Speakers
Description
Cell division is driven by the mitotic spindle, a self-organized macromolecular machine that assembles in prophase. During metaphase, it comprises microtubule (MT) kinetochore fibers connecting chromosomes to the bipolar MT network of the spindle, and astral MTs extending from the poles to the cell cortex. With the onset of anaphase, the mitotic spindle undergoes significant rearrangement. Shortening of kinetochore fibers moves the sister chromatids toward the poles, separating the chromosomes. Simultaneously, several motor and MT-associated proteins relocate to interpolar MTs, bundling their antiparallel plus ends at the central spindle (or midzone), essential to maintain proper spindle architecture and ensure accurate chromosome segregation. In vitro studies using purified proteins have identified the central spindle proteins PRC1 and KIF4A as a minimal set of components capable of organising MTs into bundles with central antiparallel MT plus-end overlaps that resemble anaphase midzone bundles. Their organisation was previously visualised using fluorescence microscopy. To gain higher-resolution insights into their three-dimensional architecture, we modified the procedure of minimal midzone bundle self-organisation to allow their visualisation by cryo-electron tomography (cryo-ET). We reduced the size of the reconstituted minimal midzone bundles while preserving other characteristic midzone features. Preliminary data indicate that we can now successfully image these reconstituted anaphase-like bundles using cryo-ET. This approach will enable a more detailed analysis of microtubule arrangements in reconstituted dense microtubule networks moving forward.