MOTOR PROTEIN FUNCTION DURING MITOSIS

有丝分裂纺锤体是一个双极蛋白质机器， (MT)基于马达来协调染色体分离。 我们建议 评估三种MT相关马达蛋白的功能， KRP 130/KLP 61 F、ncd和细胞质动力蛋白，具有推定的有丝分裂作用 在果蝇中。 基于这些马达的交联能力 微管，我们假设它们通过介导 特定定向MT之间的相互作用。 而且我们 假设这些马达具有不同的但可能重叠的 可能反映在空间和时间上的功能 它们在有丝分裂纺锤体中的定位差异。 测试 这些假设，我们将追求三个具体目标。 第一，我们 将使用高分辨率免疫显微镜配合详细的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， 与它们相互作用的MT的极性取向。 在 其次，我们将研究MT-MT传输和捆绑性质 通过将纯化的马达全酶与 纯化MT的制备。 将特别注意 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有丝分裂纺锤体的形成， 各种有丝分裂马达 然后我们将加入纯化的KRP 130/KLP 61 F， NCD和细胞质动力蛋白，单独或组合， 确定他们的能力，以弥补由此产生的缺陷，在主轴 阵 总的来说，这些研究将使我们能够分配特定的 功能，并确定如何的功能， 多个电动机被协调。

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.