Microtubule Associated Proteins in Mitosis

斯洛博达9727818这个项目的目标是了解一种名为p62的蛋白质在细胞分裂的两个关键过程中的作用，即有丝分裂和胞质分裂。细胞分裂是生命的一个基本特征：这是所有生物体自我繁殖和多细胞生物体从单细胞受精卵发育而来的过程。胞质分裂是细胞分裂成两个独立细胞的过程。有丝分裂是细胞质分裂前的一个复杂过程，在这个过程中，分裂细胞的染色体被分离并平均分配到注定成为子细胞的区域。为了完成这些任务，细胞建立了一个细胞骨架。对于有丝分裂，框架被称为有丝分裂纺锤体，因为它在显微镜下看起来很像，它主要由微管组成。对于细胞质分裂，在动物细胞中，骨架由高度组织的肌动蛋白微丝组成，直接位于细胞皮质的质膜下。斯洛博达博士的实验室之前的工作表明，p62蛋白参与了细胞质分裂和有丝分裂后期A阶段(分离的染色体从分裂细胞的中央向有丝分裂纺锤体的两极或尖端移动的阶段)。该蛋白在物理上与纺锤体密切相关，在后期开始时，可以通过显微注射针对p62的抗体来抑制有丝分裂。后期A的一个基本特征是微管的解体，而一种已知的防止微管解体的药物紫杉醇，以类似于抗p62抗体的方式影响有丝分裂。因此，p62似乎在允许或介导有丝分裂调节的微管拆解中发挥了一定的作用。这种蛋白质也集中在分裂细胞的皮质中，并已被证明在皮质被诱导收缩时发生了磷酸化。从海胆卵中克隆了p62基因，并对其进行了序列测定，并在细菌中表达了该蛋白。为了这个项目的目的，将开发额外的试剂(纯化的p62和额外的抗体)。纯化的p62将用于体外研究其对微管稳定性、与有丝分裂器结合和皮质收缩的影响。从这些研究中获得的信息将与完整细胞中的实验信息相结合，使用抗体来定位和抑制p62的功能。定点突变将被用来改变p62的磷酸化位点，以确定这些位点在这些过程中的作用(S)。此外，还将在其他提供基因操纵机会的系统中寻找p62同源基因。支持该项目的一般假设是，p62在有丝分裂中发挥关键作用，特别是在A后期，不仅通过允许微管分解发生，而且通过诱导分裂沟的活动。了解p62的特性以及p62与有丝分裂和胞质分裂机制的其他组件的功能相互作用将有助于更好地理解这些至关重要的细胞过程。

Sloboda 9727818 The goal of this project is to understand the role of a protein termed p62 in two key processes of cell division, mitosis and cytokinesis. Cell division is an essential feature of life: it is the process whereby all organisms reproduce themselves and whereby multicellular organisms develop from a single-celled zygote. Cytokinesis is the process in which the cell cleaves itself into two separate cells. Mitosis is the complex process immediately preceding cytokinesis, in which the chromosomes of the dividing cell are separated and equally apportioned into the regions destined to become the daughter cells. A cytoskeletal framework is erected by the cell in order to accomplish these tasks. For mitosis, the framework is called the mitotic spindle because of its appearance under the microscope, and it consists primarily of microtubules. For cytokinesis, in animal cells the framework consists of highly organized actin microfilaments directly under the plasma membrane in the cortex of the cell. Prior work from Dr. Sloboda's laboratory has implicated the p62 protein in both cytokinesis and in the anaphase A stage of mitosis (the stage in which the separated chromosomes move away from the middle of the dividing cells, toward the poles, or pointed ends, of the mitotic spindle apparatus). The protein is physically closely associated with the spindle, and mitosis can be inhibited at the start of anaphase by microinjection of antibodies specific for p62. An essential feature of anaphase A is the disassembly of microtubules, and a drug known to prevent microtubule disassembly, taxol, affects mitosis in a manner analogous to the anti-p62 antibodies. Thus, p62 appears to play some role in allowing or mediating the mitotically-regulated disassembly of microtubules. The protein is also concentrated in the cortex of dividing cells, and has been shown to undergo phosphorylation when the cortex is induced to contract. The cDNA for p62 from sea urchin eggs has been cloned and sequenced, and the protein has been expressed in bacteria. For the purposes of this project, additional reagents (purified p62 and additional antibodies) will be developed. Purified p62 will be used for in vitro studies of its effect on microtubule stability, binding to the mitotic apparatus, and cortical contraction. The information obtained from these studies will be combined with information from experiments in intact cells, using antibodies to localize and to inhibit the functions of p62. Site directed mutagenesis will be used to alter the phosphorylation sites in p62 to determine the role(s) of these sites in these processes. Also, p62 homologues will be sought in other systems that offer the opportunity of genetic manipulation. The general hypothesis underlying this project is that p62 plays a pivotal role in mitosis, particularly during anaphase A, not only by allowing microtubule disassembly to occur but also by inducing the activity of the cleavage furrow. Understanding the characteristics of p62 and the functional interactions of p62 with other components of the machinery of mitosis and cytokinesis will lead to a greater understanding of these critically important cellular processes.