Figure 5. WAPL-1 antagonizes binding of COH-3/4 cohesin to axial elements.

(A) Projections of pachytene nuclei stained with the indicated antibodies and DAPI. In all cases, WT and wapl-1 examples were acquired with the same exposure settings and images are non-deconvolved projections adjusted with same settings to allow visual comparisons in staining intensity. The intensities of SMC-3 and COH-3/4 are increased in wapl-1 mutants, while REC-8 staining appears similar in WT and wapl-1 nuclei. Scale bar = 5 µm. (B) Quantification of mean fluorescence intensity per nucleus of REC-8, COH-3/4 and SMC-3 in transition zone (TZ), early pachytene (EP) and late pachytene (LP) nuclei. Between 15 and 20 nuclei per germ line, from a minimum of 5 germ lines were analyzed per genotype and stage. Differences indicated with asterisks are significant (p<0.0001, t-test), error bars= SEM. (C) Line profile quantification to compare the intensity of cohesin at axial elements versus inter-chromosome domains. Top left-hand side panel: example of a SMC-3 and DAPI-stained pachytene nucleus showing the intensities of DAPI (blue) and SMC-3 (green) along the depicted line. The line profile of SMC-3 intensity is shown in the top right-hand panel, ΔF indicates the increment in staining between the peak (axial element) and the valley (inter chromosome domain as determined by lack of DAPI staining). Graph: Plotting of individual ΔF values from late pachytene nuclei of wapl-1 mutants and WT stained with REC-8, COH-3/4 or SMC-3 antibodies. Between 12 and 32 nuclei from different germ lines were analyzed per genotype. Mean value and SEM are indicated. Proportional increase between the mean ΔF value in wapl-1 and WT: 27% for REC-8 (p= 0.0008, t-test), 114% for COH-3/4 (p<0.0001, t-test), 59% for SMC-3 (p<0.0001, t-test). (D) Western blots of triton soluble and insoluble (DNA bound) protein fractions from WT and wapl-1 mutant worms probed with anti-COH-4 (see Figure 5—figure supplement 5 for additional controls), anti-HAL-2 (marker for soluble fraction), and anti-H3 (marker for DNA-bound fraction) antibodies. Asterisk indicates a non-specific band recognized by anti-COH-4 antibodies (see Figure 5—figure supplement 5). Note that COH-4 signal in WT extracts is higher in the soluble than in the DNA fraction, while in wapl-1 mutant extracts COH-4 intensity is higher in DNA-bound than in the soluble fraction. (E) Quantification of relative intensity of anti-COH-4 signal in the soluble and DNA-bound fractions. Three westerns were included in the analysis.

DOI: http://dx.doi.org/10.7554/eLife.10851.019

Figure 5—figure supplement 1. Controls showing specificity of anti-COH-3/4 antibodies.

Projections of pachytene nuclei stained with the anti-COH-3/4 antibodies and DAPI. Note that signal from anti-COH-3/4 antibodies only disappears in germ lines from coh-3 coh-4 double mutants, demonstrating that the antibodies recognize both COH-3 and COH-4.

DOI: http://dx.doi.org/10.7554/eLife.10851.020

Figure 5—figure supplement 2. A different anti-COH-3 antibody also shows increased staining intensity in pachytene nuclei of wapl-1 mutant germ lines.

WT and wapl-1 examples were acquired with the same exposure settings and images are non-deconvolved projections adjusted with same display settings to allow visual comparisons in staining intensity. Chromatin was stained with DAPI (blue). Graph shows quantification of mean fluorescence intensity in pachytene nuclei stained with anti-COH-3 antibodies. Differences between WT and wapl-1 are significant (p<0.0001, t-test), error bars = SEM.

DOI: http://dx.doi.org/10.7554/eLife.10851.021

Figure 5—figure supplement 3. A REC-8::GFP transgene shows similar staining intensity in pachytene nuclei of WT and wapl-1 mutant germ lines.

WT and wapl-1 examples were acquired with the same exposure settings and images are non-deconvolved projections adjusted with same display settings to allow visual comparisons in staining intensity. Chromatin was stained with DAPI (blue). Graph shows quantification of mean fluorescence intensity in pachytene nuclei stained with anti-GFP antibodies. Differences between WT and wapl-1 are not significant, error bars = SEM.

DOI: http://dx.doi.org/10.7554/eLife.10851.022

Figure 5—figure supplement 4. Controls demonstrating the functionality of the REC-8::GFP transgene.

Expression of the REC-8::GFP transgene shown in Figure 4—figure supplement 3 rescues the defects in chiasma formation of rec-8 mutants, demonstrating that the REC-8::GFP fusion protein provides full REC-8 function. Scale bar = 5 µm.

DOI: http://dx.doi.org/10.7554/eLife.10851.023

Figure 5—figure supplement 5. Control western blots for anti-COH-4 antibodies.

Left-hand side western blot: whole worm extracts from indicated genotypes labeled with anti-COH-4 antibodies. A band of the expected molecular weight for COH-4 is present in WT and coh-3 mutant extracts, but not in coh-4 single or coh-3 coh-4 double mutant extracts, demonstrating that the antibodies specifically recognize COH-4. Asterisks indicate two non-specific bands recognized by the antibodies as they are present in extracts from coh-4 single and coh-3 coh-4 double mutants. Actin antibodies are used as loading control. Right-hand side western blot: Protein fractionation (soluble and DNA bound) from WT and coh-4 mutant extracts labeled with anti-COH-4 antibodies. Note that the band corresponding to COH-4 is only detected in WT (arrowheads), with the signal being stronger in the soluble fraction. Two non-specific bands are detected in the soluble fraction of both WT and coh-4 mutant extracts (asterisks). Anti-HAL-2 antibodies were used as a marker for the soluble protein fraction and anti-histone H3 antibodies were used as a marker for the DNA-bound protein fraction.

DOI: http://dx.doi.org/10.7554/eLife.10851.024