Microtubule-Based Motors in Chromosome Segregation

染色体分离中基于微管的马达

• 批准号: 9507008
• 负责人: William Saunders
• 金额: $ 28.7万
• 依托单位: University of Pittsburgh
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-15 至 1996-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9507008&HistoricalAwards=false
• 关键词: Microtubule; Based; Motors; Chromosome; Segregation

9507008 Saunders Recent results have indicated that spindle function in various eukaryotes is controlled by a relative balance between inwardly and outwardly-directed spindle motors. The outwardly-directed motors have recently been identified. The experiments outlined in this proposal are designed to identify the rest of the "inwardly-directed " force generating proteins and to examine the function of these motors in chromosome segregation. Library induced suppression of the kar 3 ts phenotype and the identification of mutants whose spindles do not collapse are used to identify novel members of this group. Mutational analysis and genetic combinations are used to determine if these newly identified motors act antagonistically to the known outwardly-directed motors. New mutants of inwardly-directed motors will be combined with a kar3 disruption allele to determine the "synthetic" phenotype of loss-of-function of all members of this group of mitotic motors. The goal of these mutational analysis experiments will be to determine the functional role of these inwardly-directed motors. The suggested role of these proteins as kinetochore motors an/or inhibitors of spindle elongation is investigated by examination of the effect of loss-of-function on chromosome segregation and the timing and rate of spindle elongation. For this analysis both traditional fixed cell immunocytochemistry and newly-developing real-time analysis of live cells with chromatin and green fluorescent protein- labeled tubulin staining is used. %%% All organisms are composed of microscopic components called cells, analogous to the bricks in a brick building. When the organism grows, or following injury, the mass of organism in most cases is increased not by expansion of the size of existing cells, but by creation of new cells. New cells are created by division of existing cells. The newly dividing cells can grow in size until a second round of division can occur creating four cells where one existed originally. In many unicellular organisms this process will continue indefinitely until limited by availability of nutrients and space. In larger multicellular organisms this process is carefully controlled to prevent the excess proliferation associated with cancer and other growth abnormalities. When cells divide, it is necessary that each daughter cell contain all the components required for survival. Many of the cell components are apparently randomly partitioned, but some of the components are unique and both of the daughter cells requires a certain number of each type. The most well-known example of these unique components are the chromosomes. Each chromosome contains the information to produce proteins, the building blocks of the cell. The amount of each protein made is generally proportional to the number of chromosomes present. Therefore it is critical to that organism that the cells get the right number of each type of chromosome. To achieve this, eukaryotic (non-bacterial) cells utilize a complex and transitory structure called the spindle. The spindle is made of fibers that assemble during cell division. The chromosomes attach to the spindle fibers and are moved to the daughter cells at division in an exquisitely timed and orchestrated process. Recently, this laboratory and others have identified small molecular motors that act within the cell to separate the chromosomes to the daughter cells. The motors are related to other motors discovered previously to move other cellular components. Three different types of motors were identified that can separate the chromosomes, and each can perform the task fairly well by itself. Surprisingly, a second type of motor was identified that acts in an opposite manner to pull chromosomes together. The role of this motor is unknown but it may act to delay the activity of the chromosome separating motors until the appropriate time for cell division. This project identifies and examines thes e motors which pull the chromosomes together. When all of the motors are identified, then their precise role in cell division is determined. This is done by inactivating the motors and asking what the cell is unable to do. Specifically, spindle assembly and stability and chromosome separation are carefully scrutinized. If the cell is unable to perform these function or performs in an abnormal manner, it will suggest when and where the motors work. Understanding the timing and activity of these motors allows a basic understanding of how the cell divides. ***

最近的研究结果表明，各种真核生物的纺锤体功能是由向内和向外的纺锤体马达之间的相对平衡控制的。向外定向的马达最近已被确定。本提案中概述的实验旨在确定其余的“向内定向”力产生蛋白质，并检查这些马达在染色体分离中的功能。文库诱导的k3ts表型抑制和纺锤体不塌陷的突变体的鉴定被用来鉴定这一群体的新成员。突变分析和基因组合被用来确定这些新发现的马达是否对已知的外向马达起拮抗作用。向内定向马达的新突变体将与kar3断裂等位基因结合，以确定这组有丝分裂马达所有成员功能丧失的“合成”表型。这些突变分析实验的目标将是确定这些向内定向马达的功能作用。通过检查功能丧失对染色体分离和纺锤体伸长的时间和速率的影响，研究了这些蛋白质作为着丝点马达和/或纺锤体伸长抑制剂的作用。在这种分析中，使用了传统的固定细胞免疫细胞化学和新发展的实时活细胞分析，用染色质和绿色荧光蛋白标记的微管蛋白染色。所有的生物体都是由称为细胞的微观成分组成的，类似于砖房中的砖块。在大多数情况下，当生物体生长或受伤后，生物体的质量不是通过扩大现有细胞的大小，而是通过产生新细胞来增加的。新细胞是由现有细胞分裂产生的。新分裂的细胞可以在大小上增长，直到第二轮分裂发生，在原来存在一个细胞的地方产生四个细胞。在许多单细胞生物中，这一过程将无限地持续下去，直到受到可用的营养物质和空间的限制。在较大的多细胞生物中，这一过程受到严格控制，以防止与癌症和其他生长异常相关的过度增殖。当细胞分裂时，每个子细胞必须包含生存所需的所有成分。许多细胞成分显然是随机分割的，但有些成分是唯一的，两个子细胞每种类型都需要一定数量的成分。这些独特成分中最著名的例子是染色体。每条染色体都包含产生蛋白质的信息，蛋白质是细胞的组成部分。每种蛋白质的数量通常与存在的染色体数量成正比。因此，对生物体来说，细胞获得每种类型染色体的正确数量是至关重要的。为了达到这个目的，真核（非细菌）细胞利用一种复杂而短暂的结构，称为纺锤体。纺锤体是由细胞分裂时聚集的纤维组成的。染色体附着在纺锤体纤维上，并在分裂过程中以一个精确的时间和精心安排的过程移动到子细胞。最近，这个实验室和其他实验室已经确定了在细胞内起作用的小分子马达，将染色体分离到子细胞。这些马达与先前发现的用于移动其他细胞成分的其他马达有关。三种不同类型的马达可以分离染色体，每一种都能很好地独立完成这项任务。令人惊讶的是，第二种类型的马达被发现以相反的方式将染色体拉到一起。该马达的作用尚不清楚，但它可能延迟染色体分离马达的活动，直到适当的时间进行细胞分裂。这个项目识别和检查这些把染色体拉在一起的马达。当所有的马达都被识别出来时，它们在细胞分裂中的确切作用就被确定了。这是通过使马达失活并询问细胞不能做什么来完成的。具体来说，纺锤体组装和稳定性和染色体分离是仔细审查。如果电池不能执行这些功能或以不正常的方式执行，它将提示电机工作的时间和地点。了解这些马达的时间和活动可以让我们对细胞如何分裂有一个基本的了解。＊＊＊