MOTOR PROTEIN FUNCTION DURING MITOSIS

有丝分裂纺锤体是一种使用微管的双极蛋白质机器 基于(MT)的电机来协调染色体分离。我们建议 评估三种MT相关马达蛋白的功能， KRP130/KLP61F、NCD和细胞质动力蛋白，推测具有有丝分裂作用 在果蝇身上。基于这些马达的交联性 微管，我们假设它们的功能是通过 特定定向MT之间的相互作用。此外，我们 假设这些马达有不同但可能重叠的 可能在空间和时间上反映的功能 它们在有丝分裂纺锤体内的定位差异。为了测试 在这些假设下，我们将追求三个具体目标。首先，我们 将结合详细的MT使用高分辨率免疫显微镜检查 果蝇有丝分裂纺锤体的极性分析 这些马达在有丝分裂的不同阶段的定位和 它们相互作用的MT的极性方向。在 其次，我们将研究MT-MT的输运和捆绑特性 通过将纯化的马达全酶与 纯化的MTS的制备。我们会特别注意 交联型MT之间的极性关系。在第三部分，我们将 免疫耗竭小鼠无细胞提取液中有丝分裂纺锤体形成的研究 各种有丝分裂的马达。然后我们将重新添加纯化的KRP130/KLP61F， NCD和细胞质动力蛋白，单独或组合，以 确定他们弥补心轴缺陷的能力 队形。总体而言，这些研究将使我们能够指定具体的 各个电机的功能，并确定 多台马达协调一致。

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.