Abstract
During mitosis, the spindle undergoes morphological and dynamic changes. It reorganizes at the onset of the anaphase when the antiparallel bundler PRC1 accumulates and recruits central spindle proteins to the midzone. Little is known about how the dynamic properties of the central spindle change during its morphological changes in human cells. Using gene editing, we generated human cells that express from their endogenous locus fluorescent PRC1 and EB1 to quantify their native spindle distribution and binding/unbinding turnover. EB1 plus end tracking revealed a general slowdown of microtubule growth, whereas PRC1, similar to its yeast orthologue Ase1, binds increasingly strongly to compacting antiparallel microtubule overlaps. KIF4A and CLASP1 bind more dynamically to the central spindle, but also show slowing down turnover. These results show that the central spindle gradually becomes more stable during mitosis, in agreement with a recent "bundling, sliding, and compaction" model of antiparallel midzone bundle formation in the central spindle during late mitosis.
Highlights
During cell division, the mitotic spindle that is responsible for segregating the two sets of chromosomes to the new daughter cells, undergoes dramatic morphological changes
To visualize how the microtubule network reorganizes in human cells during mitosis under close-to-native conditions, we generated hTERT-RPE1 cell lines in which Protein regulator of cytokinesis 1 (PRC1) and EB1 were fluorescently tagged at their endogenous locus using CRISPR/Cas9 gene editing
Confocal microscopy imaging demonstrated that endogenously expressed EB1 with a N-terminal monomeric green fluorescent protein (mGFP) tag preferentially localized to growing microtubule ends both in interphase and during mitosis (Video 1), as previously observed for overexpressed C-terminally tagged EB1 [57] or N-terminally tagged EB3 [42]. mGFP-EB1 accumulated to spindle poles as noted previously [63]
Summary
The mitotic spindle that is responsible for segregating the two sets of chromosomes to the new daughter cells, undergoes dramatic morphological changes. During anaphase which lasts ~10 min in animal cells, chromosomes are pulled to the spindle poles, whereas the spindle dynamically reorganizes and elongates [1]. As the overall microtubule network becomes less dynamic, the spindle concentrates antiparallel microtubule overlaps in its centre, a region called the midzone, that is critically important for spindle stability and function, as well as for intracellular signalling events ensuring correct cytokinesis [7, 8]. These antiparallel microtubule overlaps are formed and stabilised by microtubule-crosslinking proteins. All midzone proteins generate central antiparallel microtubule overlaps which have a length of ~2 μm in animal cells [16, 17, 29, 30]
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