Three-dimensional structure of kinetochore-fibers in human mitotic spindles

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.

Self-organization of kinetochore-fibers in human mitotic spindles

“…We defined KMTs as MTs with one end near a kinetochore in the reconstructions and assigned the plus end to the end at the kinetochore and the minus end to the opposite end of the MT ( Figure 1A ). KMT minus ends are located throughout the spindle, with approximately 51% of them more than 1.7μm away from the pole, as found in Kiewisz et al, 2022 ( Figure 1B ). We defined the location of the pole as the center of the mother centriole.…”

“…We first analyzed a recent cellular tomography electron microscopy (EM) reconstruction data set which captured the trajectories of every MT in the mitotic spindle of three HeLa cells ( Kiewisz et al, 2022 ). We defined KMTs as MTs with one end near a kinetochore in the reconstructions and assigned the plus end to the end at the kinetochore and the minus end to the opposite end of the MT ( Figure 1A ).…”

“…( B ) The frequency of 3D minus end distance from the pole. Inset: the fraction of minus ends within 1.7µm of the pole (as shown in Kiewisz et al, 2022 ). ( C ) A sample k-Fiber.…”

“…Black line: dual-exponential fit to the fit height of the peak over time. ( F ) Sample ultrastructure from a 3D spindle reconstructed by electron tomography ( Kiewisz et al, 2022 ). KMTs are shown in red, non-KMTs yellow.…”

“…In a companion paper, we used serial-section electron tomography to reconstruct the locations, lengths, and configurations of MTs in metaphase spindles in HeLa cells ( Kiewisz et al, 2022 ). These whole spindle reconstructions can unambiguously identify which MTs are bound to the kinetochore and measure their lengths, providing a remarkable new tool for the study of KMTs.…”