Structural Engineering Principles of Centrosome Assembly by Super Resolution Microscopy Imaging

我的研究计划旨在通过分析其大分子组装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，它组织微管细胞骨架。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围绕中心粒的无定形电子致密物质。近中心粒物质在100多年前就被发现了，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能够以纳米精度确定蛋白质位置的超分辨率成像和分析策略，这种创新方法导致在中心粒附近的中心粒周围物质中发现了一种未知的支架，我们将其命名为相间-中心粒周围物质层。我的发现对科学知识的不同层次和领域产生了影响：1）它阐明了细胞功能的基本分子机制; 2）它概述了一种新的，广泛适用的研究细胞中大分子组装结构的策略; 3）它改变了教科书对细胞器的看法。我在该领域的认可已经通过邀请演讲（国家癌症研究所，国立卫生研究院），我关于新兴技术和方法的出版物（细胞生物学百科全书，细胞生物学方法），我的咨询活动赠款委员会（香港），评论（细胞生物学趋势）和许多合作。在这个NSERC计划中，我将建立在我最近的研究结果，以确定如何pericentriolar材料的形式和控制其结构。我们计划关注三个基本问题：*1）在体内，中心粒周围物质层是如何组织的？我们计划通过相关的超分辨率显微镜/电子显微镜方法，以纳米分辨率确定中心粒周围物质界面层已知组分的3D图。** 2)哪些是重建间期中心粒周围物质层的最基本成分？我们正在进行实验，在细胞和体外纯化的成分，以确定所需的最小机械重建的pericentriolar材料的相间层。**3）无中心体微管阵列中的中心粒周围物质组分的组织是什么？我们正在研究中心粒周围物质蛋白质如何改变其分子结构以在核膜上组织微管。

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.**