Abstract
The two Condensin complexes in human cells are essential for mitotic chromosome structure. We used homozygous genome editing to fluorescently tag Condensin I and II subunits and mapped their absolute abundance, spacing and dynamic localization during mitosis by fluorescence correlation spectroscopy-calibrated live cell imaging and superresolution microscopy. While ~35,000 Condensin II complexes are stably bound to chromosomes throughout mitosis, ~195,000 Condensin I complexes dynamically bind in two steps, in prometaphase and early anaphase. The two Condensins rarely co-localize at the chromatid axis, where Condensin II is centrally confined but Condensin I reaches ~50% of the chromatid diameter from its center. Based on our comprehensive quantitative data, we propose a three-step hierarchical loop model of mitotic chromosome compaction: Condensin II initially fixes ~450 kb average sized loops at the chromatid axis whose size is then reduced by Condensin I binding to ~90 kb in prometaphase and ~70 kb in anaphase, achieving maximum chromosome compaction upon sister chromatid segregation.
Abbreviations
- AB
- antibody
- APD
- avalanche photodiode
- FCS
- fluorescence correlation spectroscopy
- FWHM
- full width at half maximum
- GaAsP
- Gallium arsenide phosphide
- HK
- HeLa Kyoto
- IN
- input
- IP
- immunoprecipitation / eluate
- NEBD
- nuclear envelope breakdown
- Noc
- nocodazole
- ON
- overnight
- POI
- protein of interest
- ROI
- region of interest
- SB
- Southern Blot
- SMC
- structural maintenance of chromosomes
- SN
- supernatant
- STED
- stimulated emission depletion
- WB
- Western Blot
- ZFN
- zinc finger nuclease