Continued analysis of mitotic spindle architecture in mammalian cells

有丝分裂纺锤体是一种三维（3D）微管（MT）为基础的装置，用于细胞分裂，以确保染色体的忠实分离。以前，我们可以证明中期HeLa细胞的染色体与纺锤极半直接相连。着丝点纤维（k-纤维）在其极端面端显示出增宽，可能介导着丝点微管（kmt）锚定到非kmt网络。我们可以进一步证明，国民党在中期遵循非国民党的明确轨迹。现在我们的目标是将我们的结构分析扩展到第二种非癌症来源的细胞系，以揭示两种不同哺乳动物细胞类型中纺锤体组织的异同。采用高压冷冻、连续切片电子断层扫描和MT超微结构三维定量相结合的方法，我们提出重建RPE1细胞中期纺锤体。初步重建表明，与HeLa细胞相比，RPE1细胞显示到达纺锤极的kmt数量增加。总的来说，与HeLa细胞相比，RPE1细胞的中期纺锤体不那么圆，k-纤维中的kmt似乎更直，更稳定（通过光镜观察）。同时，我们的目标是分析单极RPE1纺锤体中k-纤维的超微结构。出乎意料的是，我们在每个单极细胞中观察到一个“迷你纺锤”，它似乎被径向向外指向aster几何形状的mt所包围。光镜进一步显示，kmt从外缘的染色体沿较短的轨迹向纺锤体中心生长。有趣的是，大多数kmt停在aster和“迷你纺锤体”结构域壁之间的边界上，不与中心体直接接触。然后，我们将使用这种单极RPE1细胞诱导过渡到双极性，以更好地了解KMTs与非KMTs的相互作用。特别是，分析这种单极到双极的转变，在MT组织有望提高我们的理解形成良好定义的非国民党轨迹主轴装配过程中。总而言之，RPE1细胞中纺锤体的超微结构信息将使我们能够完善哺乳动物纺锤体中k纤维自组织的生物物理模型。根据FAIR（可查找性、可访问性、互操作性、重用/再现性）原则，我们还建议为我们的电子层析图和相应的三维重建建立一个试点数据管理系统。最后，我们的目标是开发更多先进的可视化工具，以便更好地将我们的发现传达给科学界和公众。

The mitotic spindle is a three-dimensional (3D) microtubule (MT)-based apparatus used during cell division to ensure the faithful segregation of chromosomes. Previously, we could show that the chromosomes in HeLa cells in metaphase are semi-directly linked to the spindle poles. The kinetochore fibers (k-fibers) showed a broadening at their pole-facing ends, likely to mediate an anchoring of the kinetochore microtubules (KMTs) to the non-KMT network. We could further show that the KMTs follow well-defined trajectories of non-KMTs in metaphase. It is now our goal to extend our structural analyses to a second, non-cancer derived cell line to uncover the similarities and differences in spindle organization in two different mammalian cell types. Applying a fully established approach by combining of high-pressure freezing, serial-section electron tomography and 3D quantification of MT ultrastructure, we propose to reconstruct metaphase spindles in RPE1 cells. Preliminary reconstructions have indicated that RPE1 cells show an increase in the number of KMTs reaching the spindle poles compared to HeLa cells. Overall, metaphase spindles are less rounded and KMTs in the k-fibers appear to be straighter and more stable (as observed by light microscopy) in RPE1 versus HeLa cells. In parallel, we aim to analyze the ultrastructure of k-fibers in monopolar RPE1 spindles. Unexpectedly, we observed a “mini spindle” in each monopolar cell that appears to be surrounded by MTs pointing radially outwards in an aster geometry. Light microscopy further indicated that the KMTs grow from chromosomes at the outer edge towards the spindle center along aster trajectories. Interestingly, the majority of the KMTs stop at the boundary between the aster and the “mini spindle” domain wall and do not make direct contact with the centrosomes. We will then use such monopolar RPE1 cells to induce a transition to bipolarity to better understand the interaction of KMTs with non-KMTs. Particularly, an analysis of such a monopolar-to-bipolar transition in MT organization is expected to enhance our understanding of the formation of the well-defined non-KMT trajectories during spindle assembly. All in all, ultrastructural information on spindles in RPE1 cells will allow us to refine our developed biophysical model on k-fiber self-organization in mammalian spindles. In accord with the FAIR (findability, accessibility, interoperability, reuse/reproduce) principles, we also propose to establish a pilot data management system for our electron tomograms and the corresponding 3D reconstructions. Last not least, we aim to develop additional advanced visualization tools to better communicate our findings both to the scientific community as well as to the public.