Functional characterization of the SAS-4-related protein CPAP in centrosome biology of human cells

The centrosome is an organelle that resides at the center of most animal cells and comprises two microtubule-based centriole cylinders surrounded by pericentriolar material (PCM). The centrosome plays a fundamental role for nucleating and organizing a radial array of cytoplasmic microtubules during interphase and promoting the assembly of the mitotic spindle during mitosis. In proliferating cells, the centrosome duplicates once per cell cycle, a process that involves notably the formation of one procentriole at the base of each centriole. The procentriole then grows until it reaches the same size of its associated centriole. The formation of supernumerary centrosomes causes severe problems, since this can lead to multipolar spindle formation, chromosome missegregation and genomic instability, which are hallmarks of cancer cells. The mechanisms regulating centrosome duplication are still incompletely understood. We have studied the role of the centrosomal P4.1-associated protein (CPAP) in human cells. We found that this protein is required for efficient growth of cytoplasmic microtubules from centrosomes as well as for centrosome duplication. There, CPAP is required at an early step during procentriole assembly, after the incorporation of HsSAS-6 but before that of Centrin. Importantly, we found also that the overexpression of CPAP leads to abnormal elongation of procentrioles and centrioles, indicating that the levels of CPAP determine centriole length. Excess CPAP levels furthermore lead to the formation of multiple procentrioles along overly elongated centrioles, multipolar spindle formation and cell division errors. Therefore, proper regulation of proteins such as CPAP that set centriole length contributes to ensure genome integrity. Overall, we gained important insights into the functions of CPAP at the centrosome and identified a novel control mechanism of centriole length. This will be relevant for a better understanding of how centrosomes function in the progression of cancer and other diseases

Spindle positioning in human cells relies on proper centriole formation and on the microcephaly proteins CPAP and STIL

Patients with MCPH (autosomal recessive primary microcephaly) exhibit impaired brain development, presumably due to the compromised function of neuronal progenitors. Seven MCPH loci have been identified, including one that encodes centrosome protein 4.1 associated protein (CPAP; also known as centromere protein J, CENPJ). CPAP is a large coiled-coil protein enriched at the centrosome, a structure that comprises two centrioles and surrounding pericentriolar material (PCM). CPAP depletion impairs centriole formation, whereas CPAP overexpression results in overly long centrioles. The mechanisms by which CPAP MCPH patient mutations affect brain development are not clear. Here, we identify CPAP protein domains crucial for its centriolar localization, as well as for the elongation and the formation of centrioles. Furthermore, we demonstrate that conditions that resemble CPAP MCPH patient mutations compromise centriole formation in tissue culture cells. Using adhesive micropatterns, we reveal that such defects correlate with a randomization of spindle position. Moreover, we demonstrate that the MCPH protein SCL/TAL1 interrupting locus (STIL) is also essential for centriole formation and for proper spindle position. Our findings are compatible with the notion that mutations in CPAP and STIL cause MCPH because of aberrant spindle positioning in progenitor cells during brain development

Overly Long Centrioles and Defective Cell Division upon Excess of the SAS-4-Related Prootein CPAP

SummaryThe centrosome is the principal microtubule organizing center (MTOC) of animal cells [1]. Accurate centrosome duplication is fundamental for genome integrity and entails the formation of one procentriole next to each existing centriole, once per cell cycle. The procentriole then elongates to eventually reach the same size as the centriole. The mechanisms that govern elongation of the centriolar cylinder and their potential relevance for cell division are not known. Here, we show that the SAS-4-related protein CPAP [2] is required for centrosome duplication in cycling human cells. Furthermore, we demonstrate that CPAP overexpression results in the formation of abnormally long centrioles. This also promotes formation of more than one procentriole in the vicinity of such overly long centrioles, eventually resulting in the presence of supernumerary MTOCs. This in turn leads to multipolar spindle assembly and cytokinesis defects. Overall, our findings suggest that centriole length must be carefully regulated to restrict procentriole number and thus ensure accurate cell division