Functional properties of centrosomes in somatic cells

描述（由申请人提供）：本研究的主要目的是阐明中心体在脊椎动物体细胞中的功能，以及实现这些功能的分子机制。为了实现这些目标，我们使用了一种独特的方法，其中中心体（用荧光蛋白标记）被激光微束消融。这种方法使我们能够设计缺乏中心体的细胞，并将它们的行为与具有这种细胞器的基因相同的细胞进行比较。此外，我们将激光显微手术与siRNA和细胞渗透性化合物灭活单个蛋白质结合起来。这种协同方法提供了其他方法无法获得的新信息。我们目前的研究主要集中在两个方面。首先，我们正在测试一个假设，即在有丝分裂纺锤体组装过程中，中心体和中心体独立机制之间的不适当平衡导致染色体不稳定。我们通过高分辨率显微镜跟踪人类癌细胞中单个染色体的行为，并研究当中心体被激光消融时这种行为是如何变化的（目的1）。我们还研究了染色体行为如何响应与中心体无关的微管组织（TPX2和HURP）有关的关键分子的扰动或错误染色体取向的纠正（Aurora B和kinesin 13s） （Aim 2）。最后，我们使用激光显微手术、细胞渗透性化学抑制剂和siRNA来研究“中心粒脱离”的分子和结构要求，“中心粒脱离”是中心体周期中的一个关键事件，必须适当控制以防止中心粒在同一细胞周期中重复重复（目的3）。我们的观察表明，子中心粒在达到一定的成熟水平之前不能脱离。因此，中心粒脱离的控制涉及一个以前被忽视的中心体固有的机制。我们目前正在研究负责前戊烯醇成熟的分子机制。

DESCRIPTION (provided by applicant): The broad objectives of the research proposed here are to elucidate the functions of the centrosome in vertebrate somatic cells, and the molecular mechanisms by which it accomplishes these functions. To achieve these goals we use a unique approach in which the centrosome (labeled with fluorescent proteins) is ablated by a laser microbeam. This approach allows us to engineer cells that lack the centrosome, and compare their behavior with genetically identical cells that possess this organelle. Further, we combine laser microsurgery with inactivation of individual proteins by siRNA and cell-permeable compounds. This synergistic methodology provides new information unobtainable by other means. Our current research focuses on two areas. First, we are testing the hypothesis that improper balance between the contributions of centrosomal and centrosome- independent mechanisms during the assembly of mitotic spindle assembly leads to chromosomal instability. We follow the behavior of individual chromosomes in human cancer cells by high-resolution microscopy and investigate how this behavior changes when the centrosomes are laser ablated (Aim 1). We also study how chromosome behavior changes in response to perturbations of key molecules implicated in the centrosome- independent microtubule organization (TPX2 and HURP) or in the correction of erroneous chromosome orientations (Aurora B and kinesin 13s) (Aim 2). Finally, we use laser microsurgery, cell-permeable chemical inhibitors, and siRNA to study molecular and structural requirements for 'centriole disengagement', a key event in the centrosome cycle that must be properly controlled to prevent repetitive re-duplication of centrioles in the same cell cycle (Aim 3). Our observations suggest that daughter centrioles cannot disengage until they reach a certain level of maturation. Thus, control of centriole disengagement involves a previously overlooked mechanism that is intrinsic to the centrosome. We currently investigate molecular mechanisms responsible for procentriole maturation.