MITOTIC SPINDLES IN NEMATODES: COMPARATIVE BIOPHYSICS TO EVOLUTIONARY BIOLOGY

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 有丝分裂纺锤体可以在组织和生物体之间表现出显著的变异性，但 目前对这种多样性的进化基础知之甚少。因此，目前尚不清楚为什么不同真核生物的中期纺锤体表现出不同的形态，并且这些纺锤体的体积差异超过1000倍。在这里，我们提出了一项比较研究，以调查形成有丝分裂纺锤体的进化力量。我们将在有丝分裂中使用单细胞线虫胚胎来解决三个广泛的问题： 1)主轴动力学和结构的变化程度如何？我们将通过研究线虫门中的约60个物种--包括线虫属的所有已知物种--来研究小的和大的系统发育距离。这项工作将使用一些光学显微镜技术来确定线虫纺锤体变异的范围。 2)这种变异的生物物理基础是什么？我们将对约10个物种的微管动力学和纺锤体结构进行深入分析。荧光技术(如果在非模型物种中可能)，薄层和三维电子显微镜，以及物理和遗传的扰动实验(如果可行)，将被用来测量微管的动力学、数量和行为。我们将开发生物物理模型来解释微管行为的差异如何导致纺锤体结构的差异。 3)这种变异的进化基础是什么？我们将使用表型进化模型来深入了解：(I)系统发育影响的程度；(Ii)纺锤体特征对细胞大小、核型、生活史和其他属性的依赖；以及(Iii)选择和漂移在形成纺锤体的各种组件中的重要性。对一个物种秀丽线虫的平行研究将提供对进化力量的更详细的分析：我们将使用突变累积线来测量自发突变如何修改有丝分裂纺锤体，我们将通过将这些线与大约40个不同的野生分离物进行比较来确定这种变异性是如何在进化过程中形成的。这种独特的比较方法结合了分子方法、定量显微镜和数学建模，以开发一个全面的模型，说明有丝分裂纺锤体是如何通过突变、选择、漂移和细胞生物物理的进化来塑造的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Mitotic spindles can show remarkable variability between tissues and organisms, but there is currently little understanding of the evolutionary basis of this diversity. Thus it is unclear why metaphase spindles in different Eukaryotes exhibit a range of morphologies and the volumes of these spindles vary over one thousand fold. Here, we propose a comparative study to investigate the evolutionary forces that shape the mitotic spindle. We will use one-cell nematode embryos in mitosis to address three broad questions: 1) What is the extent of variation in spindle dynamics and structure? We will investigate both small and large phylogenic distances by studying ~60 species from across the nematode phylum - including all known species of the Caenorhabditis genus. This work will use a number of light microscopy techniques to determine the range of spindle variation in nematodes. 2) What is the biophysical basis of this variation? We will perform an in-depth analysis of microtubule dynamics and spindle architecture in ~10 select species. Fluorescence techniques (when possible in nonmodel species), thin-section and 3-D electron microscopy, and perturbations experiments, both physical and genetic (when feasible), will be used to measure the dynamics, number and behavior of microtubules. We will develop biophysical models to explain how differences in microtubule behavior lead to differences in spindle architecture. 3) What is the evolutionary basis of this variation? We will use models of phenotypic evolution to gain insight into: (i) the extent of phylogenetic effects; (ii) the dependence of spindle features on cell size, karyotype, life history, and other attributes; and (iii) the importance of selection and drift in shaping various components of the spindle. A parallel study on one species, Caenorhabditis elegans, will provide a more detailed analysis of evolutionary forces: we will use mutation accumulation lines to measure how spontaneous mutations modify the mitotic spindle, and we will determine how this variability is shaped over evolution by comparing these lines with ~40 different wild isolates. This unique comparative approach combines molecular methods, quantitative microscopy, and mathematical modeling, to develop a comprehensive model of how the mitotic spindle is shaped through evolution by mutation, selection, drift, and cellular biophysics.