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Fig. 1 | BMC Biology

Fig. 1

From: The ESCRT-III isoforms CHMP2A and CHMP2B display different effects on membranes upon polymerization

Fig. 1

Interaction of CHMP2A versus CHMP2B with charged model membranes. a Confocal images of 10% PI (4,5)P2-GUVs incubated with 500 nM CHMP2B-ΔC (first line) (called CHMP2B) and MBP-CHMP2A-ΔC (third line) (called CHMP2A) alone or in combination with 2 μM CHMP3 (second and fourth lines, respectively). A single confocal plane is shown. Scale bar, 10 μm. Note that in the case of MBP-CHMP2A-ΔC (third line), the laser intensity in the protein channel has been increased (as visible by the higher background intensity) to detect protein binding on the GUV membrane. b Effect of CHMP3 on MBP-CHMP2A-ΔC and CHMP2B-ΔC binding to 10% PI (4,5)P2-GUVs (same conditions as in a). The fluorescence intensity was measured from the analysis of spinning disk microscopy images using the Cell Profiler software. The fluorescence intensity of MBP-CHMP2A-ΔC+CHMP3 and CHMP2B-ΔC+CHMP3-covered vesicles was normalized to the intensity of MBP-CHMP2A-ΔC and CHMP2B-ΔC-covered vesicles, respectively. **p-value < 0.01; ***p-value < 0.001 (Student’s t-test). N = 48. c Quantification by FACS of the fluorescence intensities of MBP-CHMP2A-ΔC ± CHMP3, CHMP2B-ΔC ± CHMP3 and CHMP3 co-polymers bound to 10% PI(4,5)P2-containing GUVs. The concentrations of CHMP2A/B and CHMP3 proteins are, respectively, 500 nM and 2 μM. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001 (Student’s t-test). N = 4 (number of FACS experiment with about 104 counted events per experiment, per condition). d Quantification of CHMP2B-FL, CHMP2B-ΔC, and MBP-CHMP2A-ΔC (CHMP2A) + CHMP3 binding to GUVs containing DOPS and different PIPs by flow cytometry (FACS). Equimolar amount of DOPS and different PIPs (2% mol/mol of total lipids) have been used. Note that data on CHMP2B-ΔC binding to DOPS, PI(4,5)P2, and PI(3,4)P2 were already published [48]. Binding efficiencies were normalized to the fluorescence intensity of DOPS-containing vesicles (see Figure S2.D). These values were then normalized to the total amount of charge for each lipid composition. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001 (Student’s t-test). N = 6 (number of FACS experiment with about 104 counted events per experiment, per condition). e Resonance frequency shift ϑ5 in the QCM-D experiments when CHMP2B-ΔC is bound to the different types of supported lipid bilayers (light gray 30% DOPS, 70% DOPC; gray 40% DOPS, 60% DOPC; light blue 10% DOPS, 10% PI(4,5)P2, 80% DOPC, magenta 10% DOPS, 10% PI(3,4,5)P3, 80% DOPC). *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001 (Student’s t-test). N = 5

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