Structural Engineering Principles of Centrosome Assembly by Super Resolution Microscopy Imaging

我的研究项目旨在通过分析细胞的大分子组合来确定细胞功能的工程原理。我把注意力集中在中心体上，这是一种对人类发育至关重要的细胞器，负责组织微管细胞骨架。中心体由两个大结构组成：中心粒，一个由微管叶片包围的9倍对称圆柱形结构，以及中心粒周围物质，传统上被描述和理解为包围中心粒的无定形、电子密集的物质。心泡周围物质是在100多年前发现的，但在了解其组织方面没有取得重大进展。这种知识的缺乏部分是由于缺乏有效的方法来研究细胞内大分子组装的结构组织。我最近与其他科学家合作设计了一种超分辨率成像和分析策略，能够以纳米精度确定蛋白质的位置。这种创新的方法导致在中心粒附近的心泡周围材料中发现了一个未知的支架，我们将其命名为间相-心泡周围材料层。我的发现对科学知识的不同层面和领域产生了影响：1)它揭示了细胞功能的基本分子机制；2)概述了一种新的、广泛适用的研究细胞中大分子组装结构的策略；3)它改变了教科书上对细胞器的看法。

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.