Dictyostelium discoideum RnoA Interprets cAMP Mediated Signals to Influence Actin Organization
Item
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Title
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Dictyostelium discoideum RnoA Interprets cAMP Mediated Signals to Influence Actin Organization
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Identifier
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d_2009_2013:36b4d737bc5c:10880
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identifier
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11126
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Creator
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Garcia, Rebecca,
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Contributor
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Derrick Brazill
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Date
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2011
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biochemistry | Cellular biology | Molecular biology
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Abstract
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Tight control of actin cytoskeletal dynamics is essential for proper cell function. ARNO, a guanine nucleotide exchange factor for Arf, is associated with actin cytoskeletal regulation but its exact role is unknown. To explore ARNO's role in this regulation and in actin mediated processes, the Dictyostelium discoideum homolog RnoA was examined. RnoA is involved in development, as the reduction of RnoA by antisense prolongs aggregation, delaying development. Also, RnoA overproduction arrests development at the mound stage. This arrest is rescued by the addition of wild type cells. In these chimeric mixtures, rnoA overexpressing cells (RnoA OE) preferentially sort to the stalk, suggesting RnoA plays a role in cell sorting. RnoA antisense and RnoA OE cells fail to stream during aggregation. Chemotaxis assays reveal these mutants do not chemotax toward cAMP, indicating RnoA is part of the cellular response to cAMP. This defect is specific to cAMP-directed chemotaxis, as both RnoA mutants effectively chemotax to folate and exhibit normal cell motility. The chemotactic defects of the RnoA mutants may be due to an impaired cAMP response as evidenced by altered cell polarity and F-actin polymerization after cAMP stimulation. RnoA OE cells have increased filopodia compared to wild type cells, implying altered F-actin localization. Thus, RnoA likely organizes F-actin during development. Phospholipase D (PLD), the enzyme responsible for phosphatidic acid production, and paxillin, a cytoskeletal adaptor protein, are also involved in actin cytoskeletal organization. Given their communal association with the actin cytoskeleton, we explored the interactions between RnoA and the D. discoideum PLD and paxillin homologs, PldB and PaxB, respectively. PaxB and PldB regulate calcium dependent and calcium independent cell-cell cohesion, respectively. Cells lacking PaxB are known to have reduced cell-substrate adhesion. We find that overexpression of PldB rescues the adhesion defect of paxB null cells, implying a genetic interaction between paxB and pldB. Co-immunoprecipitation studies suggest RnoA, PldB, and PaxB physically interact. This interaction does not depend on PaxB, PldB, or F-actin organization. Taken together, the results suggest that RnoA, in complex with PldB and PaxB, coordinates F-actin organization during actin mediated processes such as adhesion.
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Type
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dissertation
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Source
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2009_2013.csv
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degree
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Ph.D.
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Program
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Biochemistry
