The centrosome is the main microtubule organizing center (MTOC) in animal cells. It consists of a pair of centrioles surrounded by pericentriolar material (PCM) that nucleates and anchors microtubules. The binding of PCM to centrioles, PCM self-assembly and PCM transport by the microtubule-based motor dynein are the prerequisites for centrosome assembly, but the mechanisms of PCM self-assembly in interphase cells are poorly understood. Moreover, the relative importance of different molecular pathways of PCM assembly varies between different cell systems and different phases of the cell cycle. Previous studies have mostly focused on mitotic cells, but systematic investigations have not been performed in interphase cells. In addition, γ-tubulin ring complex (γ-TuRC) as template for microtubule assembly largely determines the function of MTOC, but the relative importance of different centrosomal components in microtubule nucleation and maintenance of microtubule networks has not been systematically investigated. Moreover, it is unclear whether γ-TuRC can nucleate microtubules and maintain microtubule arrays in mammalian cells in the absence of γ-TuRC adaptors. In this thesis, we address these questions. In addition to the PCM, the other major component of the centrosome are centrioles, microtubule-based organelles whose formation and elongation are regulated by a large number of specific proteins. In vitro reconstitution experiments showed that two centriole biogenesis factors, CPAP and CP110, cooperatively regulate microtubule plus-end growth in vitro, but the importance of their interaction for centriole formation has not been examined on cells. In this thesis, we investigated the role of the CPAP-CP110 interaction in regulating centriole elongation.
In Chapter 2, we reported that when centrioles are lost due to either depletion or inhibition of PLK4, self-clustering of PCM proteins is sufficient to form a compact acentriolar MTOC (caMTOC) and thus organize a dense radial microtubule array in interphase cells. We showed that such self-clustering of PCM is dynein-dependent and requires pericentrin, CDK5RAP2, ninein and γ-tubulin, but not CEP192, NEDD1 and CEP152. We found that the formation of caMTOC is sensitive to the non-centrosomal MTOC pathways such as AKAP450-dependent PCM recruitment to the Golgi apparatus and CAMSAP2-mediated stabilization of free microtubule minus ends. We also reproduced the findings of our cellular experiments using computer simulations.
In Chapter 3, we describe tools for comprehensive and systematic functional analysis of the complex regulatory pathways of microtubule nucleation and anchoring at mammalian centrosomes using a cellular model that simultaneously lacks multiple PCM components. We found that in cells lacking the non-centrosomal microtubule minus-end stabilization pathways, different γ-TuRC adaptors exert additive effects on microtubule nucleation and attachment at the centrosome, and knocking them out in interphase cells prevents γ-TuRC from generating a dense microtubule array, even though all positive regulators of microtubule dynamics, are still present.
In Chapter 4, we examined the importance of CPAP-CP110 interaction in cells by replacing wild-type CPAP with CPAP mutants that cannot bind CP110. Our data indicate that centrioles, albeit shorter ones, could still form in the presence of the CPAP mutant, demonstrating that the CPAP-CP110 interaction is not essential for centriole formation, but promotes centriole elongation.
