CENTROSOME ASSEMBLY AND FUNCTION

中心体在细胞中发挥着几种基本作用，包括纺锤体组装和分子马达驱动过程中微管的成核和组织。 它们还锚控制中心体和纺锤体功能的重要调节活动，并且可能通过不能正确分离染色体的功能失调的纺锤体的组织而促成肿瘤发生。 我们研究的总体目标是了解中心体功能的分子基础。 我们的总体策略是集中在pericentrin的功能，一个中心体蛋白，我发现在六年前，使用分子，生物化学和形态学的策略相结合。 在过去的预算期间，我们在了解中心周围蛋白功能方面取得了重大进展。 我们确定，pericentrin形成一个复杂的微管成核蛋白，包括γ微管蛋白，并在中心体与γ微管蛋白密切相关。 我们发现，pericentrin直接与细胞质动力蛋白轻中间链1和动力蛋白介导的组装pericentrin和γ微管蛋白到中心体。 此外，围中心蛋白过表达破坏动力蛋白定位，导致纺锤体缺陷并产生非整倍体细胞。 与J. Scott博士（Vollum Inst.）合作，我们发现中心周蛋白将激酶A锚定在中心体上，这种相互作用对纺锤体的功能很重要。基于上述观察，我们建立了一个模型，其中中心周蛋白用于特异性运输、锚和组织中心体的重要功能和调节活动。 在下一个预算期间，我们将继续使用体内方法和体外重建来研究中心周围蛋白和中心周围蛋白相互作用蛋白在中心体和纺锤体功能中的作用。 在这个建议的第一个目标，我们将调查的作用，pericentrin在组装的微管成核复合物到中心体。 我们将使用从非洲爪蟾卵制备的细胞质提取物来测试含有中心周围蛋白的蛋白复合物在体外介导γ微管蛋白复合物组装到中心体上的能力。 第二个目标是确定的意义之间的相互作用的pericentrin和动力蛋白轻中间链1。 具体来说，我们将使用一个显性负形式的pericentrin解偶联的pericentrin动力蛋白轻中间链的相互作用，并检查这种相互作用中的作用，中心体组装和纺锤体组织。 第三个目的是确定和表征其他的围中心蛋白相互作用的蛋白质，并表征一种新的中心粒蛋白质，如围中心蛋白，被确定使用自身免疫血清。

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.