Microtubule based spindles are a complex three-dimensional apparatus that functions to ensure faithful segregation of chromosomes during cell division in mitosis and meiosis. Our current understanding of spindle architecture is mainly based on a plethora of information derived from light microscopy with rather few insights about spindle ultrastructure obtained from 3D electron microscopy. Recently, serial-section electron tomography has delivered detailed reconstructions of complete mitotic and meiotic spindles in the early C. elegans embryo. We set out to reconstruct the complex spatial arrangements of microtubules in C. elegans spindles to understand how spindles are assembled and how forces are generated to faithfully segregate chromosomes. Recent studies have highlighted the significance of the spindle midzone, the region positioned between chromosomes, in ensuring proper chromosome segregation. By combining advanced 3D electron tomography and cutting-edge light microscopy we have discovered a previously unknown role of the regulation of microtubule dynamics within the spindle midzone of C. elegans. Using Fluorescence recovery after photobleaching and a combination of second harmonic generation and two-photon fluorescence microscopy, we found that the length of the antiparallel microtubule overlap zone in the spindle midzone is constant throughout anaphase, and independent of cortical pulling forces as well as the presence of the microtubule bundling protein SPD-1. Further investigations of SPD-1 and the chromokinesin KLP-19 in C. elegans suggest that KLP-19 regulates the overlap length and functions independently of SPD-1. Our data shows that KLP-19 plays an active role in regulating the length and turn-over of microtubules within the midzone as well as the size of the antiparallel overlap region throughout mitosis. Depletion of KLP-19 in mitosis leads to an increase in microtubule length in the spindle midzone, which also leads to increased microtubule-microtubule interaction, thus building up a more robust microtubule network. The spindle is globally stiffer and more stable, which has implications for the transmission of forces within the spindle affecting chromosome segregation dynamics. Our data shows that by localizing KLP-19 to the spindle midzone in anaphase microtubule dynamics can be locally controlled allowing the formation of a functional midzone. Lastly, we have taken first steps to use Cryo-electron microscopy of focused ion-beam lamellae to obtain further detailed insight into the microtubule morphology in the spindle [1].
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Zimyanin et al. (2024) studied this question.