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Kinetochores can organize the mitotic spindle without assistance from chromosome arms or centrosomes, report O'Connell et al.
Several pathways contribute to assembly of the microtubule-based mitotic machinery that segregates chromosomes. Microtubules grow from centrosomes and capture chromosomes by attaching to kinetochore complexes at their centromeres. Microtubules originating near the chromosomes are also involved, and can even form a spindle in the complete absence of centrosomes. The integration of these pathways is thought to rely on a gradient of RanGTP, a small GTPase activated by a nucleotide exchange factor present on chromatin that liberates microtubule nucleation and stabilization factors in the vicinity of chromosomes.
O'Connell et al. were thus puzzled by the organization of spindles in HeLa cells undergoing mitosis with unreplicated genomes (MUG), a situation in which kinetochores and centromeric DNA are separated from all other chromatin. This unusual event—induced by prolonged treatment with hydroxyurea—results in the formation of a robust spindle prominently centered on kinetochores, while the bulk of the chromatin is pushed out to the cell periphery. This sidelined chromatin still produced high levels of RanGTP but the kinetochores of MUG cells lay outside this region, indicating that they need not reside within the gradient peak to organize and attach to spindle microtubules. Knocking down a key kinetochore protein called Nuf2 did prevent spindle assembly, highlighting the importance of kinetochores in this process.
This suggests that kinetochores are the dominant factor in building mitotic spindles. Human cells undergoing MUG provide a unique system to reveal spatial cues that direct microtubule growth in vivo, says author Chris O'Connell. Previously, these experiments could only be conducted in vitro, such as assays with egg extracts.