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Myosin Dynamics in Drosophila Neuroblasts Lead to Asymmetric CytokinesisConnell, Marisa 11 July 2013 (has links)
Cells divide to create two daughter cells through cytokinesis. Daughter cells of different sizes are created by shifting the position of the cleavage furrow. The cleavage furrow forms at the position of the metaphase plate so in asymmetric cytokinesis the spindle is shifted towards one pole. Unlike most systems, Drosophila neuroblasts have a centrally localized metaphase plate but divide asymmetrically. Drosophila neuroblasts divide asymmetrically due to the presence of a polarized myosin domain at the basal pole during mitosis. I investigated the mechanism by which the basal myosin domain produces asymmetric cytokinesis and the pathway regulating this domain.
We tested several mechanisms by which the basal myosin domain could lead to asymmetric cytokinesis. Based on surface area and volume measurements, I demonstrated that asymmetric addition of new membrane is not involved. I determined that neuroblasts exhibit asymmetric cortical extension during anaphase with the apical pole extending 2-3 times more than the basal pole. Mutants that lose basal myosin extend equally at both poles supporting this model. Mutants that retain apical myosin exhibited symmetric cortical extension but still divided asymmetrically, demonstrating that asymmetric cortical extension is not required for asymmetric cytokinesis. Observations of the mitotic spindle show that the cleavage furrow forms at a position biased towards the basal pole when compared to the position of the metaphase plate even though this position is still equidistant between the centrosomes. I observed that midzone components shift basally in a basal domain dependent manner suggesting that contraction of the basal domain leads to new microtubule-cortex interactions at a position away from the spindle midzone.
I demonstrated that the basal domain is regulated by the heterotrimeric G protein, Gβ13F, which is activated by Pins. In Gβ mutants, the localization of all basal components (myosin, anillin, and pavarotti) is lost and the cells divide symmetrically. Although the basal domain is contiguous with equatorial myosin, it is not regulated by the same pathway and photobleaching experiments indicate that they exhibit different behaviors during anaphase suggesting a difference in temporal regulation.
This dissertation includes previously published coauthored material.
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Comparative analyses of cell wall integrity signaling and cytokinesis regulation in the yeasts Saccharomyces cerevisiae and Kluyveromyces lactisHeppeler, Nele 12 October 2011 (has links)
Cell division and the maintenance of cellular integrity are key features of life itself and some vital aspects of these processes have been studied in this thesis, using the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis as model eukaryotes.
In the first part of the thesis, three putative negative regulators of the cell wall integrity (CWI) signal transduction pathway were investigated, which have been isolated in previous genetic screens. Whereas the FIG4 gene seems to encode a protein which could be distantly related to CWI signaling, NTA1 and SET4 gene products were not found to have a major influence, as judged from phenotypes of deletion mutants,
overproducers, and epistasis analyses with different CWI pathway mutants. In general, the data indicated rather indirect connections of all three protein functions
with the maintenance of cellular integrity. Therefore, this line of research was
discontinued, in order to investigate more closely the regulation of cytokinesis in the
dairy yeast K. lactis.
In this second and major part of the thesis the homologue of a recently found
cytokinesis regulator in S. cerevisiae (INN1, accordingly designated as KlINN1), was
cloned and characterized. It could be shown, that the gene is essential and that the
encoded protein is species-specific, i.e. KlINN1 does not complement the lethality of
a Scinn1 deletion mutant and vice versa. Analyses of hybrid proteins demonstrated
that this specificity is most likely mediated by the C2-domain of the protein, which is thought to interact with membrane lipids. In S. cerevisiae, Inn1 interacts through its proline-rich motifs located in the C-terminal half of the protein with the cytokinesis regulators Hof1 and Cyk3. They both carry a SH3-domain which has been shown to mediate the interaction with Inn1. Consequently, the two encoding genes, KlHOF1
and KlCYK3, were also characterized in K. lactis. In contrast to S. cerevisiae, where
the homologues seem to exert somewhat redundant functions and only a double
deletion is lethal, each of the genes is essential in K. lactis. The exact nature of their roles in cytokinesis of this yeast remains to be determined. Unexpectedly, attempts to confirm an interaction between the proline-rich motifs of KlInn1 and the SH3-domains of KlHof1 and KlCyk3 in a yeast two-hybrid assay failed so far, but this line of research will be followed up in future experiments. In order to compare the timing of cytokinesis and the localization of the above mentioned regulators between S. cerevisiae and K. lactis, another homologue of a protein involved in cytokinesis in S. cerevisiae was investigated in K. lactis.
Preliminary evidence from deletion of the KlMYO1 gene, which encodes a likely
component of the contractile actomyosin ring (CAR) in K. lactis, indicates that this
yeast may predominantly engage in a CAR-independent pathway of cytokinesis.
Nevertheless, similar to S. cerevisiae GFP-fusions of KlInn1, KlHof1, KlCyk3, and
KlMyo1 all were shown here to localize to the bud neck during cytokinesis in K. lactis.
In summary, components identified to play a crucial role in yeast cytokinesis in
S. cerevisiae display a similar localization in K. lactis, but may differ considerably in
their detailed functions in vivo. This thesis represents the first detailed investigation of the molecular processes underlying cytokinesis in K. lactis and provides the basis for elaborate future studies.
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Anillin, An Organizer of CytokinesisHeshmati, Fatemeh 30 October 2012 (has links)
Anillin is a highly conserved multi-domain cytoskeletal protein that provides a spatial and temporal scaffold for contractile ring proteins to ensure successful cytokinesis. We have looked at the temporal order of anillin and septin recruitment to the cleavage furrow using time-lapse microscopy and found that anillin localizes to the furrow in early anaphase while septins appear there later in an anillin-dependent manner. We also characterized the effect of anillin depletion in different cell lines and observed that septins and myosin delocalize in the absence of anillin in Tet-ON HeLa, AD293 and ARPE-19 cells but not in wild type HeLa cells. Asymmetric furrow formation was also investigated using the epithelial cell model: MDCK cells. Depletion of anillin and SEPT9 in MDCK cells was achieved using lentivirus shRNA constructs and this revealed that anillin or SEPT9 depletion did not affect asymmetric cytokinesis, although localization of SEPT 9 was affected by anillin depletion.
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Anillin, An Organizer of CytokinesisHeshmati, Fatemeh 30 October 2012 (has links)
Anillin is a highly conserved multi-domain cytoskeletal protein that provides a spatial and temporal scaffold for contractile ring proteins to ensure successful cytokinesis. We have looked at the temporal order of anillin and septin recruitment to the cleavage furrow using time-lapse microscopy and found that anillin localizes to the furrow in early anaphase while septins appear there later in an anillin-dependent manner. We also characterized the effect of anillin depletion in different cell lines and observed that septins and myosin delocalize in the absence of anillin in Tet-ON HeLa, AD293 and ARPE-19 cells but not in wild type HeLa cells. Asymmetric furrow formation was also investigated using the epithelial cell model: MDCK cells. Depletion of anillin and SEPT9 in MDCK cells was achieved using lentivirus shRNA constructs and this revealed that anillin or SEPT9 depletion did not affect asymmetric cytokinesis, although localization of SEPT 9 was affected by anillin depletion.
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Early development of the peach (Prunus persica (L.) Batsch.) fruit and the time of endosperm cytokinesisAllison, Max L January 2011 (has links)
Digitized by Kansas Correctional Industries
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Molecular characterisation of fission yeast myosin IIMay, Karen Marie January 1997 (has links)
No description available.
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Genetic analysis of the role of pebble during cytokinesis in DrosophilaO'Keefe, Louise. January 2001 (has links) (PDF)
Errata pasted onto back page. Bibliography: p. 133-149. The RhoGEF activity of PBL is shown to be acting predominantly by the activation of Rho1 and downstream signaling pathways required for contractile ring function during cytokinesis. Genetic evidence suggests this could be through the activation of Diaphanous (an FH protein) to reorganize the actin cytoskeleton, as well as through the activation of Rho-kinase which results in the phosphorylation, and activation of myosin. Highlights a possible role for PBL during contractile ring function at a later stage that previously thought. Genetic interaction screens were employed to identify regulators of PBL activity during cytokinesis. CDK1 was identified genetically as a candidate for regulating PFB activity, but functional studies in vivo showed that this regulation was not by direct phophorylation of the PBK consensus CDK1 suites tested. Further screening has identified other possible components pf PBL signaling pathways, but a role during cytokinesis for these interactors remains to be confirmed. The eye phenotypes described provide ideal systems for the identification of components of PBL signaling pathways in Drosophila. The high level of conservation in the mechanism of cytokinesis from yeast to mammals would also suggest that the identified interactors would most likely represent components of cytokinesis pathways in all eukaryotes.
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Genetic analysis of the role of pebble during cytokinesis in Drosophila / by Louise O'Keefe.O'Keefe, Louise Veronica January 2001 (has links)
Errata pasted onto back page. / Bibliography: p. 133-149. / 149 p., [29] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The RhoGEF activity of PBL is shown to be acting predominantly by the activation of Rho1 and downstream signaling pathways required for contractile ring function during cytokinesis. Genetic evidence suggests this could be through the activation of Diaphanous (an FH protein) to reorganize the actin cytoskeleton, as well as through the activation of Rho-kinase which results in the phosphorylation, and activation of myosin. Highlights a possible role for PBL during contractile ring function at a later stage that previously thought. Genetic interaction screens were employed to identify regulators of PBL activity during cytokinesis. CDK1 was identified genetically as a candidate for regulating PFB activity, but functional studies in vivo showed that this regulation was not by direct phophorylation of the PBK consensus CDK1 suites tested. Further screening has identified other possible components pf PBL signaling pathways, but a role during cytokinesis for these interactors remains to be confirmed. The eye phenotypes described provide ideal systems for the identification of components of PBL signaling pathways in Drosophila. The high level of conservation in the mechanism of cytokinesis from yeast to mammals would also suggest that the identified interactors would most likely represent components of cytokinesis pathways in all eukaryotes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2002?
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Dissecting induction of cell cleavageAlsop, G. Bradley 04 December 2003 (has links)
Cytokinesis separates replicated chromosomes and cytoplasm into two
daughter cells. In animal cells, this is achieved by the formation of a cleavage
furrow that bisects the mitotic (or meiotic) spindle. It is known that the mitotic
apparatus defines the cell cleavage plane. However, it is not clear how the mitotic
apparatus initiates the cleavage furrow. Each part of the mitotic apparatus; namely
asters, central spindle (microtubule arrays and the spindle midzone), and
chromosomes, has been found capable of inducing a cleavage furrow in certain
cell types. Yet it is uncertain which part is the essential source of the signal and
whether all parts act in concert.
This thesis systematically examines in grasshopper spermatocytes 1) which
spindle constituent is the essential source of furrow signal; 2) the impact of
microtubules on distribution of actin filaments and positioning of cell cleavage
relative to spindle reorganization; 3) the independent role of the spindle midzone
relative to microtubules in furrow initiation and ingression. These examinations
combine micromanipulation with digital-enhanced polarization microscopy and
epifluorescence microscopy, in which mitotic spindles in living cells are
mechanically dissected and rearranged as desired as well as microfixed to evaluate
and propose models for cell cleavage.
This thesis has come to the conclusion that none of structural constituents
of the spindle apparatus is essential for cell cleavage induction except
microtubules. First, furrow induction occurs regardless of a particular spindle
constituent, so long as sufficient microtubules are present to form bipolar arrays.
Second, microtubules continuously dictate distribution of actin filaments and
positioning of cell cleavage. Asymmetric alterations of spindle microtubules
dynamically affect the location of the spindle midzone, distribution of actin
filaments, and ultimately position of the cleavage furrow in cells containing a
bipolar spindle, monopolar spindle, or half-spindle. Third, actin filaments are
distributed to the furrow region by microtubule-mediated transport, but organized
by the midzone, which is essential for furrow ingression, but not initiation. These
results suggest that during post-anaphase spindle assembly, actin filaments are
excluded by bipolar microtubule arrays to the equatorial cell cortex where they
bundle into a contractile ring with cytokinetic factors. / Graduation date: 2004
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An investigation into the mechanism of cytokinesis in the Caenorhabditis elegans embryo /Severson, Aaron Frederick, January 2001 (has links)
Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 118-127). Also available for download via the World Wide Web; free to University of Oregon users.
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