CHROMOSOME MOVEMENT IN MEIOSIS AND MITOSIS

该项目的长期目标是确定和表征减数分裂和有丝分裂中染色体运动的基础力量，包括染色体与纺锤体的相互作用以及纺锤体组装和功能中涉及的微管动力学。本研究的重点是果蝇运动蛋白家族微管运动蛋白Ncd。Ncd是一个负端定向马达，在纺锤体组装和减数分裂和有丝分裂中起重要作用。建议的研究集中在1)“颈部”，蛋白质邻近保守运动结构域的区域，决定运动方向性的机制，2)运动功能期间发生的运动构象变化，以及3)负端运动和运动解耦对细胞Ncd功能的影响。拟议研究的具体目的是：1。确定颈部运动定向功能的机制。颈部在确定马达极性方面的功能测试将通过构建突变马达和体外极性分析来进行。2. 识别在运动功能过程中参与构象改变的运动区域。将选择或设计突变体，使ATP水解脱离力产生或使马达处于ATP水解的不同状态，并在体外进行测试，以确定运动功能期间移动的结构元件。3. 确定负端极性和马达解耦对体内Ncd功能的影响。截断突变体和马达解偶联突变体将作为gfp基因融合体引入果蝇，以确定突变马达对活细胞Ncd功能和动力学的影响。这些研究解决了纺锤体产生力的分子基础和细胞分裂过程中微管马达在染色体运动中的作用。染色体分布缺陷导致非整倍体和遗传异常，并与人类恶性肿瘤有关。

The long-term objectives of this project are to identify and characterize the forces that underlie chromosome movement in meiosis and mitosis, including interactions of chromosomes with the spindle and microtubule dynamics involved in spindle assembly and function. The proposed studies focus on Ncd, a kinesin family microtubule motor protein in Drosophila. Ncd is a minus end-directed motor that plays an essential role in spindle assembly and function in meiosis and mitosis. The proposed studies focus on 1) the mechanism by which the 'neck', the region of the protein adjacent to the conserved motor domain, functions to determine motor directionality, 2) conformational changes in the motor that occur during motor function, and 3) the effects of minus-end motility and motor uncoupling on Ncd function in the cell. The specific aims of the proposed studies are to: 1. Determine the mechanism of neck function in motor directionality. Functional tests of the neck in determining motor polarity will be performed by constructing mutant motors and analyzing polarity in vitro. 2. Identify regions of the motor involved in changes of conformation during motor function. Mutants that uncouple ATP hydrolysis from force generation or trap the motor in different states of ATP hydrolysis will be selected or designed, and tested in vitro to identify structural elements that move during motor function. 3. Determine the effect of minus-end polarity and motor uncoupling on Ncd function in vivo. Truncated mutants and motor uncoupling mutants will be introduced into Drosophila as gfp gene fusions to determine the effects of the mutated motors on Ncd function and dynamics in live cells. These studies address the molecular basis of force generation in the spindle and the role of microtubule motors in chromosome movement during cell division. Defective chromosome distribution causes aneuploidy and genetic abnormalities, and is associated with malignancies in humans.