Functional characterization of the mitotic kinesin-like protein Kif18A

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MAYR, Monika Isabelle, 2010. Functional characterization of the mitotic kinesin-like protein Kif18A

@phdthesis{Mayr2010Funct-7899, title={Functional characterization of the mitotic kinesin-like protein Kif18A}, year={2010}, author={Mayr, Monika Isabelle}, address={Konstanz}, school={Universität Konstanz} }

Mitosis, the process by which identical copies of the replicated genome are distributed to newly forming daughter cells, depends upon the action of the mitotic spindle, a protein machine that uses mirotubules and microtubule-based motor proteins to assemble itself and to segregate sister chromatids. The assembly and the functions of superfamily are fundamental for the structure and function of the mitotic spindle by regulating microtubule dynamic behaviour to ensure the fast capture of chromosomes and the alignment of chromosomes at the metaphase plate.<br /><br />Our studies demonstrate that the human kinesin Kif18A is a motile microtubule depolymerase essential for chromosome congression in mammalian tissue culture cells and thus identify the human kinesin Kif18A as a dual-functional kinesin. In vitro, Kif18A is a slow plus-end-directed kinesin that possesses microtubule depolymerizing activity. Notably, Kif18A depolymerizes longer microtubules more quickly than shorter ones. In vivo, Kif18A accumulates in mitosis where it localizes close to the plus ends of kinetochore microtubules. The depletion of Kif18A induces aberrantly long mitotic spindles and loss of tension across sister kinetochores, resulting in the activation of the Mad2-dependent spindle-assembly checkpoint and thus accounts for the observed prometaphase delay. Live-cell microscopy studies revealed that Kif18A-depleted cells fail to align chromosomes correctly at the metaphase plate resulting in an elongated phase of chromosome oscillations and thus in a delay of anaphase onset. Moreover, our studies indicate that key mitotic kinases are required for the observed mitosis-specific upshift in the electrophoretic mobility of Kif18A. In addition, the C-terminus of Kif18A is phosphorylated by mitotic kinases in vitro.<br /><br />Serine to alanine substitutions at specific sites indicate that these sites are critical for the observed mitotic upshift. Most importantly, the identified phosphorylation sites in vitro are target sites in Kif18A in vivo. To examine the consequence of phosphorylation events of Kif18A in vivo, stable cellines expressing either non-phosphorylatable or phosphorylationmimicking Kif18A versions were generated. Our studies focused thereby on potential phosphorylation sites identified in vitro. Moreover, these sites in Kif18A were conserved among different species. The ability of the different Kif18A variants to complement the loss of endogenous Kif18A was assayed by transfecting HeLa cells with siRNAs targeting the ORF region of endogenous Kif18A transcripts and by tetracycline induced expression of siRNA resistant GFP-Kif18AFL constructs. These studies revealed that HeLa cells expressing, instead of the endogenous protein, Kif18A with a mutation at the identified phosphorylation sites, entered anaphase after a slight delay compared to cells expressing GFP-Kif18AWT and were characterized by defects in chromosome oscillations. 2010 eng Mayr, Monika Isabelle deposit-license 2011-03-24T17:38:22Z Functional characterization of the mitotic kinesin-like protein Kif18A Mayr, Monika Isabelle application/pdf Funktionelle Untersuchung des mitotischen Kinesins Kif18A 2012-12-08T23:25:04Z

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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