CHROMOSOME MOVEMENT IN MEIOSIS AND MITOSIS

该项目的长期目标是识别和表征减数分裂和有丝分裂过程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和参与纺锤体组装和功能的微管动力学。拟议的研究重点是NCD，一种在果蝇中的运动蛋白家族微管运动蛋白。NCD是一种负端定向的马达，在纺锤体的组装和减数分裂和有丝分裂的功能中起着至关重要的作用。建议的研究集中在1)‘颈’，即邻近保守的运动区的蛋白质区域，决定运动方向性的机制，2)运动功能过程中发生的运动的构象变化，以及3)负端运动和运动解偶联对细胞中NCD功能的影响。本研究的具体目的是：1.确定颈部功能在运动方向性中的作用机制。通过构建突变电机并在体外分析极性，将对颈部进行功能测试，以确定电机的极性。2.确定运动功能过程中与构象变化有关的运动区域。将选择或设计将ATP水解从力产生中分离出来或将马达困在不同状态的突变体，并在体外进行测试，以确定在运动功能期间移动的结构元件。3.在体内测定负端极性和运动解偶联对NCD功能的影响。截短突变体和马达解偶联突变体将以GFP基因融合的形式引入果蝇，以确定突变马达对活细胞中NCD功能和动力学的影响。这些研究涉及纺锤体中力产生的分子基础，以及微管马达在细胞分裂期间染色体运动中的作用。染色体分布缺陷会导致非整倍体和遗传异常，并与人类的恶性肿瘤有关。

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.