Journal: bioRxiv

Article Title: TMC1 and TMC2 are cholesterol-dependent scramblases that regulate membrane homeostasis in auditory hair cells

doi: 10.1101/2025.07.03.663083

Figure Lengend Snippet: A. NBD-fluorescence decrease after SDT addition to POPC/PE/PS liposomes (black) or proteo-liposomes containing CmTMC1 purified in DDM/CHS (red) or FC12 (pink) or MuTMC2 purified in DDM/CHS (blue) or FC12 (light blue). B. NBD-fluorescence decrease after SDT addition to POPC/PE/PS liposomes containing 10% cholesterol (black) or proteo-liposomes containing CmTMC1 purified in DDM/CHS (red) or FC12 (pink) or MuTMC2 purified in DDM/CHS (blue) or FC12 (light blue). C. Quantification of the SDT-(yellow) and BSA-(grey) PLS activity of CmTMC1 (left) or MuTMC2 (right) purified in the conditions represented in A and B. D. NBD-fluorescence decrease after SDT addition to POPC/PE/PS liposomes (black) or CmTMC1 proteo-liposomes (red) containing increasing amounts of cholesterol (left) and quantification of CmTMC1 PLS activity in these assays (right). E. Decrease of NBD-fluorescence after SDT addition to POPC/PE/PS liposomes (black) or MuTMC2 proteo-liposomes (blue) containing increasing amounts of cholesterol (left) and quantification of MuTMC2 PLS activity in these conditions (right). H. NBD-fluorescence decrease after SDT addition to POPC liposomes (black) or proteo-liposomes containing CmTMC1 (red) or MuTMC2 (blue). I. Decrease of NBD-fluorescence after SDT addition to POPC liposomes containing 10% cholesterol (black) or proteo-liposomes containing CmTMC1 (red) or TMC2 (blue). J. Quantification of the SDT-(yellow) and BSA-based (grey) PLS activity of CmTMC1 (left) or MuTMC2 (right) of the representative data shown in H and I. K. CmTMC1 (left) or MuTMC2 (right) PLS activity, normalized by average PLS activity at 10% cholesterol, after cholesterol addition via treatment with empty mβCD (control) or mβCD/CHO complexes with increasing cholesterol amounts. Grey bars indicate PLS activity at 0% (bottom) or 10% (top) cholesterol. Mean ± SEM is shown for C, D, E, J and K. Each dot represents one independent experiment. Two (C) or one-way ANOVA (D-E, J-K) was performed to evaluate statistical significance. P values are indicated as: ns≥0.05, *<0.05, **<0.01, ***<0.001 and ****<0.0001. See also Supplemental Fig. 6A-D.

Article Snippet: For cholesterol addition experiments, POPC liposomes and proteo-liposomes were prepared as described above, except with 18:1-12:0 NBD-PE (Avanti 819156) rather than 14:0-06:0 NBD-PE to reduce NBD lipid extraction by methyl-β-cyclodextrin (mβCD).

Techniques: Fluorescence, Liposomes, Purification, Activity Assay, Control

Journal: bioRxiv

Article Title: TMC1 and TMC2 are cholesterol-dependent scramblases that regulate membrane homeostasis in auditory hair cells

doi: 10.1101/2025.07.03.663083

Figure Lengend Snippet: NBD-fluorescence decrease after SDT addition to POPC/PE/PS liposomes (black) or MmTMC1 (red), or MmTMC2 (blue) proteo-liposomes containing 10% (A), 0% (B), or 30% cholesterol (C). D. Quantification of the MmTMC1 and MmTMC2 SDT-(yellow) and BSA-based (grey) PLS activity in the absence or presence of 10% and 30% cholesterol. Mean ± SEM is shown, and each dot represents one independent experiment. Two-way ANOVA was performed to evaluate statistical significance. E. Representative confocal images of auditory OHCs and IHCs at the middle cochlear region from P6 wild-type mice incubated in HBSS in the absence (control) or presence of benzamil, mβCD, or mβCD loaded with cholesterol (mβCD/CHO) and labeled with annexin V (magenta) and spy-555 (green) to visualize PS and F-actin, respectively. F. Quantification of background-subtracted annexin V-647 fluorescence intensity from the experimental conditions represented in E. Each dot represents the annexin V fluorescence intensity from one hair cell. Mean ± SEM is shown for n ∼ 100 OHCs and 30 IHCs from 3 tissues. One-way ANOVA was performed to evaluate statistical significance between control and treated samples. P values are indicated as: ns ≥ 0.05, ** < 0.01, and **** < 0.0001. See also Supplemental Fig. 6E-F.

Article Snippet: For cholesterol addition experiments, POPC liposomes and proteo-liposomes were prepared as described above, except with 18:1-12:0 NBD-PE (Avanti 819156) rather than 14:0-06:0 NBD-PE to reduce NBD lipid extraction by methyl-β-cyclodextrin (mβCD).

Techniques: Fluorescence, Liposomes, Activity Assay, Incubation, Control, Labeling

Journal: bioRxiv

Article Title: TMC1 and TMC2 are cholesterol-dependent scramblases that regulate membrane homeostasis in auditory hair cells

doi: 10.1101/2025.07.03.663083

Figure Lengend Snippet: A. Localization of the equivalent residues to mouse TMC1 M412 (M507), D528 (D617) and D569 (D658) in the Caenorhabditis elegans TMC1 structure (PDB ID: 7USX). The approximate localization of the plasma membrane as calculated by the PPM web server is indicated by two parallel planes of spheres. NBD-fluorescence decrease after SDT addition to POPC/PE/PS liposomes (black) or proteo-liposomes containing wild type (gray) or mutants CmTMC1 M507K (yellow), D617N (purple) or D658N (pink) in liposomes containing 0% (B), 10% (C), or 30% (D) cholesterol. E. Quantification of the SDT-based PLS activity of wild-type CmTMC1, M507K, D617N or D658N mutants in POPC/PE/PS liposomes containing 0, 10 or 30 % cholesterol. F-H. SDT (yellow)- and BSA (grey)-based PLS activity of M507K, D617N and D658N mutants relative to wild-type CmTMC1 in proteo-liposomes containing 0% (F), 10% (G) or 30% (H) cholesterol. One-way ANOVA was performed to evaluate statistical significance between wild type and mutant TMC1 relative SDT-based activity. I-L. Representative confocal images of auditory OHCs and IHCs at the middle cochlear region from Tmc1 M412K/M412K (I) or Tmc1 D569N/D569N (K) mice incubated in HBSS without (control) or with mβCD to remove cholesterol and labeled with annexin V (magenta) or spy-555 (green) to visualize PS or F-actin. J and L. Quantification of background-subtracted annexin V-647 fluorescence intensity from the experiments showed in I or K. Mean ± SEM is shown for n∼ 100 OHCs and 30 IHCs from 3 tissues. Two-way ANOVA was performed to evaluate statistical significance between control and mβCD-treated samples in IHCs or OHCs. P values are indicated as: ns≥0.05, ** <0.01, *** <0.001, and ****<0.0001.See also Supplemental Fig. 3G-H.

Article Snippet: For cholesterol addition experiments, POPC liposomes and proteo-liposomes were prepared as described above, except with 18:1-12:0 NBD-PE (Avanti 819156) rather than 14:0-06:0 NBD-PE to reduce NBD lipid extraction by methyl-β-cyclodextrin (mβCD).

Techniques: Clinical Proteomics, Membrane, Fluorescence, Liposomes, Activity Assay, Mutagenesis, Incubation, Control, Labeling