CENTROSOME ASSEMBLY AND FUNCTION

中心体在细胞中扮演着几个基本的角色，包括纺锤体组装的微管的成核和组织以及分子马达驱动的过程。它们还锚定了控制中心体和纺锤体功能的重要调控活动，并可能通过未能正确分离染色体的功能失调的纺锤体的组织来促进肿瘤的发生。我们研究的总体目标是了解中心体功能的分子基础。我们的总体策略是利用分子、生化和形态策略的组合，专注于周中心素的功能，这是我在六年前发现的一种中心体蛋白。在过去的预算期间，我们在理解近中心功能方面取得了重大进展。我们确定周中心素与微管核蛋白形成复合体，包括伽马微管蛋白，并在中心体与伽马微管蛋白紧密相连。我们发现胞质动力蛋白轻中间链1直接与胞质动力蛋白轻中间链相互作用，动力蛋白介导胞质动力蛋白和γ微管蛋白在中心体上的组装。此外，外周蛋白的过度表达扰乱了动力蛋白的定位，导致纺锤体缺陷，并创造了非整倍体细胞。在与Vollum Inst的J.Scott博士的合作中，我们证明了外周中心素将激酶A锚定在中心体上，并且这种相互作用对纺锤体的功能很重要。在上述观察的基础上，我们已经建立了一个模型，在该模型中，周丝蛋白专门运输、锚定和组织中心体上的重要功能和调节活动。在下一个预算阶段，我们将继续研究周中心素和周中心素相互作用蛋白在中心体和纺锤体功能中的作用，采用体内和体外重建的方法。在这项提议的第一个目标中，我们将研究周中心素在将微管核复合体组装到中心体上的作用。我们将使用非洲爪哇卵的细胞质提取物来测试含有周丝蛋白的蛋白质复合体在体外介导伽马微管蛋白复合体组装到中心体上的能力。第二个目标是确定周着丝点和动力蛋白轻中间链相互作用的意义。具体地说，我们将使用周着丝点蛋白的一种显性否定形式来解偶联着丝点蛋白-动力蛋白轻中间链相互作用，并研究这种相互作用在中心体组装和纺锤体组织中的作用。第三个目标是鉴定和鉴定其他与胞外凝集素相互作用的蛋白，并鉴定一种新的中心粒蛋白，它和胞外凝集素一样，是用自身免疫血清鉴定的。

Centrosomes play several fundamental roles in the cell including the nucleation and organization of microtubules for spindle assembly and molecular motor-driven processes. They also anchor important regulatory activities that control centrosome and spindle function and may contribute to tumorigenesis through the organization of dysfunctional spindles that fail to segregate chromosomes properly. The overall objective of our research is to understand the molecular basis of centrosome function. Our general strategy is to focus on the function of pericentrin, a centrosome protein I identified in six years ago, using a combination of molecular, biochemical and morphological strategies. Over the past budget period, we have made significant progress in understanding pericentrin function. We determined that pericentrin forms a complex with microtubule nucleating proteins including gamma tubulin and is in close proximity with gamma tubulin at the centrosome. We found that pericentrin interacts directly with cytoplasmic dynein light intermediate chain 1 and that dynein mediates assembly of pericentrin and gamma tubulin onto centrosomes. Moreover, pericentrin overexpression disrupts dynein localization, causes spindle defects and creates aneuploid cells. In collaboration with Dr. J. Scott (Vollum Inst.), we showed that pericentrin anchors kinase A to centrosomes and that this interaction is important for spindle function. Based on the observations outlined above, we have formulated a model in which pericentrin serves to specifically transport, anchor and organize important functional and regulatory activities at the centrosome. Over the next budget period we will continue to study the role of pericentrin and pericentrin- interacting proteins in centrosome and spindle function using both in vivo approaches and in vitro reconstitution. In the first aim of this proposal we will investigate the role of pericentrin in the assembly of microtubule nucleating complexes onto centrosomes. We will use cytoplasmic extracts prepared from Xenopus eggs to test the ability of protein complexes containing pericentrin to mediate the assembly of gamma tubulin complexes onto centrosomes in vitro. The second objective is to determine the significance of the interaction between pericentrin and dynein light intermediate chain 1. Specifically, we will use a dominant negative form of pericentrin to uncouple the pericentrin-dynein light intermediate chain interaction and examine the role of this interaction in centrosome assembly and spindle organization. The third aim is to identify and characterize other pericentrin-interacting proteins and to characterize a novel centriole protein which, like pericentrin, was identified using autoimmune sera.