Vardanyan, Nelli (2009) Functional genomics of cartwheel proteins: Bld10 localizes to parental basal bodies and forms a protein complex. PhD thesis, Universität zu Köln.
In Chlamydomonas reinhardtii basal body formation occurs in a similar manner to centriole formation. Therefore, this organism provides an excellent model system for the study of the basal bodies/centrioles. The cartwheel with its ninefold symmetry and characteristic hub and spoke structure can be observed at the onset of basal body assembly. It is a highly conserved structure that determines the ninefold symmetry of centrioles/basal bodies. Although this structure has fascinated many scientists until now little is known about the cartwheel composition and function. To obtain knowledge about the cartwheel structural composition two key approaches were employed in this study: an isolation and purification approach and an affinity-based approach. It is crucial to emphasize that to date no cartwheel structure has been purified from any eukaryote. To address this question, an attempt was made to purify the cartwheel structure biochemically by isolating the probasal bodies, which is predominantly comprised of cartwheels. It is important to stress the significance of the biochemical purification of the cartwheel structure as a novel concept, since it can provide a more direct way to study the whole cartwheel. To develop this enabling strategy three main parameters were tested: suitable Chlamydomonas cell wall deficient mutants, buffers and detergents. Despite progress made, isolation of the probasal bodies was not achieved. Bld10p (175 kDa) is the first protein known to be localized to the cartwheel. By generating antibodies against this protein I aimed on one hand to identify the interacting proteins of Bld10 and on the other hand to have a closer look at the Bld10 localization. Therefore, polyclonal antibodies (against the C-terminal, the N-terminal and middle part) were raised in rabbits. Colocalization of Bld10 together with prominent cytoskeleton proteins such as centrin and tubulin helped to better understand the localization of the Bld10. The present study reports two key findings. First, it has been determined by employing BN-PAGE followed by Western blotting that Bld10p forms a complex with molecular weight of about 600 kDa. Second, the immunolocalization data reveal Bld10p localization to parental basal bodies rather than to probasal bodies. Since the probasal bodies consist primarily of cartwheel we question the role of the Bld10 as a pivotal structural protein of the cartwheel.
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