Mechanisms underlying centriole morphogenesis

项目摘要/摘要 中心体的中心是微管组织中心，由两个中心粒组成。两个人 中心粒并不相等。两者中较年长的被称为母中心粒，在结构上与较年轻的不同， 女儿中心粒。母中心粒特有的结构赋予了初级核形成的独特能力 纤毛是一种细胞器，充当细胞的触角。因此，中心粒蛋白的缺陷可能会导致 人类的睫毛病，是由睫毛功能紊乱引起的疾病。尽管是脊椎动物的普遍特征 细胞、母子中心粒如何不同以及中心粒是如何构建的仍然是个谜。我揭开了 由CEP90、MNR和OFD1组成的蛋白质复合体(我已将其命名为DISCO，意为远端中心粒 复合体)中心粒形态发生所必需的。迪斯科舞曲成分的突变会导致Joubert和 口-指综合征，脑、面部和四肢发育障碍。通过研究这种新型的中心粒 复杂，我试图理解中心粒是如何构建的，以及它们是如何被重塑以支持纤毛的。 集合。使用扩展和结构照明显微镜(Ex-SIM)的创新组合，我将 定义这种复杂的成分是如何构成远端中心粒的，以及人类疾病与 突变破坏了这个亚室(目标1)。MNR和OFD1通过未知基因控制中心粒长度 机制。使用超分辨率成像和生化分析，我将通过 中心粒长度由MNR和OFD1确定(目标2)。CEP90和MNR也是 中心粒卫星，围绕中心体的鲜为人知的无膜颗粒。我发现 中心星卫星显示出相分离的特征。利用活体成像和体外生化 重建，我将检验CEP90，MNR和OFD1被分相运输到中心粒的假设- 分离中心粒卫星以支持纤毛发生(目标3)。在一位杰出的顾问的帮助下 委员会，我将在高级成像和生物物理技术方面进行培训，这将使我能够解决基本的 中心粒和纤毛是如何构成的问题。跨越指导阶段和独立阶段，这些 研究将阐明人类疾病相关蛋白如何构建和修饰中心粒以允许纤毛 生物发生，并为研究中心粒的作用的独立研究事业创造了坚实的基础 纤毛在人类发育和疾病中的作用。

PROJECT SUMMARY/ ABSTRACT The heart of the centrosome, the microtubule organizing center, is composed of two centrioles. The two centrioles are not equal. The older of the two, called the mother centriole, differs structurally from the younger, daughter centriole. Mother centriole-specific structures confer the unique capability to nucleate the primary cilium, an organelle that serves as the cell’s antenna. Consequently, defects in centriolar proteins can cause human ciliopathies, diseases caused by disrupted ciliary function. Despite being universal features of vertebrate cells, how the mother and daughter centrioles differ and how centrioles are built remain mysterious. I uncovered a complex of proteins comprised of CEP90, MNR and OFD1 (which I have named DISCO for DIStal Centriole cOmplex) required for proper centriole morphogenesis. Mutations in DISCO components cause Joubert and Orofaciodigital syndromes, disorders of brain, face and limb development. By studying this novel centriolar complex, I seek to understand how centrioles are built, and how they are remodeled to support cilium assembly. Using an innovative combination of expansion and structured illumination microscopy (Ex-SIM), I will define how components of this complex structure the distal centriole and how human disease-associated mutations disrupt this sub-compartment (Aim 1). MNR and OFD1 control centriole length by an unknown mechanism. Using super-resolved imaging and biochemical assays, I will uncover molecular mechanisms by which centriole length is established by MNR and OFD1 (Aim 2). CEP90 and MNR are also components of centriolar satellites, poorly understood membrane-less granules surrounding the centrosome. I have found that centriolar satellites display hallmarks of phase separation. Using live-imaging and in vitro biochemical reconstitution, I will test the hypothesis that CEP90, MNR and OFD1 are trafficked to the centriole by phase- separated centriolar satellites to support ciliogenesis (Aim 3). With the help of an outstanding advisory committee, I will train in advanced imaging and biophysical techniques that will allow me to address fundamental questions on how centrioles and cilia are built. Spanning both the mentored and independent phases, these studies will illuminate how human disease-associated proteins build and modify centrioles to allow cilium biogenesis, and create a strong foundation for an independent research career studying the role of centrioles and cilia in human development and disease.