Centrosomes are microtubule nucleating and organizing organelles that directly affect the interphase microtubule array, mitotic spindles and cilia. Despite their significance in development and linkage to diseases such as cancer, ciliopathies and birth defects, fundamental aspects of centrosome function and duplication remain poorly understood. For example, some proteins within the organelle act as barriers to its duplication while others inhibit the assembly of cilia, and, thus, must be eliminated at specific points in the cell cycle to enable the step-wise progression of these events. At present, it is assumed that diffusion accounts for the exchange of most centrosomal proteins. However, trafficking of centrosomal proteins toward or away from centrosomes is a plausible but relatively unexplored mechanism for controlling centrosome duplication and ciliogenesis. Previously, we showed that the endocytic regulatory protein, EHD1, is a regulator of ciliogenesis by mediating the fusion of distal appendage vesicles on centrosomes to form the ciliary vesicle. Our data support an additional role for EHD1 in controlling the centrosome duplication cycle, by promoting the redistribution of Cep215, CP110 and pericentrin (PCNT) from centrosomes to the spindle midbody during mitosis, effectively removing these protein barriers of centrosome duplication. Our findings also implicate several EHD1-interacting partners, including the retromer and MICAL-L1. Remarkably, we now find that MICAL-L1 is anchored to the centrosome/centrioles by binding to tubulins, highlighting the possibility that it may serve as a recruiter of EHD1 to the centrosome. The objective of this application is to determine whether vesicular trafficking is a novel fundamental regulator of the centrosome duplication cycle and cilia formation. Our central hypothesis is that the EHD1/MICAL-L1/retromer recycling complex promotes a vesicular transport mechanism to strip specific regulatory proteins from centrosomes, thereby enabling key steps in centrosome duplication and ciliogenesis.
Our specific aims are: 1.) Elucidate the mechanism by which endocytic vesicles remove centriolar and PCM proteins from centrosomes. We hypothesize that EHD1 (present of the cytoplasmic face of endocytic vesicles) docks with the distal appendages of centrioles, potentially via MICAL-L1, interacts with centriolar protein CP110 and PCM proteins Cep215 and PCNT, and facilitates the redistribution of these regulatory proteins to the spindle midbody as vesicular cargo. 2.) Interrogate the mechanisms by which EHD1 and its endocytic interaction partners regulate ciliogenesis. We will test the hypothesis that a EHD1/MICAL-L1/retromer complex coordinate the steps leading to ciliogenesis. Impact: Understanding the mechanisms of these processes forges a novel link between endocytic trafficking and centrosome biology, and will guide future studies to explore the etiology of centrosome dysfunction during tumorigenesis and ciliopathy.
Despite its significance during development and the direct linkage between the centrosome and numerous diseases (including cancer and ciliopathies), key aspects of centrosome function and duplication remain poorly understood. The knowledge to be gained from undertaking this proposal will lead to a significantly enhanced understanding of the mechanisms by which centrosomes duplicate and cilia are generated, and will have an important bearing on health and disease.
