Self-organization of kinetochore-fibers in human mitotic spindles

Abstract

During eukaryotic cell division, chromosomes are linked to microtubules (MTs) in the spindle by a macromolecular complex called the kinetochore. The bound kinetochore microtubules (KMTs) are crucial to ensuring accurate chromosome segregation. Recent reconstructions by electron tomography (Kiewisz et al. 2021) captured the positions and configurations of every MT in human mitotic spindles, revealing that roughly half the KMTs in these spindles do not reach the pole. Here, we investigate the processes that give rise to this distribution of KMTs using a combination of analysis of large-scale electron tomography, photoconversion experiments, quantitative polarized light microscopy, and biophysical modeling. Our results indicate that in metaphase, KMTs grow away from the kinetochores along well-defined trajectories, with the speed of the KMT minus ends continually decreasing as the minus ends approach the pole, implying that longer KMTs grow more slowly than shorter KMTs. The locations of KMT minus ends, and the turnover and movements of tubulin in KMTs, are consistent with models in which KMTs predominately nucleate de novo at kinetochores in metaphase and are inconsistent with substantial numbers of non-KMTs being recruited to the kinetochore in metaphase. Taken together, this work leads to a mathematical model of the self-organization of kinetochore-fibers in human mitotic spindles.

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

“…In this parallel study, we supplemented our electron tomography data on the KMT length distribution with light microscopic data. Essentially, our 3D reconstructions show a distribution of KMT length in metaphase that is strikingly similar to the distribution plot of KMT length as obtained by biophysical modeling in combination with light microscopy (see Figure 8B-D in Conway et al, 2022 ). All this shows that light and electron microscopy produces truly complementary data, although completely different methods of sample preparation and data analysis have to be applied.…”

“…Such liquid crystals can be characterized by the degree of local MT alignment, expressed by the nematic order parameter. Interestingly, the analyzed spindles show a high nematic order parameter (S = 0.81 ± 0.02) near the chromosomes, whereas the nematic order parameter (S = 0.54 ± 0.02) is lower at the spindle poles ( Conway et al, 2022 ). Along this line, KMTs in our electron tomography study are well aligned in the middle of the spindle, while the order of the KMTs in the k-fibers is progressively lost at positions closer to the spindle poles.…”

“…In the HeLa spindles, this was 1.7 µm from the mother centriole. We applied the same cutoff in a parallel study on the dynamics of mammalian k-fibers (see Figure 1 in Conway et al, 2022 ).…”

“…Counting the total number of KMTs and non-KMTs in our spindles, we show that only ~14% of all MTs in the reconstructed spindles were KMTs. However, this percentage in the total number of all MTs corresponds to ~25% of the tubulin mass as measured in parallel by light microscopy ( Conway et al, 2022 ). Comparing the average length of KMTs and non-KMTs, we also find that KMTs are on average twice as long as non-KMT.…”

“…As previously noted, we have combined our 3D reconstructions with additional live-cell imaging and biophysical modeling in a parallel publication ( Conway et al, 2022 ). Combining data on the length and the position of KMT minus ends in spindles (as obtained here by electron microscopy), and the turnover and movement of tubulin in KMTs as generated by light microscopy, a model was proposed in which KMTs predominantly nucleate de novo at kinetochores, with KMTs growing towards the spindle poles.…”