Functional analysis after rapid degradation of condensins and 3D-EM reveals chromatin volume is uncoupled from chromosome architecture in mitosis

Abstract

The requirement for condensin in chromosome formation in somatic cells remains unclear, as imperfectly condensed chromosomes do form in cells depleted of condensin by conventional methodologies. In order to dissect the roles of condensin at different stages of vertebrate mitosis, we have established a versatile cellular system that combines auxin-mediated rapid degradation with chemical genetics to obtain near-synchronous mitotic entry of chicken DT40 cells in the presence and absence of condensin. We analyzed the outcome by live- and fixed-cell microscopy methods, including serial block face scanning electron microscopy with digital reconstruction. Following rapid depletion of condensin, chromosomal defects were much more obvious than those seen after a slow depletion of condensin. The total mitotic chromatin volume was similar to that in control cells, but a single mass of mitotic chromosomes was clustered at one side of a bent mitotic spindle. Cultures arrest at prometaphase, eventually exiting mitosis without segregating chromosomes. Experiments where the auxin concentration was titrated showed that different condensin levels are required for anaphase chromosome segregation and formation of a normal chromosome architecture.This article has an associated First Person interview with the first author of the paper.

Ki-67 and condensins support the integrity of mitotic chromosomes through distinct mechanisms

“…S2G). Consistent with this, a very recent study in which the AID system was applied to deplete SMC2 from chicken DT40 cells has reported similar spindle phenotypes (Samejima et al, 2018).…”

The dynamics of centromere motion through the metaphase-to-anaphase transition reveal a centromere separation order

“…One of the crucial tasks of mitosis is to correctly segregate DNA, compactly packaged into chromosomes, to the two daughter cells. Upon mitotic entry, chromatin undergoes a 2-3 fold compaction and a dramatic re-organisation into a higher-order structure to form rod-shaped chromosomes [1][2][3][4] . After nuclear envelope disassembly, the chromosomes then attach to the mitotic spindle via kinetochores during prometaphase and congress to the equator of the spindle where they form the metaphase plate.…”

“…The condensin protein complexes are fundamental in establishing and regulating chromosome morphology during mitosis 2,6,7 . Eukaryotes have two condensin complexes -condensin I and condensin II -which fulfil non-overlapping roles throughout the cell cycle, are differentially regulated and have differing chromosome binding profiles during the cell cycle 4,8,9 .…”

Transcription factor Sp1 regulates mitotic fidelity through Aurora B kinase-mediated condensin I localization

“…Rapid Sp1 depletion results in micronuclei formation, increased mitotic duration, aberrant metaphase alignment, and monopolar spindle formation. These phenotypes are all associated with defective chromosome condensation (Hirota et al, 2004;Martin et al, 2016;Samejima et al, 2018;Samoshkin et al, 2009). We therefore hypothesized that Sp1 regulates chromosome condensation during mitosis.…”