Investigation of human centrosomes - with a special focus on the function of Cep152 and Cep192 in centriole duplication

Centrioles are symmetrical, barrel-shaped, microtubule-based structures, that serve as building platforms for the formation of centrosomes and cilia. In dividing cells, they recruit a matrix of proteins called pericentriolar material (PCM) to form the centrosome, the major microtubule-organizing centre in animal cells. In differentiating cells, centrioles also function as basal bodies for the formation of flagella and cilia. To ensure the proper segregation of centrioles during cell division, centriole duplication is tightly controlled and coordinated with the cell cycle. To date, several factors have been identified in human cells that are recruited in a consecutive fashion to nascent centrioles to template the outgrowth of one procentriole orthogonally to the pre-existing one during each cell cycle. In addition to structural components, these duplication factors include the kinase Plk4 (polo-like kinase 4), which is pivotal for the initiation of centriole duplication. Here, we have taken two approaches to investigate centrosomes in more detail: first, the human centrosomal proteins Cep192 and Cep152 were functionally characterized; second, the localization of key centrosomal and centriolar components was analyzed using super-resolution three-dimensional structured illumination microscopy (3D-SIM). 1. Previously, Cep192 and Cep152 had been identified as novel centrosomal proteins in a proteomic screen. Homologues of Cep152 in flies and zebrafish had been implicated in PCM recruitment and centrosome duplication, but Cep152 had not been investigated in humans. Likewise, the worm homologue of Cep192 also functions in PCM recruitment and centrosome duplication. However, in humans its role in centriole duplication remained controversial. Here, we have established that Cep152 is dispensable, whereas Cep192 is essential for PCM recruitment in mitotic cells. This functional difference is further illustrated by their differential subcellular localizations during interphase and mitosis. The stable centrosomal integration of Cep152 depended on Cep192, whereas Cep192 localized independently of Cep152. Furthermore, both Cep152 and Cep192 were required for proper centriole duplication, thereby clarifying the controversy about the implication of Cep192 in this process. We also show that Cep192 and Cep152 co-operate in the centriolar recruitment of the kinase Plk4. Concomitantly, centriole duplication was only inhibited to a similar extent as in Plk4- or Sas-6-depleted cells, if both Cep152 and Cep192 were depleted. In agreement, we have identified and characterized interactions of Plk4 with the N termini of both Cep152 and Cep192. Finally, not only the recruitment of Plk4, but also of other duplication factors such as CPAP and Sas-6 was impaired in Cep152- and/ or Cep192-depleted cells. We have also addressed the regulation of Cep152 and Cep192. Centrosomal levels of both proteins increased towards mitosis. Similarly, cytoplasmic Cep152 levels peaked when cells approached mitosis, whereas Cep192 levels were stable. Hence, we show that both Cep152 and Cep192 function as centriole duplication factors. Both proteins exert a partly redundant function and their co-operation orchestrates recruitment of Plk4 as well as other centriole duplication factors and thus canonical centriole duplication. 2. Using 3D-SIM we have analysed the spatial relationship of 18 centriole and PCM components of human centrosomes at different cell cycle stages. During mitosis, PCM proteins formed extended networks with interspersed gamma-Tubulin. In interphase, most proteins were arranged at specific and defined distances from the walls of centrioles, resulting in ring-like staining. Additionally, orientation of Cep152 with its C-terminus close to centriole walls and its N-terminus facing outwards was visualised using site-specific antibodies against either terminus of the protein. At the distal end of centrioles, appendage proteins formed rings with several density masses, usually with a multiplicity below that expected from the 9-fold symmetry of centrioles. Although Cep164 remained centriolar throughout the cell cycle, the number of discernible density masses was clearly reduced during mitosis. At the proximal end, Sas-6 formed a dot at the site of daughter centriole formation, consistent with its role in cartwheel formation. Plk4 and STIL co-localized with Sas-6, but the bulk of the cartwheel protein Cep135 was associated with mother centrioles. Remarkably, Plk4 formed a dot on the surface of the mother centriole even before Sas-6 staining became detectable, indicating that Plk4 constitutes an early marker for the site of nascent centriole formation.