Intrinsic Spindle Mechanisms to Insure Bipolarity

描述（由申请人提供）：有丝分裂期间染色体的准确分离需要双极纺锤体的正确组装。这个过程中的错误可能导致非整倍体，这是恶性转化的先兆，也是出生缺陷的主要原因。纺锤体组装源于微管和瞬时相关微管运动蛋白之间的随机相互作用。这项工作的总体目标是更好地理解这些相互作用和由它们产生的力如何有助于主轴装配和功能。本研究基于对爪蟾卵提取物中纺锤体的观察，重点研究了促进纺锤体融合的动力蛋白依赖机制。该提案的具体目标是1)表征动力蛋白依赖的微管捆绑和微管正端力产生的机制，以及2)确定反平行微管相互作用和负端定向力产生所需的额外分子成分。高分辨率多模式数字显微镜将用于分析荧光标记的微管和微管运动蛋白在微管“桥”中观察到的反平行相互作用，形成融合纺锤体之间。纺锤体融合将在爪蟾提取物中进一步表征，这些提取物被认为涉及反平行微管相互作用和力产生的已知运动和非运动微管蛋白。动力蛋白和新发现的蛋白质对纺锤体张力和稳态长度维持的贡献将通过测量在药物抑制、功能阻断抗体添加或非洲爪蟾卵提取物中免疫消耗后着丝点间拉伸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功能纺锤体形成的基本机制的集体理解，并可能最终导致新的治疗策略，以减少这些疾病的患病率和发生率。

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.