Figure 1. A system to acutely remove... | Rockefeller University Press

Figure 1.

$Figure 1. A system to acutely remove variable cohesin amounts from metaphase chromosomes. (A) Experimental approach used to titrate the amount of TEV-resistant cohesin complexes (green circles) upon TEV-induced removal of TEV-cleavable cohesin fraction (brown circles). Variable ratios of these two cohesin versions allow testing of how different cohesin levels sustain chromatid cohesion. (B) Probabilistic models for functional cohesive links (y) relative to cohesin left on chromosomes (x) based on possible models for cohesion. Single ring model: y = x. Handcuff model: y = (x/100)2, assuming random and independent distribution of TEV-resistant/TEV-cleavable rings. Dashed lines correspond with the cohesin level threshold identified in this study. (C) Representative chromosome spreads of Rad21WT-EGFP (green) and DNA (Hoechst) from the established strains. Bars, 2 µm. (D) Relative mean fluorescence intensity of Rad21WT-EGFP within chromosomal area (Hoechst staining) measured in different strains and normalized for 100% Rad21WT. n = 27, 28, 22, 26, 36, 29, and 21 individual chromosomes from at least three independent embryo spreads. Mean ± SD; see also Fig. S1.$

A system to acutely remove variable cohesin amounts from metaphase chromosomes. (A) Experimental approach used to titrate the amount of TEV-resistant cohesin complexes (green circles) upon TEV-induced removal of TEV-cleavable cohesin fraction (brown circles). Variable ratios of these two cohesin versions allow testing of how different cohesin levels sustain chromatid cohesion. (B) Probabilistic models for functional cohesive links (y) relative to cohesin left on chromosomes (x) based on possible models for cohesion. Single ring model: y = x. Handcuff model: y = (x/100)², assuming random and independent distribution of TEV-resistant/TEV-cleavable rings. Dashed lines correspond with the cohesin level threshold identified in this study. (C) Representative chromosome spreads of Rad21^WT-EGFP (green) and DNA (Hoechst) from the established strains. Bars, 2 µm. (D) Relative mean fluorescence intensity of Rad21^WT-EGFP within chromosomal area (Hoechst staining) measured in different strains and normalized for 100% Rad21^WT. n = 27, 28, 22, 26, 36, 29, and 21 individual chromosomes from at least three independent embryo spreads. Mean ± SD; see also Fig. S1.