Molecular Analysis of Centriole Assembly and Function

Centrioles perform two distinct functions in eukaryotic cells: 1) they recruit pericentriolar material to form centrosomes that organize the microtubule cytoskeleton, and 2) they template cilia, cellular projections that perform critical sensory and motile functions. Centrosome and cilia abnormalities have been linked to aneuploidy and tumorigenesis as well as developmental disorders including ciliopathies and microcephaly. Despite their relevance to human physiology and pathology, centrioles have remained poorly understood at the molecular level, largely due to the technical challenges posed by the small size of this cellular organelle. The goal of this proposal is to use a combination of biochemical, cell biological and genetic approaches in the nematode C. elegans to investigate the fundamental and conserved molecular mechanisms underlying centriole assembly and function. The proposed research is divided into three aims. The first aim focuses on centriole assembly and specifically the role of the centriolar components SAS-4, -5 and -6 in this process. Experiments proposed in this aim include a molecular characterization of these proteins, their interactions with each other, -tubulin, and a/ß-tubulin in formation of the nine-fold symmetric centriole structure. The aim further includes an examination of the in vivo consequences of disease-associated mutations in SAS-4 found in human patients. The second aim seeks to characterize the role of centrioles as centrosome organizers, by identifying components of the pericentriolar matrix that forms around centrioles and examining the dynamics of this process using a live imaging approach. These methods will further be used to study the involvement of microtubule motors in centrosome assembly as well as to identify factors responsible for centrosome disassembly. The final aim centers on the role of centrioles in initiating ciliogenesis, building on the identification of the hydrolethalus syndrome protein HYLS-1 as a molecular link between the centriole assembly machinery and ciliogenesis. Experiments in this aim include a delineation of the molecular events underlying the conversion of centrioles into basal bodies, focusing on proteins that, like HYLS-1, interact directly with the core centriole structure. The approaches briefly outlined above aim to capitalize on the advantages of C. elegans as an experimental model to further our understanding of centriole assembly and function in centrosome and cilia formation, fundamental cellular activities which are of key relevance to human development and disease. An important part of this work will be to apply our findings to vertebrate and clinical models, in collaboration with experts in the field.

Cells are the fundamental building block of all life on Earth. The human body is made up of 100 trillion cells, which arise from a single fertilized egg cell through a series of cell divisions followed by a complex pattern of interactions between the resulting daughter cells. Two cellular organelles play a particularly important role in organismal development: 1) the centrosome, which directs the assembly of the mitotic spindle coordinating the process of cell division and the segregation of the genetic material encoded on chromosomes, and 2) cilia, cellular projections that act as antennae in intercellular communication, thereby controlling the formation of tissues and organs. Both centrosomes and cilia form from the same template, the centrioles, tiny structures found deep within the cytoplasm of the cell. The goal of this START project was to use a combination of biochemical, cell biological and genetic approaches in model organisms, particularly the nematode worm C. elegans, to examine the molecular mechanisms underlying centriole assembly and function. This work was divided into three complementary aims. The first aim focused on centriole assembly and in particular the mechanisms underlying the cohesion of the pair of centrioles within each centrosome as well as the molecular basis of their remarkable stability. The second aim sought to characterize centriole function in centrosome assembly. Examining the dynamics of the assembly process in the C. elegans embryo revealed highly unusual properties of the centrosomal material which we are currently investigating by biochemical and biophysical means. We further used laser ablation to acutely remove centrioles in the context of the growing centrosome and thereby uncovered an essential role for these structures in later stages of cell division. Lastly, we studied the role of centrioles in cilium biogenesis. Here, we investigated the assembly of structures that form directly on the centriole template. We further took advantage of the naturally occurring degeneration of centrioles in the worm to define the contribution of centrioles to later stages of ciliogenesis. In surprising contrast to centrosomes, here centrioles appear only to kick-start the process of assembly, but play no direct part in later stages. Centrosome and cilium anomalies in humans have been linked to tumorigenesis and cancer, as well as developmental disorders including ciliopathies and microcephaly. The goal of this work was to investigate the fundamental features of these biomedically important cellular organelles. One of the follow up projects in the lab now also involves the study of cilium dysfunction in human patients, in collaboration with experts in the field.