Intrinsic Spindle Mechanisms to Insure Bipolarity

描述(由申请人提供)：在有丝分裂过程中染色体的准确分离需要两极纺锤体的正确组装。这一过程中的错误可能导致非整倍体，这是恶变的先兆，也是出生缺陷的主要原因。纺锤体组装是微管和瞬时相关的微管马达蛋白随机相互作用的结果。这项工作的广泛目标是更好地了解这些相互作用和它们产生的力是如何对主轴组装和功能做出贡献的。这项研究是基于对非洲爪哇卵提取液中纺锤体的观察，并专注于促进纺锤体融合的动力蛋白依赖机制。这一建议的具体目的是1)表征动力蛋白依赖的微管捆绑和微管正端的力产生的机制，以及2)确定反平行微管相互作用和负末端定向的力产生所需的额外分子成分。高分辨率多模数字显微镜将被用来分析荧光标记的微管与微管马达蛋白在微管中的反平行相互作用，观察到微管在融合纺锤体之间形成了桥。纺锤体融合将在非洲爪哇提取物中进一步表征，这些提取物耗尽了已知的运动和非运动微管蛋白，被认为参与了反平行微管相互作用和力的产生。动力蛋白和新发现的蛋白质对内在纺锤体张力和稳态长度维持的贡献将通过测量药物抑制、功能阻断抗体添加或免疫耗竭后动粒间伸展和纺锤体长度的变化来评估。有丝分裂纺锤体组装和功能的错误与癌症和出生缺陷有关。这项研究将增加我们对有助于形成功能性纺锤体的基本机制的集体理解，并最终可能导致减少这些疾病的患病率和发生的新的治疗策略。

DESCRIPTION (provided by applicant): Accurate segregation of chromosomes during mitosis requires the proper assembly of a bipolar spindle. Errors in this process can result in aneuploidy, a harbinger of malignant transformation and a major cause of birth defects. Spindle assembly results from stochastic interactions between microtubules and transiently associated microtubule motor proteins. The broad goal of this work is to better understand how these interactions and the forces generated by them contribute to spindle assembly and function. This research is based upon observations of spindles in Xenopus egg extracts and is focused on a dynein-dependent mechanism that facilitates spindle fusion. The specific aims of this proposal are 1) to characterize the mechanism of dynein-dependent microtubule-bundling and force generation at microtubule plus ends, and 2) to identify additional molecular components required for antiparallel microtubule interactions and minus end-directed force generation. High resolution multimode digital microscopy will be used to analyze antiparallel interactions of fluorescently labeled microtubules and microtubule motor protein dynamics in the microtubule "bridge" observed to form between fusing spindles. Spindle fusion will be further characterized in Xenopus extracts depleted of known motor and non-motor microtubule proteins thought to be involved in antiparallel microtubule interaction and force generation. The contribution of dynein and newly identified proteins to intrinsic spindle tension and steady state length maintenance will be evaluated by measuring changes in interkinetochore stretch and spindle length following pharmacological inhibition, function blocking antibody addition, or immunodepletion in Xenopus egg extracts. Errors in mitotic spindle assembly and function have been linked to cancer and birth defects. This research will increase our collective understanding of the basic mechanisms that contribute to the formation of a functional spindle and may ultimately lead to novel therapeutic strategies to reduce the prevalence and occurrence of these afflictions.