Structural Engineering Principles of Centrosome Assembly by Super Resolution Microscopy Imaging

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My research program is aimed at determining the engineering principles of cell function through the analysis of its macromolecular assemblies. I focused my attention on the centrosome, an organelle critical for human development, which organizes the microtubule cytoskeleton. Centrosomes are composed of two large structures: the centriole, a 9-fold symmetric cylindrical structure encircled by microtubule blades, and the pericentriolar material, traditionally described and understood as an amorphous, electron-dense material that surrounds the centrioles. The pericentriolar material was discovered over 100 years ago, but there had been no significant advances in understanding its organization. This lack of knowledge is in part due to the shortage of effective methods to study the structural organization of macromolecular assemblies within the cell. I recently designed, in collaboration with other scientists, a super-resolution imaging and analysis strategy capable of determining the position of proteins with nanometer precision.This innovative approach led to the discovery of an unknown scaffold in the pericentriolar material in the proximity of the centriole, which we named interphase-pericentriolar material layer. My discovery had an impact on different levels and areas of scientific knowledge: 1) It shed light on a fundamental molecular mechanism for cell function; 2) It outlined a novel, broadly applicable strategy for studying the structure of macromolecular assemblies in cells; and 3) It changed the textbook view of a cellular organelle.***My recognition in the field is already evidenced by invitations to lecture (National Cancer Institute, National Institutes of Health), my publications on emerging technologies and methods (Encyclopedia in Cell Biology, Methods in Cell Biol.), my consulting activity to grants committees (Hong Kong), reviews (Trends in Cell Biology) and numerous collaborations. In this NSERC program, I will build on my recent findings to determine how the pericentriolar material form and control its structure. We plan to focus on three fundamental questions:***1) How is the pericentriolar material layer organized in vivo? We plan to determine the 3D map with nanometer resolution of known components of the pericentriolar material interphase layer by correlative super-resolution microscopy/electron microscopy methods.******2) Which are the minimal components to reconstitute the interphase pericentriolar material layer? We are performing experiments in cells and in vitro with purified components to identify the minimal machinery required to reconstitute the interphase layer of the pericentriolar material. ******3) What is the organization of the pericentriolar material components in acentrosomal microtubule arrays? We are examining how pericentriolar material proteins change their molecular architecture to organize microtubules at the nuclear envelope.**