Browsing by Subject "Cell Division"
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Item Determinants Influencing Polar Flagellar Biosynthesis and Cell Division in Campylobacter jejuni(2011-08-18) Balaban, Murat; Hendrixson, David R.Campylobacter jejuni is a worldwide leading cause of bacterial gastrointestinal disease. The natural habitat of this organism is the gastrointestinal tracts of warm-blooded animals, especially poultry, where the bacterium promotoes a harmless commensal colonization. The abundance of C. jejuni in poultry creates a risk for food-borne infections to human populations. Flagellar motility by C. jejuni is required to colonize both human and animal hosts. For motility, C. jejuni produces amphitrichous flagella, resulting in the formation of a single flagellum at both poles. This work explored factors that regulate numerical and spatial parameters for amphitrichious flagellation. Two factors that have been identified to control flagellar placement and numbers in polarly-flagellated bacteria are the FlhF GTPase and the FlhG ATPase. FlhF has been shown to be required for regulation of flagellar gene expression and flagellar placement in some Pseudomonas and Vibrio species. Characterization of FlhF in C. jejuni was accomplished by creating point mutants in C-terminal GTPase domain of FlhF to decrease its GTPase activity. GTPase mutants, unlike mutants that lack FlhF, did not have a significant reduction in sigma54-dependent flagellar gene expression. Instead, a significant proportion of the population produced flagella at lateral sites or produced multiple flagella at a pole, whereas wild-type bacteria produced single polar flagella. Further experiments suggested that FlhF functions downstream of the FlgSR-flagellar export apparatus (FEA) pathway to activate sigma54-dependent flagellar gene expression. Thus, our data suggested that FlhF and its GTPase activity are required for distinct processes in flagellar gene regulation. FlhG has been shown to control flagellar numbers in Pseudomonas and Vibrio species. We examined flhG mutants and confirmed that FlhG regulates flagellar numbers. C. jejuni flhG mutants also demonstrated a minicell phenotype, which is the result of division erroneously occurring at polar regions. Further examination revealed that FlhG and the flagellar base components compose a novel division inhibition system to spatially prevent polar division and encourage septation at the cellular midpoint for symmetrical division. This work greatly extends our understanding of factors that govern spatial and numerical patterns of polar flagellation and has identified an unprecedented system to spatially regulate division in bacteria.Item Insulin-Like Growth Factor-Binding Protein 2 Supports Hematopoietic Stem Cell Expansion: From In Vitro to In Vivo(2011-08-10) Huynh, Hoang Dinh; Zhang, Chengcheng "Alec"Successful hematopoietic stem cell (HSC) transplantation is often limited by the numbers of HSCs, and robust methods to expand HSCs ex vivo are needed. We previously showed that angiopoietin-like proteins (Angptls), a group of growth factors isolated from a fetal liver HSC supportive cell population, improved ex vivo expansion of HSCs. Here we demonstrate that insulin-like growth factor binding protein 2 (IGFBP2), secreted by a tumorigenic cell line, also enhanced ex vivo expansion of mouse HSCs. As measured by competitive repopulation analyses, there was a 48-fold increase in numbers of long-term repopulating mouse HSCs after 21 days of culture. This is the first demonstration that IGFBP2 stimulates expansion or proliferation of murine stem cells. Our finding also suggests that certain cancer cells synthesize proteins that can stimulate HSC expansion. To further study the function of IGFBP2 in vivo, we showed that IGFBP2-null mice have fewer HSCs than wild-type mice. While IGFBP2 has little cell-autonomous effect on HSC function, we found decreased in vivo repopulation of HSCs in primary and secondary transplanted IGFBP2-null recipients. Importantly, bone marrow stromal cells that are deficient for IGFBP2 have significantly decreased ability to support the expansion of repopulating HSCs. To investigate the mechanism by which IGFBP2 supports HSC activity, we demonstrated that HSCs in IGFBP2-null mice had decreased survival and cycling, downregulated expression of anti-apoptotic factor Bcl-2, and upregulated expression of cell cycle inhibitors p21, p16, p19, p57, and PTEN. Moreover, we found that the C-terminus, but not the RGD domain, of extrinsic IGFBP2 was essential for support of HSC activity. Defective signaling of the IGF type I receptor did not rescue the decreased repopulation of HSCs in IGFBP2-null recipients, suggesting that the environmental effect of IGFBP2 on HSCs is independent of IGF-IR mediated signaling. Therefore, as an environmental factor, IGFBP2 supports the survival and cycling of HSCs.Item Studies of Aurora and Polo Kinases During Cell Division in C. elegans(2005-04-29) Rogers, Eric Jason; Lin, RueylingAccurate chromosome segregation during cell division requires the precisely regulated release of chromosome cohesion. In mitosis, sister chromatids are linked by chromosome cohesion until the proteolysis of the cohesion Scc1 by separase triggers the separation of sister chromatids at anaphase. Chromosome dynamics during meiosis are more complex, as homologous chromosomes separate in anaphase I, whereas sister chromatids remain attached until anaphase II. In meiosis, separase must cleave the cohesin REC-8 in a stepwise manner to separate homologs in meiosis I and then sister chromatids in meiosis II. However, the mechanisms regulating the selective and sequential release of meiotic chromosome cohesion are unclear. Using C. elegans, we investigated the roles of Aurora and Polo kinases during the release of meiotic chromosome cohesion. We found that the Aurora B kinase AIR-2 is localized to sub-chromosomal regions representing the last points of contact between homologous chromosomes in meiosis I and between sister chromatids in meiosis II. Depletion of AIR-2 by RNA interference (RNAi) prevented both chromosome separation and REC-8 removal during meiosis. We showed AIR-2 phosphorylated REC-8 at a major amino acid in vitro (T625). The depletion of two phosphatases, GSP-1 and GSP-2, altered the localization pattern of AIR-2, such that AIR-2 is detected throughout the chromosome. Concurrently, there was a chromosome-wide reduction in REC-8 and sister chromatids precociously separated at anaphase I. We propose that AIR-2 promotes the selective release of meiotic chromosome cohesion via the phosphorylation of REC-8 at specific chromosomal locations and that GSP-1/2 antagonize AIR-2 activity. We also described that the Polo-like kinase PLK-1 is required for the release of meiotic chromosome cohesion during meiosis II. Depletion of PLK-1 by RNAi did not block the separation of homologous chromosomes, but the resulting dyads fail to separate during meiosis II. Furthermore, in plk-1(RNAi) embryos, REC-8 was not removed from these dyads. PLK-1 was capable of phosphorylating REC-8 in vitro. The gsp-1/2(RNAi) phenotype of precocious loss of REC-8 at anaphase I was suppressed by the simultaneous inhibition of PLK-1. We propose PLK-1 regulates the second phase of meiotic chromosome cohesion release. In summary, we propose that both Aurora B and Polo kinases phosphorylate REC-8 in order to regulate the selective and sequential release of chromosome cohesion during meiosis in C. elegans.Item [UT Southwestern Medical Center News](2009-09-01) McKenzie, Aline