MOTOR PROTEIN FUNCTION DURING MITOSIS

有丝分裂期间的运动蛋白功能

• 批准号: 2857052
• 负责人: DAVID James SHARP
• 金额: $ 3.17万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2857052
• 关键词: Drosophilidae; Xenopus; cell cycle; immunoelectron microscopy; microtubule associated protein; protein localization; protein purification; protein reconstitution; protein structure function

The mitotic spindle is a bipolar protein machine that uses microtubule (MT) based motors to coordinate chromosome segregation. We propose to evaluate the function of three MT-associated motor proteins, KRP130/KLP61F, ncd, and cytoplasmic dynein, with putative mitotic roles in Drosophila. Based on the ability of these motors to crosslink microtubules, we hypothesize that they function by mediating interactions between specifically oriented MTs. Moreover, we hypothesize that these motors have distinct but possibly overlapping functions which are likely to be reflected in spatial and temporal differences in their localization within the mitotic spindle. To test these hypotheses, we will pursue three specific aims. In the first, we will use high resolution immunomicroscopy in concert with detailed MT polarity analyses of Drosophila mitotic spindles to determine the localization of these motors during different stages of mitosis and the polarity orientation of the MTs with which they interact. In the second, we will investigate the MT-MT transport and bundling properties of the motors in vitro by combining purified motor holoenzymes with preparations of purified MTs. Particular attention will be given to the polarity relationships between crosslinked MTs. In the third, we will study mitotic spindle formation in cell-free extracts immunodepleted of various mitotic motors. We will then add back purified KRP130/KLP61F, ncd, and cytoplasmic dynein, individually or in combinations, to determine their ability to complement the resulting defects in spindle formation. Collectively, these studies will allow us to assign specific functions to individual motors and to determine how the functions of multiple motors are coordinated.

有丝分裂纺锤体是一种使用微管的双极蛋白质机器 (MT) 基于马达来协调染色体分离。 我们建议 评估三种 MT 相关运动蛋白的功能， KRP130/KLP61F、ncd 和细胞质动力蛋白，具有假定的有丝分裂作用 在果蝇中。 基于这些电机的交联能力 微管，我们假设它们通过介导发挥作用 专门面向的 MT 之间的交互。 此外，我们 假设这些电机具有不同但可能重叠的 可能反映在空间和时间上的功能 它们在有丝分裂纺锤体内的定位存在差异。 测试 根据这些假设，我们将追求三个具体目标。 首先，我们 将使用高分辨率免疫显微镜与详细的 MT 果蝇有丝分裂纺锤体的极性分析以确定 这些马达在有丝分裂的不同阶段的定位和 与其相互作用的 MT 的极性方向。 在 其次，我们将研究 MT-MT 传输和捆绑特性 通过将纯化的运动全酶与 纯化 MT 的制剂。 将特别关注 交联 MT 之间的极性关系。 第三，我们将 研究免疫耗竭的无细胞提取物中的有丝分裂纺锤体形成 各种有丝分裂马达。 然后我们将添加回纯化的 KRP130/KLP61F， ncd 和细胞质动力蛋白，单独或组合，以 确定它们补充主轴缺陷的能力 形成。 总的来说，这些研究将使我们能够分配具体的 功能到各个电机并确定其功能如何 多个电机协调。