Centrosomin and centrosomes in cell division

描述(申请人提供)：中心粒和中心体是动物体内保守的细胞器，对细胞信号、细胞分裂和在分化细胞中的特殊作用至关重要，影响整个动物的器官功能。在中心粒/母体组装或功能中起作用的蛋白质突变，或影响从母体生长的纤毛的蛋白质突变，构成了发育和疾病基因种类不断扩大的病因学基础。其他中心体蛋白如CDK5RAP2、Pericentin、sas4和Aspm的基因突变是影响大脑和/或身体大小的相关综合征的根本原因，但对中心体功能与疾病原因之间的联系的了解尚不清楚。为了了解疾病或发育过程，我们必须首先了解受影响的蛋白质的功能。在这里，我们将重点放在中心体蛋白家族上。一个关键的突破来自于对果蝇中心体(CNN)的突变研究，表明它在有丝分裂中心体组装的早期过程中是必需的。中心体是动物细胞中主要的微管组织中心，在有丝分裂时微管有效地组装成双极纺锤体是必需的。中心体和中心粒蛋白的分子功能在很大程度上还没有确定下来；事实上，中心体“部分清单”仍在编制中。我们以果蝇为模型系统，研究了中心体蛋白(CNN)的分子功能及其影响的蛋白质和过程。通过这个模型，我们将经典的和分子遗传学方法与细胞生物学和生物化学结合起来，充分利用了这个模型提供的高效和丰富的工具箱。据估计，在果蝇中，70%的人类疾病基因与同源基因证实了它作为脊椎动物生物学模型的使用。然而，通过使用小鼠CNN家族成员CDK5RAP2的小鼠突变体，我们在这些系统之间架起了桥梁，并更好地理解了CDK5RAP2突变背后的病理，这些突变导致人类小头畸形。CNN包含两个保守的模块。N端附近的一个结构域调节中心小体的微管组装，而C末端的第二个结构域调节肌动蛋白的组织进入卵裂沟。第一个结构域的功能是将微管组装因子招募到中心体，而第二个结构域直接与中央皮质素(Centrocortin，Cen)结合，这是切割沟组装所需的一个新因子。在小鼠中，CDK5RAP2突变细胞失去了中心粒的结合和凝聚力，导致中心粒放大。在有丝分裂时，这些过剩的中心体诱导多极纺锤体组装，暗示CDK5RAP2在有丝分裂时中心体聚集中发挥作用。在CDK5RAP2突变细胞中，多个中心粒也模板多个初生纤毛。这些发现为更深入地理解中心体调控和中心体复制调控的关键过程铺平了道路。我们的具体目标是：1.确定CNN对MTOC的调控机制；2.确定Centrocortin在卵裂沟组装中的作用及其与CNN的协同作用；3.确定CDK5RAP2在小鼠体内的功能。与公共卫生相关：在中心粒和中心体起作用的基因突变会导致许多人类疾病，因为它们在如此多的发育和生理过程中具有重要意义。这些疾病包括多囊肾病、耳聋、脑积水、肥胖、小头畸形等。这项建议的目标是从分子和机制上了解中心粒和中心体，这些结构存在于苍蝇和小鼠的几乎每个细胞中，并识别常染色体隐性原发小头畸形(MCPH)的病理。

DESCRIPTION (provided by applicant): Centrioles and centrosomes are conserved organelles in animals that are essential for cell signaling, cell division, and for specialized roles in differentiated cells, impacting organ function in whole animals. Mutations in proteins that function in centriole/basal body assembly or function, or which impact the cilia that grow from basal bodies, comprise the etiological basis for an expanding category of developmental and disease genes. Mutations in genes for other centrosomal proteins like CDK5RAP2, Pericentrin, sas4 and Aspm, are the root cause of related syndromes that affect brain and/or body size, yet an understanding of the connection between function at the centrosome and the causes of disease are unclear. To understand the disease or developmental process, we must first understand the functions of the proteins affected. Here we focus on the Centrosomin family of proteins. A key breakthrough came with mutation studies of Drosophila Centrosomin (CNN), showing that it is required in an early process of mitotic centrosome assembly. Centrosomes are the major microtubule-organizing centers (MTOCs) in animal cells and are required for efficient assembly of microtubules into the bipolar spindle apparatus at mitosis. The molecular functions of centrosome and centriole proteins are largely uncharacterized; indeed, the centrosome "parts list" is still being compiled. We employ Drosophila as a model system to investigate the molecular function of Centrosomin (Cnn) and the proteins and processes it impacts. With this model we combine classical and molecular genetic approaches with cell biology and biochemistry, capitalizing on the efficiency and rich tool chest that this model affords. The estimated 70% of human disease genes with homologs in Drosophila validate its use as a model for vertebrate biology. Yet, by employing a mouse mutant for CDK5RAP2, a mouse CNN family member, we bridge these systems and achieve a closer understanding of the pathology that underlies mutations in CDK5RAP2, which cause microcephaly in humans. CNN contains two conserved modules. One domain, near the N-terminus, regulates microtubule assembly at centrosomes, while the second domain at the C-terminus regulates actin organization into cleavage furrows. The first domain functions to recruit a microtubule assembly factor to centrosomes, while the second domain binds directly to Centrocortin (Cen), a novel factor required for cleavage furrow assembly. In mice, CDK5RAP2 mutant cells lose centriole engagement and cohesion, inducing centriole amplification. At mitosis, these excess centrosomes induce multipolar spindle assembly, implicating a role for CDK5RAP2 in centrosome clustering at mitosis. The multiple centrioles also template multiple primary cilia in CDK5RAP2 mutant cells. These findings pave a path toward a deeper understanding of the key processes that centrosomes regulate and that govern the regulation of centrosome replication. Our specific aims are to: 1. Determine the mechanisms of MTOC regulation by CNN, 2. Define the role of Centrocortin and its cooperation with CNN in cleavage furrow assembly, and 3. Define the functions of CDK5RAP2 in the mouse. PUBLIC HEALTH RELEVANCE: Mutations in genes that function at centrioles and centrosomes cause a host of human disorders due to their importance in so many developmental and physiological processes. These ailments include polycystic kidney disease, deafness, hydrocephaly, obesity, microcephaly, and more. The goal of this proposal is to gain molecular and mechanistic understanding of centrioles and centrosomes, structures found in nearly every cell, in flies and mice and discern the pathology of autosomal recessive primary microcephaly (MCPH).