TY - JOUR A1 - Redemann, Stefanie A1 - Lantzsch, Ina A1 - Lindow, Norbert A1 - Prohaska, Steffen A1 - Srayko, Martin A1 - Müller-Reichert, Thomas T1 - A switch in microtubule orientation during C. elegans meiosis JF - Current Biology N2 - In oocytes of many organisms, meiotic spindles form in the absence of centrosomes [1–5]. Such female meiotic spindles have a pointed appearance in metaphase with microtubules focused at acentrosomal spindle poles. At anaphase, the microtubules of acentrosomal spindles then transition to an inter- chromosomal array, while the spindle poles disappear. This transition is currently not understood. Previous studies have focused on this inter- chromosomal microtubule array and proposed a pushing model to drive chromosome segregation [6, 7]. This model includes an end-on orientation of microtubules with chromosomes. Alternatively, chromosomes were thought to associate along bundles of microtubules [8, 9]. Starting with metaphase, this second model proposed a pure lateral chromosome-to-microtubule association up to the final meiotic stages of anaphase. Here we applied large-scale electron tomography [10] of staged C. elegans oocytes in meiosis to analyze the orientation of microtubules in respect to chromosomes. We show that microtubules at metaphase I are primarily oriented laterally to the chromosomes and that microtubules switch to an end-on orientation during progression through anaphase. We further show that this switch in microtubule orientation involves a kinesin-13 microtubule depolymerase, KLP-7, which removes laterally associated microtubules around chromosomes. From this we conclude that both lateral and end-on modes of microtubule-to-chromosome orientations are successively used in C. elegans oocytes to segregate meiotic chromosomes. Y1 - 2018 U6 - https://doi.org/10.1016/j.cub.2018.07.012 SN - 0960-9822 ER - TY - JOUR A1 - Fabig, Gunar A1 - Kiewisz, Robert A1 - Lindow, Norbert A1 - Powers, James A. A1 - Cota, Vanessa A1 - Quintanilla, Luis J. A1 - Brugués, Jan A1 - Prohaska, Steffen A1 - Chu, Diana S. A1 - Müller-Reichert, Thomas T1 - Sperm-specific meiotic chromosome segregation in C. elegans JF - eLife Y1 - 2020 U6 - https://doi.org/10.7554/eLife.50988 VL - 9 SP - e50988 ER - TY - JOUR A1 - Lindow, Norbert A1 - Brünig, Florian A1 - Dercksen, Vincent J. A1 - Fabig, Gunar A1 - Kiewisz, Robert A1 - Redemann, Stefanie A1 - Müller-Reichert, Thomas A1 - Prohaska, Steffen A1 - Baum, Daniel T1 - Semi-automatic stitching of filamentous structures in image stacks from serial-section electron tomography JF - bioRxiv N2 - We present a software-assisted workflow for the alignment and matching of filamentous structures across a 3D stack of serial images. This is achieved by combining automatic methods, visual validation, and interactive correction. After an initial alignment, the user can continuously improve the result by interactively correcting landmarks or matches of filaments. Supported by a visual quality assessment of regions that have been already inspected, this allows a trade-off between quality and manual labor. The software tool was developed to investigate cell division by quantitative 3D analysis of microtubules (MTs) in both mitotic and meiotic spindles. For this, each spindle is cut into a series of semi-thick physical sections, of which electron tomograms are acquired. The serial tomograms are then stitched and non-rigidly aligned to allow tracing and connecting of MTs across tomogram boundaries. In practice, automatic stitching alone provides only an incomplete solution, because large physical distortions and a low signal-to-noise ratio often cause experimental difficulties. To derive 3D models of spindles despite the problems related to sample preparation and subsequent data collection, semi-automatic validation and correction is required to remove stitching mistakes. However, due to the large number of MTs in spindles (up to 30k) and their resulting dense spatial arrangement, a naive inspection of each MT is too time consuming. Furthermore, an interactive visualization of the full image stack is hampered by the size of the data (up to 100 GB). Here, we present a specialized, interactive, semi-automatic solution that considers all requirements for large-scale stitching of filamentous structures in serial-section image stacks. The key to our solution is a careful design of the visualization and interaction tools for each processing step to guarantee real-time response, and an optimized workflow that efficiently guides the user through datasets. Y1 - 2020 U6 - https://doi.org/10.1101/2020.05.28.120899 ER - TY - JOUR A1 - Weber, Britta A1 - Greenan, Garrett A1 - Prohaska, Steffen A1 - Baum, Daniel A1 - Hege, Hans-Christian A1 - Müller-Reichert, Thomas A1 - Hyman, Anthony A1 - Verbavatz, Jean-Marc T1 - Automated tracing of microtubules in electron tomograms of plastic embedded samples of Caenorhabditis elegans embryos JF - Journal of Structural Biology Y1 - 2012 UR - http://www.sciencedirect.com/science/article/pii/S1047847711003509 U6 - https://doi.org/10.1016/j.jsb.2011.12.004 VL - 178 IS - 2 SP - 129 EP - 138 ER - TY - JOUR A1 - Weber, Britta A1 - Tranfield, Erin M. A1 - Höög, Johanna L. A1 - Baum, Daniel A1 - Antony, Claude A1 - Hyman, Tony A1 - Verbavatz, Jean-Marc A1 - Prohaska, Steffen T1 - Automated stitching of microtubule centerlines across serial electron tomograms JF - PLoS ONE Y1 - 2014 U6 - https://doi.org/10.1371/journal.pone.0113222 SP - e113222 ER - TY - JOUR A1 - Redemann, Stefanie A1 - Baumgart, Johannes A1 - Lindow, Norbert A1 - Shelley, Michael A1 - Nazockdast, Ehssan A1 - Kratz, Andrea A1 - Prohaska, Steffen A1 - Brugués, Jan A1 - Fürthauer, Sebastian A1 - Müller-Reichert, Thomas T1 - C. elegans chromosomes connect to centrosomes by anchoring into the spindle network JF - Nature Communications N2 - The mitotic spindle ensures the faithful segregation of chromosomes. Here we combine the first large-scale serial electron tomography of whole mitotic spindles in early C. elegans embryos with live-cell imaging to reconstruct all microtubules in 3D and identify their plus- and minus-ends. We classify them as kinetochore (KMTs), spindle (SMTs) or astral microtubules (AMTs) according to their positions, and quantify distinct properties of each class. While our light microscopy and mutant studies show that microtubules are nucleated from the centrosomes, we find only a few KMTs directly connected to the centrosomes. Indeed, by quantitatively analysing several models of microtubule growth, we conclude that minus-ends of KMTs have selectively detached and depolymerized from the centrosome. In toto, our results show that the connection between centrosomes and chromosomes is mediated by an anchoring into the entire spindle network and that any direct connections through KMTs are few and likely very transient. Y1 - 2017 U6 - https://doi.org/10.1038/ncomms15288 VL - 8 IS - 15288 ER - TY - JOUR A1 - Lindow, Norbert A1 - Brünig, Florian A1 - Dercksen, Vincent J. A1 - Fabig, Gunar A1 - Kiewisz, Robert A1 - Redemann, Stefanie A1 - Müller-Reichert, Thomas A1 - Prohaska, Steffen A1 - Baum, Daniel T1 - Semi-automatic stitching of filamentous structures in image stacks from serial-section electron tomography JF - Journal of Microscopy N2 - We present a software-assisted workflow for the alignment and matching of filamentous structures across a three-dimensional (3D) stack of serial images. This is achieved by combining automatic methods, visual validation, and interactive correction. After the computation of an initial automatic matching, the user can continuously improve the result by interactively correcting landmarks or matches of filaments. Supported by a visual quality assessment of regions that have been already inspected, this allows a trade-off between quality and manual labor. The software tool was developed in an interdisciplinary collaboration between computer scientists and cell biologists to investigate cell division by quantitative 3D analysis of microtubules (MTs) in both mitotic and meiotic spindles. For this, each spindle is cut into a series of semi-thick physical sections, of which electron tomograms are acquired. The serial tomograms are then stitched and non-rigidly aligned to allow tracing and connecting of MTs across tomogram boundaries. In practice, automatic stitching alone provides only an incomplete solution, because large physical distortions and a low signal-to-noise ratio often cause experimental difficulties. To derive 3D models of spindles despite dealing with imperfect data related to sample preparation and subsequent data collection, semi-automatic validation and correction is required to remove stitching mistakes. However, due to the large number of MTs in spindles (up to 30k) and their resulting dense spatial arrangement, a naive inspection of each MT is too time-consuming. Furthermore, an interactive visualization of the full image stack is hampered by the size of the data (up to 100 GB). Here, we present a specialized, interactive, semi-automatic solution that considers all requirements for large-scale stitching of filamentous structures in serial-section image stacks. To the best of our knowledge, it is the only currently available tool which is able to process data of the type and size presented here. The key to our solution is a careful design of the visualization and interaction tools for each processing step to guarantee real-time response, and an optimized workflow that efficiently guides the user through datasets. The final solution presented here is the result of an iterative process with tight feedback loops between the involved computer scientists and cell biologists. Y1 - 2021 U6 - https://doi.org/10.1111/jmi.13039 VL - 284 IS - 1 SP - 25 EP - 44 ER - TY - JOUR A1 - Kiewisz, Robert A1 - Baum, Daniel A1 - Müller-Reichert, Thomas A1 - Fabig, Gunar T1 - Serial-section electron tomography and quantitative analysis of the microtubule organization in 3D-reconstructed mitotic spindles JF - Bio-protocol Y1 - 2023 U6 - https://doi.org/10.21769/BioProtoc.4849 VL - 13 IS - 20 ER - TY - JOUR A1 - Okafornta, Chukwuebuka William A1 - Farhadifar, Reza A1 - Fabig, Gunar A1 - Wu, Hai-Yin A1 - Köckert, Maria A1 - Vogel, Martin A1 - Baum, Daniel A1 - Haase, Robert A1 - Shelley, Michael J. A1 - Needleman, Daniel J. A1 - Müller-Reichert, Thomas T1 - Cell size reduction scales spindle elongation but not chromosome segregation in C. elegans JF - bioRxiv N2 - 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. Y1 - 2025 U6 - https://doi.org/10.1101/2025.10.13.681585 ER - TY - JOUR A1 - Lantzsch, Ina A1 - Yu, Che-Hang A1 - Chen, Yu-Zen A1 - Zimyanin, Vitaly A1 - Yazdkhasti, Hossein A1 - Lindow, Norbert A1 - Szentgyoergyi, Erik A1 - Pani, Ariel M A1 - Prohaska, Steffen A1 - Srayko, Martin A1 - Fürthauer, Sebastian A1 - Redemann, Stefanie T1 - Microtubule reorganization during female meiosis in C. elegans JF - eLife N2 - Most female meiotic spindles undergo striking morphological changes while transitioning from metaphase to anaphase. The ultra-structure of meiotic spindles, and how changes to this structure correlate with such dramatic spindle rearrangements remains largely unknown. To address this, we applied light microscopy, large-scale electron tomography and mathematical modeling of female meiotic \textit{Caenorhabditis elegans} spindles. Combining these approaches, we find that meiotic spindles are dynamic arrays of short microtubules that turn over within seconds. The results show that the metaphase to anaphase transition correlates with an increase in microtubule numbers and a decrease in their average length. Detailed analysis of the tomographic data revealed that the microtubule length changes significantly during the metaphase-to-anaphase transition. This effect is most pronounced for microtubules located within 150 nm of the chromosome surface. To understand the mechanisms that drive this transition, we developed a mathematical model for the microtubule length distribution that considers microtubule growth, catastrophe, and severing. Using Bayesian inference to compare model predictions and data, we find that microtubule turn-over is the major driver of the spindle reorganizations. Our data suggest that in metaphase only a minor fraction of microtubules, those closest to the chromosomes, are severed. The large majority of microtubules, which are not in close contact with chromosomes, do not undergo severing. Instead, their length distribution is fully explained by growth and catastrophe. This suggests that the most prominent drivers of spindle rearrangements are changes in nucleation and catastrophe rate. In addition, we provide evidence that microtubule severing is dependent on katanin. Y1 - 2021 U6 - https://doi.org/10.7554/eLife.58903 VL - 10 SP - e58903 ER -