believed that cdc42 is mediating the protrusion by regulating actin polymerization. However, it is not clear what upstream signaling pathway regulates cdc42 activation during PBE. One possibility is calcium signaling, which occurs at fertilization, and is required for second PBE. Interestingly, the fertilization calcium transient also regulates cortical granule exocytosis/membrane retrieval, a process that also involves cdc42-mediated actin assembly. Furthermore, active cdc42 and RhoA are found in non-overlapping concentric zones in single-cell wound healing; their activation requires calcium signaling. To determine possible calcium transients during polar body emission, we employed the calcium-binding C2 domain of PKCβ in live cell imaging. Surprisingly, the most prominent C2 signal was seen after cdc42 activation and membrane protrusion. Co-localization experiments indicated that the C2 signal appeared at the cortical area marked by the contractile ring component anillin, and after partial constriction of the ring. Injection of the calcium chelator, dibromo-BAPTA, abolished the C2 signal, suggesting that it is indeed depicting a calcium transient. Dibromo-BAPTA injection also inhibited polar body abscission, as assessed by a novel abscission assay developed in our lab. We have for the first time detected a calcium signal during PBE that is essential to the last step of cytokinesis—abscission.
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Roles of the Rac/Cdc42 effector proteins Pak and PIX in cytokinesis, ciliogenesis, and cyst formation in renal epithelial cellsPuglise, Jason Matthew January 2010 (has links)
No description available.
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The effects of chromosome number changes on mitotic fidelity and karyotype stabilityNicholson, Joshua Miles 17 June 2015 (has links)
The correct number of chromosomes is important for the maintenance of healthy cells and organisms. Maintenance of a correct chromosome number depends on the accurate distribution of chromosomes to the daughter cells during cell division, and errors in chromosome segregation result in abnormal chromosome numbers, or aneuploidy. Aneuploidy is typically associated with deleterious effects on organismal and cellular fitness; however, aneuploidy has also been associated with enhanced cellular growth in certain contexts, such as cancer. Another type of deviation from the normal chromosome number can occur when entire sets of chromosomes are added to the normal (diploid) chromosome number, resulting in polyploidy. Whereas polyploidy is found in certain normal tissues and organisms, tetraploidy (four sets of chromosomes) is associated with a number of precancerous lesions and is believed to promote aneuploidy and tumorigenesis. While it is clear that chromosome mis-segregation causes aneuploidy, the effect of aneuploidy on chromosome segregation is less clear. Similarly, it is unclear whether and how tetraploidy may affect chromosome segregation. The work described here shows that aneuploidy can cause chromosome mis-segregation and induces chromosome-specific phenotypic effects. In contrast, tetraploidy does not per se induce chromosome mis-segregation, but enables the accumulation of aneuploidy thanks to a "genetic buffer" effect that allows tetraploid cells to tolerate aneuploidy better than diploid cells. / Ph. D.
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The evolution of centrosome and chromosome number in newly formed tetraploid human cellsBaudoin, Nicolaas C. 22 June 2020 (has links)
Tetraploidy – the presence of four copies of the haploid chromosome complement – is common in cancer. There is evidence that ~40% of tumors pass through a tetraploid stage at some point during their development, and tetraploid cells injected in mice are more tumorigenic than their diploid counterparts. However, the reason for this increased tumorigenicity of tetraploid cells is not well established. Most routes by which cells may become tetraploid also confer cells with double the number of centrosomes, the small membraneless organelle that organizes the cell's microtubule cytoskeleton and mitotic spindle apparatus. Centrosome number homeostasis is crucial for health, and recent studies have shown inducing extra centrosomes in cells can induce tumor formation in mice. This has led some researchers to propose that the extra centrosomes that arise together with tetraploidy may be the reason that tetraploid cells are more tumorigenic. However, several anecdotal reports have found that tetraploid clones generated and grown in vitro appear to lose their extra centrosomes. Here, I investigate the population dynamics of the loss of extra centrosomes in newly formed tetraploid cells generated via cytokinesis failure. I uncover the mechanism driving the process and build a mathematical model that captures the experimentally observed dynamics. Next, I investigate karyotypic heterogeneity in newly formed tetraploid cells and their counterparts that are grown for 12 days under standard culture conditions and find that karyotypic heterogeneity has increased after 12 days of growth after tetraploidization. The day 12 'evolved' population with increased heterogeneity formed larger colonies in soft agar than newly formed tetraploid cells or diploid parental precursors and karyotype analysis of the largest soft agar colonies revealed recurrent aneuploidies shared by a subset of colonies. Finally, I investigate the effects of different culture conditions - meant to mimic various conditions in the tumor microenvironment - on the evolution of centrosome and chromosome number in newly formed tetraploid cells and identify a small subset of conditions that altered centrosome homeostasis or the fitness of tetraploid cells. / Doctor of Philosophy / The genetic information in cancer cells is often drastically altered compared to normal cells in the body. As one important example of this, the number and structure of chromosomes - the DNA structures that hold the genetic information - is often abnormal in cancer cells. Abnormal chromosome number is closely linked with cancer development, but the details of why this leads to more cancer are not clear. One important kind of chromosome number change is when a cell undergoes incomplete cell division, and the resulting cell acquires double the number of chromosomes compared to a normal cell (known as tetraploidy). Tetraploidy occurs in close to 40% of cancers and is linked with the most aggressive cases. The abnormal cell divisions that cause tetraploidy also lead to other cellular changes. One important change is that tetraploid cells also acquire double the number of the structures that organizes cell division (centrosomes). The centrosome organizes the mitotic spindle, the major apparatus that is responsible for equally distributing chromosomes to two daughter cells during the cell division process. Extra centrosomes in cells are closely linked with cancer and can lead to additionally chromosome number changes. Researchers have believed that the extra centrosomes that are acquired with doubled chromosome number may be the major reason that tetraploid cells are linked to more aggressive cancer. However, recent studies have suggested that tetraploid cells may lose their extra centrosomes, calling into question the details of the relationship between tetraploidy and tumor formation. Here, I use human cells grown in culture to understand how extra centrosomes are lost from tetraploid cells. I find that extra centrosomes in newly formed tetraploid cells promote abnormal 'multipolar' cell divisions, in which chromosomes are segregated unevenly to three or more partitions. Such divisions are often fatal to daughter cells. In some cases, the extra centrosomes can cluster to form bipolar spindles that segregate chromosomes into two equal partitions (as is normal). When forming bipolar spindles, extra centrosomes can cluster asymmetrically (three centrosomes at one pole, one at the other) or symmetrically (two centrosomes at each pole). Tetraploid cells with a normal number of centrosomes emerge when extra centrosomes cluster asymmetrically in a bipolar spindle, yielding one tetraploid daughter cell with a normal number of centrosomes. Such cells have a fitness advantage over cells with extra centrosomes, which over time are very likely to undergo fatal multipolar divisions. Thus, cells with a normal centrosome number 'take over' the population. Next, I investigate the cancer-like properties of tetraploid cells without their extra centrosomes and find that they display increased tumor-like behavior even in the absence of extra centrosomes. Finally, I investigate whether changing the conditions in which cells are grown (in ways meant to mimic different conditions that may be experienced in the body) affects whether tetraploid cells lose their extra centrosomes. We identify a small number of conditions that do influence loss of extra centrosomes. Together, these studies illuminate important details of the relationship between tetraploidy and tumor formation. This work lays the foundation to further explore and understand the relative roles of tetraploidy, extra centrosomes, and tissue environment in cancer.
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Cell Cycle Associated Gene Expression Predicts Function in MycobacteriaBandekar, Aditya C. 07 April 2020 (has links)
While the major events in prokaryotic cell cycle progression are likely to be coordinated with transcriptional and metabolic changes, these processes remain poorly characterized. Unlike many rapidly-growing bacteria, DNA replication and cell division are temporally-resolved in mycobacteria, making these slow-growing organisms a potentially useful system to investigate the prokaryotic cell cycle. To determine if cell-cycle dependent gene regulation occurs in mycobacteria, we characterized the temporal changes in the transcriptome of synchronously replicating populations of Mycobacterium tuberculosis (Mtb). By enriching for genes that display a sinusoidal expression pattern, we discover 485 genes that oscillate with a period consistent with the cell cycle. During cytokinesis, the timing of gene induction could be used to predict the timing of gene function, as mRNA abundance was found to correlate with the order in which proteins were recruited to the developing septum. Similarly, the expression pattern of primary metabolic genes could be used to predict the relative importance of these pathways for different cell cycle processes. Pyrimidine synthetic genes peaked during DNA replication and their depletion caused a filamentation phenotype that phenocopied defects in this process. In contrast, the IMP dehydrogenase guaB2 dedicated to guanosine synthesis displayed the opposite expression pattern and its depletion perturbed septation. Together, these data imply obligate coordination between primary metabolism and cell division, and identify periodically regulated genes that can be related to specific cell biological functions.
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Microtubule arrays and cell divisions of stomatal development in ArabidopsisLucas, Jessica Regan 16 July 2007 (has links)
No description available.
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Régulation temporelle de l’abscission, la dernière étape de la division cellulaire : rôle des forces exercées au niveau du pont intercellulaire / Temporal regulation of the abscission, the last step of cell division : role of forces exerted on the intercellular bridgeJanvore, Julie 28 September 2012 (has links)
La dernière étape de la cytocinèse, l’abscission, consiste en la coupure du pont intercellulaire reliant les deux cellules filles à la suite de la contraction de l’anneau acto-myosique. Comme toutes les étapes de la division cellulaire, l’abscission doit être régulée dans l’espace et dans le temps afin qu’elle intervienne au bon endroit et au bon moment. Mon travail de doctorat a porté sur l’étude de la régulation dans le temps de l’abscission par l’environnement des cellules filles, en particulier par les forces de traction exercées par les cellules sur le pont intercellulaire. En utilisant une combinaison d’approches permettant de contrôler le confinement spatial 2D des cellules filles, de mesurer les forces exercées par les cellules au cours de la cytocinèse et de micro-manipuler le pont intercellulaire, j’ai montré que, de façon contre-intuitive, une tension exercée au niveau du pont retardait l’abscission et qu’au contraire la relâche de cette tension induisait l’abscission. De plus, la régulation temporelle de l’abscission par les facteurs environnementaux des cellules filles implique les protéines des « Endosomal Sorting Complex Required for Transport III » (ESCRT-III), machinerie centrale de l’abscission. Enfin, des expériences préliminaires suggèrent que cette régulation serait importante pour le maintien de l’intégrité tissulaire et la morphogenèse au cours du développement. / The last step of cytokinesis, abscission, consists in the severing of the intercellular bridge connecting the two daughter cells after the contraction of the acto-myosin ring. As any other step of cell division, abscission has to be regulated both in time and space in order to take place at the proper time and proper place. During my PhD, I studied the temporal regulation of the abscission by the cell micro-environment, particularly by the traction forces exerted by the cells on the intercellular bridge. I used a combination of approaches to control the daughter cells 2D spatial confinement, to measure the forces exerted by the cells throughout cytokinesis, and to micro-manipulate the intercellular bridge. Counter-intuitively, a tension exerted on the intercellular bridge delayed abscission while a release of tension in the bridge induced abscission. Moreover, the temporal regulation of abscission by the environment of the daughter cells implies the Endosomal Sorting Complex Required for Transport III (ESCRT-III), the main abscission machinery. Finally, preliminary experiments suggest that this mechanism could be important for tissue integrity and morphogenesis.
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Experiments concerning the mechanism of cytokinesis in Caenorhabditis elegans embryos / Experimente zur Untersuchung der Zytokinese in Caenorhabditis elegansBringmann, Henrik Philipp 31 January 2007 (has links) (PDF)
In my thesis I aimed to contribute to the understanding of the mechanism of cytokinesis in C. elegans embryos. I wanted to analyze the relative contributions of different spindle parts – microtubule asters and the midzone - to cytokinesis furrow positioning. I developed a UV laser-based severing assay that allows the spatial separation of the region midway between the asters and the spindle midzone. The spindle is severed asymmetrically between one aster and the midzone. I found that the spindle provides two consecutive signals that can each position a cytokinesis furrow: microtubule asters provide a first signal, and the spindle midzone provides a second signal. The use of mutants that do not form a midzone suggested that the aster-positioned furrow is able to divide the cell alone without a spindle midzone. Analysis of cytokinesis in hypercontracile mutants suggests that the aster-positioned cytokinesis furrow and the midzone positioned furrow inhibit each other by competing for cortical contractile elements. I then wanted to identify the molecular pathway responsible for cytokinesis furrow positioning in response to the microtubule asters. To this end, I performed an RNAi screen, which identified a role for LET-99 in cytokinesis: LET-99 appeared to be required for aster-positioned cytokinesis but not midzone-positioned cytokinesis. LET-99 localizes as a cortical band that overlaps with the cytokinesis furrow. Mechanical displacement of the spindle demonstrated that the spindle positions cortical LET-99 at the site of furrow formation. The furrow localization of LET-99 depended on G proteins, and consistent with this finding, G proteins are also required for aster-positioned cytokinesis. (Anlage: Quick time movies, 466, 67 MB)
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The involvement of ARF6 in rapid membrane recycling during Drosophila spermatocyte cytokinesis / Die Bedeutung von ARF6 für das rapide Membranrecycling während der Cytokinese der Spermatocyten von DrosophilaFoster, Naomi 14 February 2007 (has links) (PDF)
Cytokinesis involves constriction of the cell at the equator. Without decreasing in volume, a spherical cell requires a net increase in the surface area during this constriction. The constriction is driven by formation of an actomyosin contractile ring, and the surface increase by addition of membrane during the formation of the cleavage furrow. Both events depend on the central spindle microtubules at the midzone of the spindle and, in particular, on the centralspindlin protein complex. The communication between the central spindle microtubules and the actomyosin ring involves binding of a GAP and a GEF for RhoA to the centralspindlin kinesin Pavarotti/MKLP1. However, it is still unclear which molecular machinery connects the mitotic spindle to membrane trafficking during cleavage furrow ingression. ARF6 is a member of the ARF family of small GTPases, and previous studies suggest that it is an important regulator of membrane trafficking through the endocytic pathway, and cortical Actin remodelling. I generated an arf6 null mutant in Drosophila. arf6 null mutants survive to adulthood without obvious morphological defects, indicating that ARF6 is not required for Drosophila somatic development. However, ARF6 is required for cytokinesis in Drosophila spermatocytes. The centralspindlin kinesin Pavarotti, identified as an ARF6 interactor in a Yeast-2-Hybrid assay, binds ARF6 in GST pulldowns, and interacts genetically with the arf6 mutant. ARF6 localizes to the plasma membrane and a population of early and recycling endosomes. During cytokinesis, ARF6 is enriched on recycling endosomes at the central spindle. arf6 mutants form a cleavage furrow during cytokinesis, which later regresses. Cytokinesis in arf6 mutant spermatocytes lacks the rapid plasma membrane expansion observed during normal divisions. The results of this study suggest that ARF6 might promote rapid recycling of endosomal membrane stores at the central spindle to the plasma membrane during cytokinesis. ARF6 might be recruited to the central spindle via its interaction with Pavarotti, and act as part of the molecular link between the central spindle cytoskeleton and the rapid plasma membrane addition necessary for cytokinesis. Für die Ansicht der quick-time-Movies mit der Endung "avi" ist die Installation des "Apple QuickTime-Players" erforderlich.
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