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.

总结 在真核细胞分裂之前，纺锤体分离染色体。马达蛋白、交联剂和 相关蛋白组织纺锤体微管以组装双极纺锤体。在纺锤体蛋白中， 驱动蛋白-5马达是特别重要的，因为它们对于在大多数或- 生物体已知四聚体驱动蛋白-5交联在微管中心的重叠反平行微管， 纺锤体，然后向每对微管的正端迈进。这产生的力量滑动分开antipar- 使微管分开，从而分开有丝分裂纺锤体极。与这一模型相矛盾的是， 驱动蛋白-5s在沿着微管的两个方向上运输，并定位在微管负端附近，部分通过 结合-微管蛋白。这两个不太清楚的机制可能是重要的主轴极分离 建立一个双极纺锤体，但他们是否是必不可少的是未知的。此外，驱动蛋白-5C末端 尾部是纺锤体组装的重要磷酸化热点，但磷酸化改变的性质， 情况尚不清楚。这一证据指出了我们对纺锤体组装机制的理解中的一个关键空白。 在初步工作中，我们做了两个关键的观察：第一，驱动蛋白-5/Cut7在分裂时双向移动- 酵母纺锤体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时，可能会发生。因此，驱动蛋白-5的两个不寻常的性质可能发挥作用， 在定位电机中起关键作用，以使主轴磁极分离时能够产生适当的力。此外，Phos- C-末端尾部的磷酸化可以调节这些功能。我们假设驱动蛋白-5双向 运动和纺锤极定位使得能够产生力来分离纺锤极，并且细胞能够重新生长。 这个发动机的方向性和定位 为了验证这些假设，我们将使用跨学科的方法，结合驱动蛋白-5扰动， 有丝分裂酵母，定量光学显微镜和图像分析，以及计算建模。到 确定使驱动蛋白-5/Cut7方向性偏向纺锤体正或负末端的细胞线索 微管，我们将测试的假设，（1）拥挤的微管晶格结合蛋白，（2） 马达交联状态，和/或（3）磷酸化改变体内Cut7方向偏向性。结果将是一个系统- 建议的手柄细胞的热比较可用于在纺锤体上引导Cut7。来确认他的亲属 驱动蛋白-5/Cut7纺锤极束缚和双向运动对纺锤极分离的贡献， 我们将测量和建模细胞中的纺锤体组装和驱动蛋白-5定位， 电动机主轴极栓系和方向偏置。此外，我们将定义Cut7尾部及其phos的作用， 在纺锤极栓系中的磷酸化。本工作将系统地测试的假设，直接主轴极 结合和/或双向运动对于纺锤体组装是必需的。该项目的结果将确定 有丝分裂纺锤体组装过程中纺锤体-极分离所需的驱动蛋白-5特性。