In contrast, tomographic analysis of RPE1 cells revealed 12.6 ± 1.7 KMTs per k-fiber ( O’Toole et al., 2020). Nevertheless, by applying serial thin-section TEM it was reported that k-fibers in PtK1 cells are composed of about 20 KMTs ( McDonald et al., 1992 McEwen et al., 1997). However, these prior studies did not present comprehensive 3D reconstructions of mammalian mitotic spindles. Other studies used scanning electron microscopy to analyze the ultrastructure of mitotic spindles ( Hoffman et al., 2020 Nixon et al., 2017 Nixon et al., 2015). Some studies used serial thin-section transmission electron microscopy (TEM) ( Khodjakov et al., 1997 Mastronarde et al., 1993 McDonald et al., 1992 McEwen and Marko, 1998 Sikirzhytski et al., 2014) or partial 3D reconstruction by electron tomography ( O’Toole et al., 2020 Yu et al., 2019).
Earlier 3D studies on mammalian spindles applied several techniques. However, our understanding of the ultrastructure of KMTs in mammalian k-fibers is rather limited due to a low number of three-dimensional (3D) studies on spindle organization. The regulation of KMT dynamics in mitotic spindles has been studied in great detail in a number of different systems, including the early Caenorhabditis elegans embryo, Xenopus egg extracts and mammalian tissue culture cells ( DeLuca et al., 2006 Dumont and Mitchison, 2009 Farhadifar et al., 2020 Inoué and Salmon, 1995 Kuhn and Dumont, 2019 Long et al., 2020). These MTs are called kinetochore microtubules (KMTs) and function to establish a physical connection between the chromosomes and the rest of the spindle ( Flemming, 1879 Khodjakov et al., 1997 Maiato et al., 2004 Musacchio and Desai, 2017 Rieder, 1981 Rieder and Salmon, 1998). While mitotic spindles can contain thousands of MTs, only a fraction of those highly dynamic filaments is associated with the kinetochores ( Redemann et al., 2017). Editor's evaluationĬhromosome segregation during cell division is carried out by microtubule (MT)-based spindles ( Anjur-Dietrich et al., 2021 McIntosh et al., 2013 Oriola et al., 2018 Prosser and Pelletier, 2017). 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. 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). 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. We found that k-fibers exhibit remarkable variation in circumference and KMT density along their length, with the pole-proximal side showing a broadening. Our comprehensive reconstructions allowed us to analyze the three-dimensional (3D) morphology of k-fibers and their surrounding MTs in detail. 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. 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. During cell division, kinetochore microtubules (KMTs) provide a physical linkage between the chromosomes and the rest of the spindle.
0 kommentar(er)
