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.

Pik1p, one of three phosphatidylinositol 4-kinases in the fission yeast, <i>Schizosaccharomyces pombe</i>, was found previously to interact with Cdc4p, a myosin essential light chain that is required for cytokinesis. The involvement of pik1 in cell cycle control was investigated. A fluorescently tagged Pik1p fusion protein was associated with Golgi throughout the cycle, and was found at the medial division plane of the cell during late cytokinesis. This latter distribution has not been reported previously. Gene deletion in diploid cells and tetrad analysis revealed that pik1 is essential for cell viability and is required for spore germination. The terminal phenotype of a temperature-sensitive, loss-of-function allele (pik1-td) indicated that pik1 is involved in cytokinesis: particularly for suppression of secondary septum material deposition, for suppression of initiation of supernumerary septa, and for cell separation. Contractile ring formation was normal in pik1-td cells at the restrictive temperature although the pattern of F-actin patches was disrupted. The F-actin patches were dispersed throughout the cytoplasm. Accumulation of extra inner membranous or vesicle-like structures was observed in these cells. The <i>S. pombe</i> nmt1 promoter and attenuated versions of it were found to be useful for complementation studies in <i>S. cerevisiae</i>. Heterologous expression of <i>S. pombe</i> pik1 complemented the essential functions of a temperature-sensitive allele (pik1﷓101) of its orthologue in <i>Saccharomyces cerevisiae</i> that were lost at the restrictive temperature. A residue required for <i>S. pombe</i> Pik1p lipid kinase activity, D709, was also required for this complementation. A residue, R838, which is required for interactions between Pik1p and Cdc4p was not required for this complementation. The timing and localization of Pik1p to the division plane of the cell late in cytokinesis combined with analysis of the terminal phenotype of a loss-of-function allele, indicate that Pik1p and/or its derived phosphoinositides are required for regulation of septation and cell separation. Pik1p may be involved in the transport, possibly via vesicular transport, of enzymes required for hydrolysis of the primary septum. It may be involved in signaling pathways that lead to the initiation of septation and to the cessation of the deposition of secondary septum material.

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.

Pik1p, one of three phosphatidylinositol 4-kinases in the fission yeast, <i>Schizosaccharomyces pombe</i>, was found previously to interact with Cdc4p, a myosin essential light chain that is required for cytokinesis. The involvement of pik1 in cell cycle control was investigated. A fluorescently tagged Pik1p fusion protein was associated with Golgi throughout the cycle, and was found at the medial division plane of the cell during late cytokinesis. This latter distribution has not been reported previously. Gene deletion in diploid cells and tetrad analysis revealed that pik1 is essential for cell viability and is required for spore germination. The terminal phenotype of a temperature-sensitive, loss-of-function allele (pik1-td) indicated that pik1 is involved in cytokinesis: particularly for suppression of secondary septum material deposition, for suppression of initiation of supernumerary septa, and for cell separation. Contractile ring formation was normal in pik1-td cells at the restrictive temperature although the pattern of F-actin patches was disrupted. The F-actin patches were dispersed throughout the cytoplasm. Accumulation of extra inner membranous or vesicle-like structures was observed in these cells. The <i>S. pombe</i> nmt1 promoter and attenuated versions of it were found to be useful for complementation studies in <i>S. cerevisiae</i>. Heterologous expression of <i>S. pombe</i> pik1 complemented the essential functions of a temperature-sensitive allele (pik1﷓101) of its orthologue in <i>Saccharomyces cerevisiae</i> that were lost at the restrictive temperature. A residue required for <i>S. pombe</i> Pik1p lipid kinase activity, D709, was also required for this complementation. A residue, R838, which is required for interactions between Pik1p and Cdc4p was not required for this complementation. The timing and localization of Pik1p to the division plane of the cell late in cytokinesis combined with analysis of the terminal phenotype of a loss-of-function allele, indicate that Pik1p and/or its derived phosphoinositides are required for regulation of septation and cell separation. Pik1p may be involved in the transport, possibly via vesicular transport, of enzymes required for hydrolysis of the primary septum. It may be involved in signaling pathways that lead to the initiation of septation and to the cessation of the deposition of secondary septum material.

Thesis (M.S.)--The University of North Carolina at Greensboro, 2008. / Directed by Paul Steimle; submitted to the Dept. of Biology. Title from PDF t.p. (viewed Mar. 19, 2010). Includes bibliographical references (p. 30-31).

Dictyostelium DdINCENP is a chromosomal passenger protein associated with centromeres, the spindle midzone and poles during mitosis and the cleavage furrow during cytokinesis. Disruption of the single DdINCENP gene revealed important roles for this protein in mitosis and cytokinesis. DdINCENP null cells lack a robust spindle midzone and are hypersensitive to microtubule depolymerizing drugs suggesting that their spindles may not be stable. Furthermore DdCP224, a protein homologous to the microtubule-stabilizing protein TOGp/XMAP215, was absent from the spindle midzone of DdINCENP null cells. Overexpression of DdCP224 rescued the weak spindle midzone defect of DdINCENP null cells. While not required for the localization of the myosin II contractile ring and subsequent formation of a cleavage furrow, DdINCENP is important for the abscission of daughter cells at the end of cytokinesis. The localization of DdINCENP at the cleavage furrow is modulated by myosin II. Loss of myosin II restricted the localization of DdINCENP to a narrow zone at the cleavage furrow. Kif12, a homolog of mitotic kinesin like protein (MKLP), was essential for relocalization of DdINCENP from the central spindle to the cleavage furrow. Furthermore, Kif12 was also localized at the cortex of the cleavage furrow and its localization during cytokinesis closely resembled that of DdINCENP, suggesting a possible interaction between them. The correct localization of DdINCENP during cytokinesis also required its N-terminal sequence. DdINCENP1-500 was found at the cleavage furrow and interacted with the actin cytoskeleton. Domain analysis of DdINCENP also revealed that its DdINCENP1-500 was sufficient to rescue the weak spindle defect of DdINCENP null cells. / text

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.

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.

Sterols are lipid components of eukaryotic membranes. Alterations of membrane sterol composition perturb the execution of cell division, which in diverse eukaryotes can have severe consequences for development of the organism. Partitioning of the cytoplasm during cell division occurs at the final stage of cell division named cytokinesis. In somatic plant cells, cytokinesis is initiated by fusion of membrane vesicles in the plane of cell division resulting in a transient compartment termed the cell plate. Cell plate maturation relies on temporal and spatial orchestration of membrane fusion and endocytosis. Impaired vesicle fusion or defects in endocytosis result in cytokinetic defects. In Arabidopsis thaliana, the KNOLLE and DYNAMIN-RELATED PROTEIN 1A (DRP1A) contribute to cytokinesis. KNOLLE mediates fusion of vesicles at the plane of cell division while DRP1A appears to be involved in cell plate maturation through its role in clathrin-mediated endocytosis. This thesis shows that KNOLLE is specifically restricted to the cell division plane through sterol-dependent endocytosis that involves a clathrin- and DRP1A-mediated mechanism. Sterols affect internalization of KNOLLE through their role in lateral membrane organization by keeping diffusion of KNOLLE to lateral membranes in check via its endocytic removal. It is shown that the cell plate represents a high-lipid-order membrane domain that depends on the correct composition and the right concentration of sterols. Accumulation of DRP1A at the cell plate requires correct sterol concentration and composition similar to high-lipid order. Conversely, high-lipid-order at the cell plate relies on DRP1A activity suggesting a feedback between DRP1A function and lipid order establishment. Finally, it is shown that sterols are also present at the tonoplast of dividing and elongated root cells. Taken together, the results reveal that formation of the cell plate in Arabidopsis thaliana depends on an intricate interplay between cytokinetic vesicle fusion, sterol-dependent lateral membrane and high-lipid-order domain organization as well as endocytic machinery function.

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.