Panel A shows fluorescence lifetime measured at different osmolarities. Panel B shows relative lysosomal sodium concentration of WT BMDMs upon hypotonic shock (100 mOsm). Panel C shows Galectin-3 staining in WT and TMEM63A cells under hypotonic stress, quantified in panel D. Panel E shows SRG and isosurface imaging under isotonic and hypotonic conditions in WT and TMEM63A KO macrophages. Panel F shows cell volume changes upon hypotonic shock over time with or without DIDS. Panel G shows Galectin-3 staining of WT BMDMs treated with DCPIB, DIDS, or ouabain before hypotonic shock, quantified in Panel H. Panel I shows Galectin-3 and ALIX staining in WT and TMEM63A KO BMDMs upon hypotonic shock, quantified in Panel J. Panel K shows Galectin-3 staining of TMEM63A KO BMDMs treated with 0.25 mM LLOMe in isotonic (330 mOsm) or hypertonic (600 mOsm) solution, quantified in Panel L. Panel M shows a model illustrating TMEM63A and Lyso-VRAC regulating osmotic balance of lysosomes.
TMEM63A relieves high membrane tension to protect lysosomes from rupture. (A) Lifetime fluorescence of Lyso-Flipper WT RAW264.7 cells before and 5 min after hypotonic shock. Each dot represents one field of 4–8 cells. n = 3. (B) Relative [Na+]lyso of WT BMDM determined using Natrium Green, normalized to 10-kDa dextran, both pulsed for 16 h and chased for 1 h. All data points represent a field containing 4–8 cells. n = 3. (C and D) Lysosome damage was determined for WT and TMEM63A KO BMDM upon 10-min exposure to hypotonic (100 mOsm) solutions. n = 3. (E) SRG-loaded WT BMDM lysosomes undergoing RVD and return to tubulation versus rupture in TMEM63A KO BMDM. xy images shown in black and white. Surface rendering of xyz images (blue) illustrates tubular versus spherical shapes of the lysosomes. (F) Cell volume percentage of WT RAW264.7 cells ±250 µM DIDS in hypotonic solutions for 3, 10, and 15 min. Normalized to 0 min measurement. n = 3. (G and H) Lysosome damage determined for WT BMDM treated with 100 µM DCPIB for 5 min, 250 µM DIDS for 5 min, or 2 mM ouabain for 4 h prior to 10 min exposure to hypotonic (100 mOsm) solutions. n = 3. (I and J) ALIX recruitment determined for WT and TMEM63A KO BMDM upon 10-min exposure to hypotonic (100 mOsm) solutions. n = 3. (K and L) Shifting TMEM63A KO BMDM to hypertonic (600 mOsm) solutions at the time of LLOMe delivery. Each data point represents a field containing 4–10 cells. n = 3, scale bars 10 µm. (M) Model.