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 - 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 - 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 - Laguillo-Diego, Alejandra A1 - Kiewisz, Robert A1 - Martí-Gómez, Carlos A1 - Baum, Daniel A1 - Müller-Reichert, Thomas A1 - Vernos, Isabelle T1 - MCRS1 modulates the heterogeneity of microtubule minus-end morphologies in mitotic spindles JF - Molecular Biology of the Cell N2 - 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. Y1 - 2022 U6 - https://doi.org/10.1091/mbc.E22-08-0306-T VL - 34 IS - 1 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 - 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 - 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 - Kiewisz, Robert A1 - Fabig, Gunar A1 - Conway, William A1 - Baum, Daniel A1 - Needleman, Daniel A1 - Müller-Reichert, Thomas T1 - Three-dimensional structure of kinetochore-fibers in human mitotic spindles JF - eLife N2 - 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. Y1 - 2022 U6 - https://doi.org/10.7554/eLife.75459 VL - 11 SP - e75459 ER -