CENTROSOME ASSEMBLY AND FUNCTION

中心体在细胞中起着几个基本的作用，包括成核和微管的组织、纺锤体组装和分子马达驱动的过程。它们还锚定控制着中心体和纺锤体功能的重要调控活动，并可能通过组织不能正确分离染色体的功能失调纺锤体来促进肿瘤的发生。我们研究的总体目标是了解中心体功能的分子基础。我们的总体策略是关注周心蛋白的功能，这是我六年前发现的一种中心体蛋白，结合了分子，生化和形态学的策略。在过去的预算期内，我们在了解心周功能方面取得了重大进展。我们确定，中心蛋白与微管成核蛋白（包括γ -微管蛋白）形成复合物，并与中心体上的γ -微管蛋白非常接近。我们发现周心蛋白直接与细胞质动力蛋白轻中间链1相互作用，动力蛋白介导周心蛋白和γ微管蛋白在中心体上的组装。此外，周心蛋白过表达会破坏动力蛋白的定位，导致纺锤体缺陷并产生非整倍体细胞。在与j.s cott博士（Vollum研究所）的合作中，我们发现心粒周围蛋白将激酶A锚定在中心体上，这种相互作用对纺锤体功能很重要。基于上述观察，我们制定了一个模型，其中中心蛋白在中心体中专门运输、锚定和组织重要的功能和调节活动。在接下来的预算期内，我们将继续使用体内方法和体外重建来研究中心蛋白和中心蛋白相互作用蛋白在中心体和纺锤体功能中的作用。在本研究的第一个目的中，我们将研究中心蛋白在微管成核复合物组装到中心体上的作用。我们将使用从爪蟾卵中提取的细胞质提取物来测试含有周心蛋白的蛋白质复合物在体外介导γ小管蛋白复合物在中心体上组装的能力。第二个目标是确定周心蛋白和动力蛋白轻中间链1之间相互作用的意义。具体来说，我们将使用一种显性负形式的中心蛋白来解耦中心蛋白-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。第三个目的是鉴定和表征其他中心粒蛋白相互作用的蛋白，并表征一种新的中心粒蛋白，与中心粒蛋白一样，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.