Ankara : The Department of Molecular Biology and Genetics and the Graduate School of Engineering and Science of Bilkent Univ., 2013.Thesis (Ph. D.) -- Bilkent University, 2013.Includes bibliographical references leaves 114-124.Cytokinetic abscission is the cellular process leading to physical separation of two
postmitotic sister cells by severing the intercellular bridge. During cell division several
functional complexes accumulate at the bridge connecting the two sister cells. The most
noticeable structural component of the intercellular bridge is a transient organelle termed
as midbody. This novel organelle is localized at a central region, which marks the site of
cytokinetic abscission. Despite its major role in completion of cell divison, our
understanding of spatiotemporal regulation of midbody assembly is incomplete. In this
thesis work, we first characterizated the coiled-coil domain-containing protein-124
(Ccdc124), a eukaryotic protein conserved from fungi-to-man, at the molecular level.
We identified that at the sub-cellular level Ccdc124 is localized at centrosomes and the
midbody depending on stages of the cell cycle. In interphase cells, as well as in mitosis,
the protein is localized to centrosomes. However at later stages of cytokinesis (lateanaphase/
telophase) Ccdc124 translocates to the midbody. Knockdown of Ccdc124 in
human HeLa cells leads to accumulation of enlarged and multinucleated cells; however,
centrosome maturation was not affected. Similarly, in preliminary in vivo assays
involving down-regulation of Ccdc124-homologue in zebra fish early embryos, we
observed multinuclear embryonic cells. Furthermore, we have validated a previously
observed in vitro interaction in our laboratory between Ccdc124 and the Ras guanine
nucleotide exchange factor 1B (RasGEF1B) by co-immunoprecipitation assays. As
RasGEF1B is strictly a Rap2 GTP-binding protein specific nucleotide exchange factor,
this result has suggested a possible involvement of Rap2 in cytokinesis related events.
Thus, subsequently, we assessed the sub-cellular localization of Rap2 in synchronized
cells during cytokinesis. We found that even though it does not play a role in cell
division, Rap2 is localized to the midbody. This result establishes a functional link
between cytokinesis and activation of localized Rap2 signaling at the midbody. Data
presented in this thesis work indicate that Ccdc124 is a novel factor operating both for
proper progression of late cytokinetic stages in eukaryotes, and for establishment of
Rap2 signaling dependent cellular functions proximal to the abscission site.Akıllılar, Pelin TelkoparanPh.D
