Peroxisome calcium uptake is dependent on ER-peroxisome membrane contact

Figures

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  Fig. 1. Peroxisomal Ca2+ dynamics are reduced in the absence of ACBD5. A: Experimental paradigm for analysis of peroxisomal Ca2+ by FRET sensor D3cpV-PTS1 in response to triggering of SOCE. B-D: Western Blot validation of siRNA-mediated knock-down. Representative images with quantification of band intensity of protein of interest relative to housekeeping control. Bar plot with mean ± SD. B: PXMP2 (N = 3); C: PEX11β (N = 3); D: ACBD5 (N = 4). E: Peroxisomal Ca2+ dynamics as D3cpV-PTS1 FRET ratio upon histamine-stimulation and Ca2+ supplementation of siPXMP2 treated cells. Mean ± SEM. N = 3 experiments, n = 221 cells (siPXMP2) and 381 cells (NC), measured over 100 imaging cycles (5 s). F-H: Quantification of (E) as baseline FRET ratio (F), ER-based response (G) and PM-based response (H). I: Peroxisomal Ca2+ dynamics of siPEX11β treated cells. Mean ± SEM from N = 6 experiments, n = 284 cells (siPEX11β) and 653 cells (NC), measured over 100 imaging cycles (5 s). J-L: Quantification of (I) as baseline FRET ratio (J), ER-based increase (K) and PM-based increase (L). M: Peroxisomal Ca2+ dynamics of siACBD5 treated cells. Mean ± SEM from N = 3 experiments, n = 149 cells (siACBD5) and 160 cells…

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  Fig. 2. Tethering constructs force association of peroxisomes to the ER. A: Peroxisome-ER contact with physiological tethers, ACBD5 depletion, and expression of artificial tethering constructs. B: Schematic domain structure of tethers. C, D: Tertiary protein structure of tetherlong (C) and tethershort (D) construct as predicted by AlphaFold2 with different domains indicated by color. Size estimations in nm of linkers and full tether construct were derived from PyMol. E: Images from live cell imaging of HeLa cells co-transfected with D3cpV-PTS1and tetherlong or tethershort. 40x magnification, scale bar 100 μm. F: Peroxisomal Ca2+ measurements of cells co-transfected with D3cpV-PTS1 and tetherlong or tethershort. Curves represent FRET ratios over time (mean ± SEM), measured over 100 imaging cycles (3 sec). G-I: Quantification of (F) as Ca2+ baseline (G), ER-based Ca2+ response (H) and PM-based Ca2+ response (I). Tukey’s box plots. N = 4, n = 87 (no tether), 78 (tetherlong), 100 (tethershort). P-values calculated by one-way ANOVA with Tukey’s HSD

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  Fig. 3. Expression of PO-ER tether restores peroxisomal Ca2+ uptake upon ACBD5 knock-down. A: Peroxisomal Ca2+ measurements of cells co-transfected with D3cpV-PTS1 and tetherlong and siRNA targeting ACBD5. Curves represent FRET ratios over time (mean across all cells ± SEM), measured over 100 imaging cycles (3 sec). B-D: Quantification of (A) as Ca2+ baseline (B), ER-based Ca2+ response (C) and PM-based Ca2+ response (D). N = 3, n = 160 (NC), 149 (ACBD5), 61 (NC + tetherlong), 50 (ACBD5 + tetherlong). Data represented as mean ± SD. P-values calculated by two-sample t-test. E: Peroxisomal Ca2+ response in cells co-transfected with D3cpV-PTS1 and tethershort and ACBD5 knock-down. FRET ratios over time (mean across all cells ± SEM), measured over 100 imaging cycles (3 sec). F-H: Quantification of (E) as Ca2+ baseline (F), ER-based Ca2+ response (G) and PM-based Ca2+ response (H). N= 3, n = 160 (NC), 149 (ACBD5), 64 (NC + tethershort), 59 (ACBD5 + tethershort). Tukey’s box plots. P-values calculated by two-sample t-test with Welch correction

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  Fig. 4. Increase in peroxisomal motility upon disruption of the PO-ER contact site and Ca2+-dependent decline in peroxisome motility of ACBD5-depleted cells. siRNA transfection 72 hours, HaloTag-SKL transfection 24 hours, labeling with HaloTag ligand for 1 hour before live cell recording of peroxisome motility. A: Representative immunofluorescence images of peroxisomes in HeLa cells treated siRNA targeting ACBD5. Cells immunostained against PMP70 (green), nuclei labeled with DAPI (blue). 63x magnification, scale bar = 50 μm. B: Quantification of the number peroxisomes per cell n = 95 cells (NC), n = 70 cells (siACBD5), Tukey’s box plots, p-values obtained with two-sided t-test with Welch correction. C: Average size of peroxisomes per cell counted in (B) in μm², n = 95 cells (NC), n = 70 cells (siACBD5), Tukey’s box plots, p-values obtained with two-sided t-test with Welch correction. D: Mean track speed in baseline conditions (DMEM with 10% FBS). Data obtained in N = 3 independent experiments, n = 27 (NC), n = 31 (siACBD5). Each cell represents an average of all (100-300) peroxisomal tracks within one cell. Tracks shorter than minimum duration of 5 seconds were excluded from analysis. E:…

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  Fig. 5. Independent effects of siACBD5, SOCE stimulation, and microtubule disruption. A: Nocodazole treatment disrupts the microtubule network. Immunofluorescence of HeLa cells treated with 10 µM nocodazole. Microtubules were immunostained for α-tubulin (green) and peroxisomes were immunostained for PEX14 (red). 63x magnification, scale bar 100 μm (top panels) and 20 μm (lower panels). B: Mean speeds of all tracked peroxisomes within a cell, filtered to include tracks with at least 10 s duration. Cells were treated with siACBD5 or scrambled control (NC), 10 µM nocodazole or DMSO, and then stimulated according to the Ca2+-mobilization protocol. N = 3 independent experiments, n = 9–10 cells per condition. Tukey’s box plots. C: Linear mixed-effects model based on (B). Representing the full interaction model (‘full interaction’, black), and the two last model reduction containing no interactions (‘no interactions’, orange), and a combination of both stimulation steps into one category (‘histamine + Ca2+ combined’, violet). ‘Intercept’ represents the mean speed [nm/s] of all control conditions (NC siRNA, DMSO treatment, and EGTA buffer). Estimated fixed effects (β ± 95% CI) from full and…