Fig. 6: ZDHHC6-mediated CLIMP-63 S-acylation controls ER morphology.

a Confocal images of HeLa cells expressing ZDHHC6-myc immunolabelled for myc (blue), BAP31 (magenta), and CLIMP-63 (green). Red arrow and inset show dilated ER in ZDHHC6-myc expressing cells. White arrowhead shows bystander cell. b Quantification of the percentage of ZDHHC6-myc expressing cells with ER dilation in control or shCLIMP-63 HeLa cells. Results are mean ± SEM (n = 3 counting total: Control, 129 cells; shCLIMP-63, 226 cells; ***p = 0.0005, obtained by unpaired, two-tailed student’s t test. c Same as in b in shCLIMP-63 cells co-overexpressing or not myc-ZDHHC6 with HA-CLIMP-63 WT or C100A. Results are mean ± SEM (n = 3) counting total: HA-CLIMP-63-WT, 137 cells, HA-CLIMP-63-WT + ZDHHC6, 79 cells, HA-CLIMP-63-C100A, 145 cells, HA-CLIMP-63-C100A + ZDHHC6, 182 cells (****p < 0.0001, obtained by one-way ANOVA, Tukey’s multiple comparison). d Computational simulation of CLIMP-63 depalmitoylation (left), Higher-order assembly (middle), and protein stability (right) upon normal (blue) and slower (orange) depalmitoylation kinetics. Median shown by solid lines, 1^st and 3^rd quartile by shaded interval. e, f Quantification of CLIMP-63. e ³H-palmitate decay or f apparent decay in shCLIMP-63 cells expressing HA-CLIMP-63 WT or CC, pulsed with ³H-palmitate pulse (2 h) or ³⁵S metabolic (20 min) and followed by the indicated chase period. Results set to 100% for T = 0 min are mean ± SD, n = 3. g Western blots of surface biotinylated proteins and total cell extracts (TCE) from shCLIMP-63 cells expressing HA-CLIMP-63 WT, C100A or CC mutant. LRP6 and actin/GAPDH are positive and negative controls, respectively. Surface CLIMP-63 results normalised to WT are mean ± SEM (n = 7), (****p < 0.0001, obtained by unpaired, two-tailed student’s t test.). h Western blot analysis of fractionated cell lysates from cells transfected as in e (DRMs in fraction 2 are marked by caveolin). HA-CLIMP-63-CC in each fraction was compared to WT HA-CLIMP-63 levels obtained in parallel experiments depicted in Supplementary Fig. 2h. Results are mean ± SEM (n = 3), (*p = 0.0255, obtained by two-way ANOVA, Sydak’s multiple comparison). i Confocal images and quantification of the percentage of cells with ER dilation in shCLIMP-63 HeLa cells transfected with RFP-CLIMP-63 WT or CC, immunolabelled for calnexin. Results are mean ± SEM (n = 3), WT: 91 cells, CC: 60 cells (***p = 0.0001, obtained by unpaired, two-tailed student’s t test.). j, k Airyscan-confocal images and quantification of the percentage of cells with dilated ER in KO-ZDHHC6 cells transfected with HA-CLIMP-63 WT or CC plus myc- WT-ZDHHC6 or inactive mutant ZDHHS6 immunolabelled for HA, myc and ER marker Bip. Results are mean ± SEM (n = 3) with >200 cells counted per condition (*p = 0.0179 and *p = 0.0179 obtained by two-way ANOVA, Sydak’s multiple comparison). Red arrow and inset show dilated ER in ZDHHC6-myc expressing cells. Unless otherwise indicated all means were derived from biologically independent experiments. Simulated data was derived from the simulation of n = 100 models. further details of the in silico labelling experiments can be found in the supplementary information – supplementary methods section. All scale bars: 10 μm.