Continued analysis of mitotic spindle architecture in mammalian cells

有丝分裂纺锤体是一种基于微管（MT）的三维（3D）装置，在细胞分裂期间用于确保染色体的忠实分离。以前，我们可以证明，在中期HeLa细胞的染色体是半直接连接到纺锤体极。动粒纤维（k-纤维）在其面向极点的末端显示出加宽，可能介导了动粒微管（KMT）锚定到非KMT网络。我们可以进一步表明，KMT遵循明确定义的轨迹，在中期的非KMT。现在我们的目标是将我们的结构分析扩展到第二个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相似性和差异。应用一个完全建立的方法相结合的高压冷冻，连续切片电子断层扫描和3D量化的MT超微结构，我们建议重建中期纺锤体RPE 1细胞。初步的重建表明，RPE 1细胞显示出增加的数量KMT达到纺锤体极相比，HeLa细胞。总的来说，中期纺锤体不太圆，K-纤维中的KMT似乎更直，更稳定（如通过光学显微镜观察到的）在RPE 1与HeLa细胞中。同时，我们的目标是分析单极RPE 1纺锤体中的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“迷你纺锤体”，它似乎被星形几何中径向向外指向的MT所包围。光学显微镜进一步表明，KMT从外边缘的染色体沿着紫菀轨迹沿着向纺锤体中心生长。有趣的是，大多数KMT停在星形和“小纺锤体”结构域壁之间的边界处，并且不与中心体直接接触。然后，我们将使用这种单极RPE 1细胞诱导向双极性的转变，以更好地理解KMT与非KMT的相互作用。特别是，这样的单极到双极的MT组织过渡的分析，预计将提高我们的理解，形成明确的非KMT轨迹在主轴组装。总之，RPE 1细胞中纺锤体的超微结构信息将使我们能够完善我们开发的哺乳动物纺锤体中K纤维自组织的生物物理模型。根据雅阁的FAIR（可查找性、可访问性、互操作性、重用/复制）原则，我们还建议为我们的电子断层图像和相应的3D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，以便更好地向科学界和公众传达我们的发现。

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.