Mechanisms of Kinesin-5 Motors in Mitotic Spindle Assembly

有丝分裂纺锤体组装中的 Kinesin-5 马达机制

• 批准号: 9751899
• 负责人: Meredith Betterton
• 金额: $ 31.83万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9751899
• 关键词: Affect; Aneuploidy; Animal Model; Antimitotic Agents; Binding; Binding Proteins; C-terminal; Cell division; Cells; Chiroptera; Chromosome Segregation; Chromosomes; Computer Simulation; Congenital Abnormality; Crosslinker; Crowding; Cues; Data; Dimensions; Eukaryotic Cell; Fission Yeast; Fluorescence Microscopy; Generations; Hot Spot; Image Analysis; In Vitro; Kinesin; Malignant Neoplasms; Measures; Microtubules; Minus End of the Microtubule; Mitosis; Mitotic; Mitotic spindle; Modeling; Motion; Motor; Movement; Organism; Outcome; Phosphorylation; Play; Positioning Attribute; Property; Proteins; Regulation; Role; Slide; Speed; Structure; Tail; Techniques; Testing; Tubulin; Work; cancer therapy; cell motility; chromosome missegregation; crosslink; genetic manipulation; imaging genetics; improved; in vivo; interdisciplinary approach; light microscopy; prevent; temporal measurement

Summary The mitotic spindle segregates chromosomes prior to eukaryotic cell division. Motor proteins, crosslinkers, and associated proteins organize spindle microtubules to assemble the bipolar spindle. Among spindle proteins, kinesin-5 motors are particularly important because they are essential to establish a bipolar spindle in most or- ganisms. Tetrameric kinesin-5s are known to crosslink overlapping antiparallel microtubules in the center of the spindle, then step toward plus ends of each microtubule in the pair. This generates force to slide apart antipar- allel microtubules and thereby separate mitotic spindle poles. Contradicting this model are observations that kinesin-5s traffic in both directions along microtubules and localize near microtubule minus ends, in part by binding -tubulin. Both of these poorly understood mechanisms may be important for spindle-pole separation to establish a bipolar spindle, but whether they are essential is unknown. Additionally, kinesin-5 C-terminal tails are a phosphorylation hotspot important for spindle assembly, but the properties altered by phosphoryla- tion are unclear. This evidence points to a key gap in our understanding of spindle-assembly mechanisms. In preliminary work, we made two key observations: first, kinesin-5/Cut7 moves bidirectionally on fission- yeast spindle microtubules at speeds similar to those measured in vitro. Second, mitotic-spindle-pole separation can occur when Cut7 remains localized at one spindle pole. Thus kinesin-5's two unusual properties may play key roles in positioning the motor to enable proper force generation for spindle pole separation. Further, phos- phorylation of the C-terminal tails may regulate these functions. We hypothesize that kinesin-5 bidirectional movement and spindle-pole localization enable force generation to separate spindle poles, and that cells regu- late this motor's directionality and localization. To test these hypotheses, we will use an interdisciplinary approach combining kinesin-5 perturbation dur- ing mitosis in fission yeast, quantitative light microscopy and image analysis, and computational modeling. To determine the cellular cues that bias kinesin-5/Cut7 directionality toward either plus or minus ends of spindle microtubules, we will test the hypotheses that (1) crowding on the microtubule lattice by binding proteins, (2) motor crosslinking state, and/or (3) phosphorylation alter Cut7 directional bias in vivo. The result will be a sys- tematic comparison of proposed handles cells might use to direct Cut7 on the spindle. To identify the relative contributions of kinesin-5/Cut7 spindle-pole tethering and bidirectional motility to spindle-pole separation, we will measure and model spindle assembly and kinesin-5 localization in cells with specific perturbations to motor spindle-pole tethering and directional bias. Further, we will define the role of the Cut7 tail and its phos- phorylation in spindle-pole tethering. This work will systematically test the hypotheses that direct spindle-pole binding and/or bidirectional motility are essential for spindle assembly. The results of this project will define the kinesin-5 properties required for spindle-pole separation during mitotic spindle assembly.

摘要 有丝分裂纺锤体在真核细胞分裂之前分离染色体。马达蛋白、交联剂和 相关的蛋白质组织纺锤体微管来组装双极纺锤体。在纺锤体蛋白中， Kinesin-5马达特别重要，因为它们对于在大多数或- 有机体。已知的四聚体激动素-5可以使重叠的反平行微管发生交联。 纺锤体，然后走向对中每个微管的正端。这会产生推开反平行线的力- 微管，从而分离有丝分裂纺锤体极。与这一模型相矛盾的是观察到的 Kinesin-5s沿微管双向传递fic，并定位于微管负端附近，部分通过 结合微管蛋白。这两种鲜为人知的机制可能对主轴-磁极分离很重要 来建立一个两极纺锤体，但它们是否必不可少还是个未知数。此外，Kinesin-5 C-末端 尾巴是一个对纺锤体组装很重要的磷酸化热点，但这种特性被磷酸化- 目前还不清楚。这一证据表明，我们对主轴装配机制的理解存在一个关键差距。 在前期工作中，我们做了两个关键的观察：fiRst，Kinesin-5/Cut7在fiSSION上双向移动- 酵母纺锤形微管的速度与体外测量的速度相似。第二，有丝分裂-纺锤体-极分离 当Cut7保持在一个主轴极点上时会发生这种情况。因此，动蛋白-5‘S的两个不同寻常的性质可能会发挥作用 在定位电机以实现适当的主轴极点分离的力产生方面起着关键作用。此外，磷酸盐- C末端的磷酸化可能调节这些功能。我们假设Kinesin-5是双向的 运动和主轴-磁极局部化能够产生力以分离主轴磁极，而细胞调节- 后来这台马达的方向性和定位。 为了验证这些假设，我们将使用一种跨学科的方法，结合Kinesin-5在 fiSSION酵母中ING有丝分裂，定量光学显微镜和图像分析，以及计算模型。至 确定使Kinesin-5/Cut7定向朝向纺锤体正端或负端的细胞信号 关于微管，我们将检验以下假设：(1)结合蛋白拥挤在微管晶格上，(2) 在体内，运动交联态和/或(3)磷酸化改变了Cut7的方向偏向。结果将是一个系统- 暂时比较细胞可能用来引导纺锤体上的Cut7的拟议手柄。确定亲属身份 Kinesin-5/Cut7主轴-极系留和双向运动对主轴-极分离的贡献 我们将测量和模拟特定fic扰动的细胞中的纺锤体组装和kinesin-5定位。 电机主轴-极系绳和定向偏置。此外，我们将Define Cut7尾巴及其磷酸盐的作用- 主轴-杆系留中的磷酸化。这项工作将系统地检验引导主轴-极的假说 结合和/或双向运动对于主轴装配是必不可少的。这个项目的结果将会是fine。 有丝分裂纺锤体组装过程中轴-极分离所需的kinesin-5特性。