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.

Overly long centrioles and defective cell division upon excess of the SAS-4-related protein CPAP

Pierre Gönczy, Gregor Kohlmaier

The centrosome is the principal microtubule organizing center (MTOC) of animal cells. 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 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

2009

Mechanisms of centrosome separation in C. elegans

Alessandro De Simone

Centrosomes are the major microtubule organizing centers of animal cells. The two centrosomes present at the onset of mitosis must separate in a timely fashion along the nuclear envelope to ensure proper bipolar spindle assembly and thus genome stability. Microtubuleassociated motors of the kinesin-5 family are required for centrosome separation in several systems, but are partially redundant or entirely dispensable in others, where the minus-end directed motor dynein plays an important role. The mechanisms by which dynein powers centrosome separation are incompletely understood. Furthermore, the nature of the symmetry-breaking mechanisms that imbalance the forces acting on centrosomes to favor theirmovement away from each other are not known. We addressed these questions using a combination of 3D time-lapse microscopy, image processing and computational modeling to dissect centrosome separation in the polarized one-cell C. elegans embryo that entirely relies on dynein for this process. First, we have characterized the quantitative features of centrosome separation in the wild-type. Next, we compared centrosome separation between wild-type and mutant/RNAi conditions. Our analysis revealed that centrosome separation is powered by the combined action of dynein at the nuclear envelope and at the cell cortex. Moreover, we demonstrated that cortical dynein requires actomyosin contractility to separate centrosomes. These observations suggest that cortical dynein acts by harnessing anterior-directed actomyosin cortical flows initiated earlier in the cell cycle by the centrosomes themselves. To confirmthismodel, we successfully tested experimentally two of its key predictions, namely that dynein complexes flow toward the anterior together with the cortex and that the velocity of centrosome separation correlates with that of the flow of the nearby cortex. Taken together, these results demonstrate that centrosome separation is driven by nuclear and cortical dynein, where the latter acts by transmitting forces produced by the cortical actomyosin flow. To test whether this model is sufficient to explain centrosome separation, we developed a 3D computationalmodel of cytoskeleton dynamics. Indeed, predicted centrosome separation agrees quantitatively with the experimental observations in wild-type and mutant/RNAi conditions. Moreover, the qualitative predictions of the model are robust for parameter changes. Furthermore, computational simulations demonstrate that forces are intrinsically organized to move centrosomes away from each other without the need of any extrinsic symmetry-breaking mechanism. Indeed, in the case of nuclear dynein-driven separation, the position of centrosomes between the nuclear envelope and the cortex results in an asymmetric microtubule aster that leads to centrosome outward movement. In the case of cortical dynein, cortical flows are triggered by centrosomes and always move away from them, such that their forces are always directed to separate centrosomes. Therefore, this separation mechanism functions irrespective of the initial position of centrosomes along the cortex. In conclusion, in this thesis we uncover a novel organizing principle in which dynein, coupled with cell geometry and flow pattern, serves to robustly separate centrosomes and thus ensure genome stability.

EPFL2016

SAS-4 is essential for centrosome duplication in C elegans and is recruited to daughter centrioles once per cell cycle

Pierre Gönczy

The mechanisms governing centrosome duplication remain poorly understood. We identified a gene called sas-4 that is essential for this process in C. elegans. SAS-4 encodes a predicted coiled-coil protein that localizes to a tiny dot in the center of centrosomes throughout the cell cycle. FRAP experiments with GFP-SAS-4 transgenic embryos reveal that SAS-4 is recruited to the centrosome once per cell cycle, at the time of organelle duplication. Additional evidence indicates that SAS-4 is recruited to the daughter centriole or a closely associated structure. These findings identify SAS-4 recruitment as a key step in the centrosome duplication cycle.

2003

Mechanisms of centrosome separation in C. elegans

Alessandro De Simone

EPFL2016