Our lab is interested in understanding the fundamentals of centrosome biogenesis.
We aim to uncovering the mechanisms that control the centriole duplication and centrosome maturation cycles. Through our own genome-wide RNAi screen and from other published screens, we now have a large number of candidate genes that play critical roles in these two cycles. What is lacking in the field is a true understanding of these proteins functions. We have completed a yeast-2-hybrid screen to identify the interactions between 22 core centrosome proteins, identifying 220 novel interactions. Over the past year, we have used the information from this screen to investigate the regulation and function of two critical centrosome scaffolding proteins Asterless (Asl, ortholog of human Cep152) and PLP (ortholog of human Pericentrin). Genetic data in Drosophila and human cells reveal a critical role for Asl in centriole duplication and a direct link to human microcephaly. Based on its localization within centrosomes, Asl was proposed to be a scaffold protein that anchors PCM proteins to the wall of centrioles. Our Y2H revealed that Asl forms a large number of interactions with centriole, PCM and regulatory proteins, supporting its role as a scaffold needed to bridge the PCM with centrioles. Testing this hypothesis was not trivial because Asl is required for centriole duplication, and therefore, loss of Asl leads to loss of the centriole pool needed for analysis. To overcome this, we took advantage of the fact that Asl is not required for maintaining centrioles once they are built. We perform an assay we call the remnant centriole assay to investigate Asl-free centrioles in zygotic asl mutant animals. In this assay we screen adult testes for centrioles that were built during embryogenesis using maternally supplied Asl. This assay is quite challenging as there are only 5-10 centriole in each testis, where normally there are thousands. Once found, these centrioles were imaged using either spinning disk confocal or structured illumination microscopy (SIM). Both our fixed and live cell analysis of these Asl-free centrioles revealed two important results: First, all the PCM proteins were recruited to asl mutant mitotic centrioles and were capable of assembling robust MT asters that contribute to forming a mitotic spindle. This result disproves our original hypothesis and the model that Asl acts as a bridge protein needed for PCM docking onto the centriole. The second major finding was that asl mutant centrioles are 5-times longer than WT, allowing us to formulate a new hypothesis for Asl function that it normally acts to limit centriole length. Using our list of Asl interactors and mutant analysis, we found that cep97 manifest the exact same centriole length defect as asl mutants. Interestingly, loss of asl or cep97 produces long centrioles in the male germline. Thus, Asl likely recruits Cep97 to limit stem cell centriole length. Importantly, these long centrioles lose distal end characteristics and render them incapable of nucleating sperm flagella. These results indicate that stem cells have adopted a specialized centriole length control mechanism;further supporting the idea that centrosome protein function can be highly tailored for each cell type. A manuscript detailing these results is under review. We have also taken advantage of our Y2H screen to investigate the regulation of the protein PLP in centrosome maturation. Based on our Y2H interactions and previous work in yeast and human cultured cells, we hypothesized that Calmodulin (CaM) is a critical regulator of PLP. We identified and disrupted the CaM binding sites within PLP, then introduced these alleles into animals lacking endogenous PLP to determine the importance of the PLP-CaM interaction. Our results show that the CaM interaction is critical for PLP function in mitosis, but dispensable for its role as a negative regulator in interphase. Our data also revealed that the PLP-CaM interaction is important for basal-body function in neurons and not in sperm. We have recently published these findings in MBoC. This work highlights our efforts and the significance of disrupting a single protein-protein interaction via point mutations. Worth noting is that these discoveries would not have been uncovered without our animal model and powerful genetic tools.
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