@article{KiewiszBaumMuellerReichertetal.2023, author = {Kiewisz, Robert and Baum, Daniel and M{\"u}ller-Reichert, Thomas and Fabig, Gunar}, title = {Serial-section electron tomography and quantitative analysis of the microtubule organization in 3D-reconstructed mitotic spindles}, volume = {13}, journal = {Bio-protocol}, number = {20}, doi = {10.21769/BioProtoc.4849}, year = {2023}, language = {en} } @article{OkaforntaFarhadifarFabigetal.2025, author = {Okafornta, Chukwuebuka William and Farhadifar, Reza and Fabig, Gunar and Wu, Hai-Yin and K{\"o}ckert, Maria and Vogel, Martin and Baum, Daniel and Haase, Robert and Shelley, Michael J. and Needleman, Daniel J. and M{\"u}ller-Reichert, Thomas}, title = {Cell size reduction scales spindle elongation but not chromosome segregation in C. elegans}, journal = {bioRxiv}, doi = {10.1101/2025.10.13.681585}, year = {2025}, abstract = {How embryos adapt their internal cellular machinery to reductions in cell size during development remains a fundamental question in cell biology. Here, we use high-resolution lattice light-sheet fluorescence microscopy and automated image analysis to quantify lineage-resolved mitotic spindle and chromosome segregation dynamics from the 2- to 64-cell stages in Caenorhabditis elegans embryos. While spindle length scales with cell size across both wild-type and size-perturbed embryos, chromosome segregation dynamics remain largely invariant, suggesting that distinct mechanisms govern these mitotic processes. Combining femtosecond laser ablation with large-scale electron tomography, we find that central spindle microtubules mediate chromosome segregation dynamics and remain uncoupled from cell size across all stages of early development. In contrast, spindle elongation is driven by cortically anchored motor proteins and astral microtubules, rendering it sensitive to cell size. Incorporating these experimental results into an extended stoichiometric model for both the spindle and chromosomes, we find that allowing only cell size and microtubule catastrophe rates to vary reproduces elongation dynamics across development. The same model also accounts for centrosome separation and pronuclear positioning in the one-cell C. elegans embryo, spindle-length scaling across nematode species spanning ~100 million years of divergence, and spindle rotation in human cells. Thus, a unified stoichiometric framework provides a predictive, mechanistic account of spindle and nuclear dynamics across scales and species.}, language = {en} } @article{LaguilloDiegoKiewiszMartiGomezetal.2022, author = {Laguillo-Diego, Alejandra and Kiewisz, Robert and Mart{\´i}-G{\´o}mez, Carlos and Baum, Daniel and M{\"u}ller-Reichert, Thomas and Vernos, Isabelle}, title = {MCRS1 modulates the heterogeneity of microtubule minus-end morphologies in mitotic spindles}, volume = {34}, journal = {Molecular Biology of the Cell}, number = {1}, doi = {10.1091/mbc.E22-08-0306-T}, year = {2022}, abstract = {Faithful chromosome segregation requires the assembly of a bipolar spindle, consisting of two antiparallel microtubule (MT) arrays having most of their minus ends focused at the spindle poles and their plus ends overlapping in the spindle midzone. Spindle assembly, chromosome alignment and segregation require highly dynamic MTs. The plus ends of MTs have been extensively investigated; instead, their minus end structure remains poorly characterized. Here, we used large-scale electron tomography to study the morphology of the MT minus ends in 3D-reconstructed metaphase spindles in HeLa cells. In contrast to the homogeneous open morphology of the MT plus ends at the kinetochores, we found that MT minus ends are heterogeneous showing either open or closed morphologies. Silencing the minus-end specific stabilizer, MCRS1 increased the proportion of open MT minus ends. Altogether, these data suggest a correlation between the morphology and the dynamic state of the MT ends. Taking this heterogeneity of the MT minus end morphologies into account, our work indicates an unsynchronized behavior of MTs at the spindle poles, thus laying the ground for further studies on the complexity of MT dynamics regulation.}, language = {en} } @article{KiewiszFabigConwayetal.2022, author = {Kiewisz, Robert and Fabig, Gunar and Conway, William and Baum, Daniel and Needleman, Daniel and M{\"u}ller-Reichert, Thomas}, title = {Three-dimensional structure of kinetochore-fibers in human mitotic spindles}, volume = {11}, journal = {eLife}, doi = {10.7554/eLife.75459}, pages = {e75459}, year = {2022}, abstract = {During cell division, kinetochore microtubules (KMTs) provide a physical linkage between the chromosomes and the rest of the spindle. KMTs in mammalian cells are organized into bundles, so-called kinetochore-fibers (k-fibers), but the ultrastructure of these fibers is currently not well characterized. Here we show by large-scale electron tomography that each k-fiber in HeLa cells in metaphase is composed of approximately nine KMTs, only half of which reach the spindle pole. Our comprehensive reconstructions allowed us to analyze the three-dimensional (3D) morphology of k-fibers and their surrounding MTs in detail. We found that k-fibers exhibit remarkable variation in circumference and KMT density along their length, with the pole-proximal side showing a broadening. Extending our structural analysis then to other MTs in the spindle, we further observed that the association of KMTs with non-KMTs predominantly occurs in the spindle pole regions. Our 3D reconstructions have implications for KMT growth and k-fiber self-organization models as covered in a parallel publication applying complementary live-cell imaging in combination with biophysical modeling (Conway et al., 2022). Finally, we also introduce a new visualization tool allowing an interactive display of our 3D spindle data that will serve as a resource for further structural studies on mitosis in human cells.}, language = {en} }