The figure presents multiple panels examining lysosomal function in WT and TMEM63A RAW264.7 macrophages. Panel A shows fluorescence lifetime measurements under control and sucrose-treated conditions, with each dot representing one field of cells. Panel B displays lysosomal proton leak over time following concanamycin A treatment in WT and TMEM63A KO cells. Panel C quantifies the rate of pH change between 2 to 8 minutes. Panel D shows WT and TMEM63A KO RAW264.7 cells under control or 0.5 mM LLOMe treatment, immunostained for LAMP-1 and LC3. Panel E quantifies galectin-3 puncta per cell in WT BMDMs treated with hypotonic shock (100 mOsm) with or without concanamycin A. Panels F and G show WT and TMEM63A KO cells under hypotonic conditions, stained for LAMP-1 and galectin-3, with quantification of galectin-3 puncta in Panel G. Panel H quantifies galectin-3 puncta in TMEM63A KO RAW264.7 cells re-expressing TMEM63A or indicated variants after hypotonic treatment.
TMEM63A activity on tensed lysosomes and resilience to hydrostatic pressure conferred by the channel. (A) Lyso-Flipper FLIM in RAW264.7 macrophages incubated with 30 mM sucrose overnight. Each dot represents one field of 4–8 cells. n = 3. (B) H+-leak determination using ratiometric measurements of Oregon Green 10-kDa dextran after concanamycin addition. n = 3. (C) Mean instantaneous rates of pH changes from 2 to 8 min. (D) Examples of WT or TMEM63A KO RAW 264.7 cells challenged with 0.5 mM LLOMe for 15 min. Cells were immunostained with LAMP-1 (green) and LC3 (magenta). (E) Quantification of galectin-3 puncta with WT BMDM pre-treated with 5 μM of concanamycin A for 30 min prior to hypotonic (100 mOsm) shock. n = 3. (F and G) WT and TMEM63A KO RAW 264.7 cells were incubated in hypotonic (100 mOsm) solutions for 15 min. Immunostained with galectin-3 (magenta) and LAMP-1 (green), quantified in G. n = 3. (H) TMEM63A KO RAW264.7 cells re-expressing an OFP version of the channel and challenged with 100 mOsm solution for 15 min. Galectin-3 puncta were quantified (each dot representing the mean of the experiment). n = 3.