Continued analysis of mitotic spindle architecture in mammalian cells

• Applicant: Professor Dr. Thomas Müller-Reichert
• Subject Area: Structural Biology ; Biophysics
• Term: since 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 258577783

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.

• DFG Programme: Research Grants