Identification and characterization of a novel mitotic kinesin Kif25

新型有丝分裂驱动蛋白 Kif25 的鉴定和表征

• 批准号: 8589818
• 负责人: Justin Decarreau
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8589818
• 关键词: ATP phosphohydrolase; Active Sites; Affect; Anaphase; Binding; Biological Assay; C-terminal; Cell division; Cells; Centrosome; Chromosomal Stability; Co-Immunoprecipitations; Defect; Development; Dominant-Negative Mutation; Family; Generations; Human; Immunofluorescence Immunologic; Investigation; Kinesin; Lead; Length; Life; Light; Location; Mad1 protein; Maintenance; Mediating; Membrane; Metaphase; Microtubules; Mitosis; Mitotic; Mitotic spindle; Molecular; Molecular Motors; Motor; Motor Activity; Neoplasm Metastasis; Phosphotransferases; Play; Positioning Attribute; Process; Property; Proteins; Regulation; Role; Running; Structure; Testing; Time; Tissues; Total Internal Reflection Fluorescent; base; cellular imaging; crosslink; mutant; novel; overexpression; public health relevance; research study; single molecule; stem cell population; tumor progression

DESCRIPTION (provided by applicant): Correct orientation of the mitotic spindle during cell division is critically important in development as well as in the regulation and maintenance of stem cell populations in tissues. Orienting the spindle in the appropriate plane of division is an active process mediated by membrane-anchored proteins and subject to regulation by mitotic kinases. Molecular motors are thought to act as force generators that both orient and anchor the spindle in the proper plane of division. We have found that depletion of a little studied kinesin family motor, Kif25, in human cells causes misorientation of the mitotic spindle and defects in spindle assembly. We have determined that Kif25 is a C-terminal kinesin motor although the structure and function of this motor are poorly understood. The focus of the current proposal is to test the hypothesis that Kif25 regulates both spindle assembly and orientation by producing force on spindle microtubules from its centrosomal location. Single molecule studies will be used to characterize the microtubule-dependent activity of this novel motor. These experiments will compliment and inform our investigations of Kif25 in live cells where we will use a combination of live and fixed cell imaging to evaluate protein depletions and dominant negative constructs, as well as the identification and characterization of Kif25 interacting partners. Overal this proposal will fully characterize Kif25 function and contribute an important new component to the machinery controlling spindle orientation in mitotic cells.

描述（由申请方提供）：细胞分裂期间有丝分裂纺锤体的正确方向对于组织中干细胞群的发育以及调节和维持至关重要。定向纺锤体在适当的分裂平面是一个主动的过程介导的膜锚定蛋白和有丝分裂激酶的调控。分子马达被认为是力的发生器，既能使纺锤体定向，又能将其锚在适当的分裂平面上。我们已经发现，在人类细胞中，很少研究的驱动蛋白家族马达Kif 25的缺失导致有丝分裂纺锤体的错误取向和纺锤体组装的缺陷。我们已经确定Kif 25是一个C-末端驱动蛋白马达，虽然这个马达的结构和功能知之甚少。目前的建议的重点是测试的假设，Kif 25调节纺锤体的组装和方向产生力的纺锤体微管从其中心体的位置。单分子研究将用于表征这种新型马达的微管依赖性活性。这些实验将补充并告知我们在活细胞中对Kif 25的研究，我们将使用活细胞和固定细胞成像的组合来评估蛋白质消耗和显性阴性构建体，以及Kif 25相互作用伴侣的鉴定和表征。总之，这一建议将充分表征Kif 25功能，并有助于一个重要的新的组成部分，在有丝分裂细胞中的纺锤体方向的机械控制。