Fig 1: TMEM63A/B are essential for mechanosensitive currents in AECs.(A) SACs were abolished in AT1 cells, AT2 cells, and LB/EL from Tmem63a/b-cDKO mice. Current amplitudes at –80 mV and –80 mmHg for AT1 and AT2 cells and –60 mV and –60 mmHg for LB/EL were used for comparison. n = 13 (Ctrl-AT1, from Ctrl-63ab mice); n = 18 (KO-AT1, from Ager-63ab); n = 25 (Ctrl-AT2, from Ctrl-63ab); n = 21 (KO-AT2, from Sftpc-63ab); n = 16 (Ctrl-LB/EL, from Ctrl-63ab); n = 13 (KO-LB/EL, from Sftpc-63ab) cells. **P < 0.01 and ***P < 0.001, by 2-tailed, unpaired Student’s t test. (B) Immunofluorescence images of endogenous TMEM63B with N-terminal 2×V5-tag–knockin (N-V5-KI) in an AT2 cell in a frozen section of mouse lung. LAMP1 is a marker of LBs. Scale bar: 5 μm. (C) Amplitudes of SACs in control and human TMEM63B-transfected (h63B-transfected) HeLa cells. Holding potential: –80 mV; pressure: –80 mmHg. n = 18 and 20 for HeLa and HeLa-h63B cells, respectively. **P < 0.01, by 2-tailed, unpaired Student’s t test. (D) Representative traces of SACs under the cell-attached configuration in a h63B-transfected HeLa cell. The pressure-current relationship is shown in the inset. (E and F) SAC traces and current-voltage relationships of inside-out recordings from h63B-transfected HeLa cells suggesting selectivity for Na+ and K+ over Ca2+.
Fig 2: Deficiency of TMEM63A/B abolishes ventilation-induced Ca2+ transients and surfactant release in AT2 cells.(A) mCherry fluorescence in AAV-infected (CAG-DIO-jGCaMP7s-mCherry–infected) lungs showing positively transduced AT2 cells (brighter spots). Sftpc-63ab represents Sftpc-CreERT2+/– 63afl/fl 63bfl/fl. Scale bar: 100 μm. (B) The densities of positively transduced AT2 cells were comparable between the control Sftpc-CreERT2 and Sftpc-63ab mice. n = 5 lung lobes. NS, nonsignificant, by 2-tailed, unpaired Student’s t test. (C) Lung inflation–induced Ca2+ transients in AT2 cells were completely abolished in all lung lobes from Sftpc-63ab mice, as revealed by jGCaMP7s fluorescence. n = 5 lung lobes. ***P < 0.001, by 1-way ANOVA with Tukey’s test. (D) Survival curves for Tmem63a/b-cDKO mice that received daily inhalation (30 min each time, 3 times/day) of aerosolized ATP (200 mM) or saline solution after tamoxifen induction. n = 3 mice. (E) ATP-induced Ca2+ response in primary AT2 cells isolated from Ctrl-63ab (63afl/fl 63bfl/fl) and Sftpc-63ab mice (n = 30 and 35 cells, respectively). (F) Cell strain–induced surfactant release occurred in AT2 cells from Ctrl-63ab, but not Sftpc-63ab, mice. Unfused LBs were stained by LysoTracker Green; LBs fused on the plasma membrane were positive for FM4-64. Arrows indicate LBs that released surfactant (FM4-64 fluorescence disappeared after strain). Scale bar: 5 μm. (G and H) Tmem63a/b-cDKO did not affect ATP-induced LB fusion but significantly attenuated cell strain–induced surfactant release. Ctrl, Ctrl-63ab; KO, Sftpc-63ab. The median and quartiles are shown by dashed and dotted lines, respectively. The numbers of cells are shown at the bottom. *P < 0.05 and ***P < 0.001, by 1-way ANOVA with Tukey’s test. (I) Reacidification of LBs after removal of ATP in AT2 cells from Sftpc-63ab mice. Overlapped LysoTracker Green and FM4-64 fluorescence (orange, indicated by the arrow) suggests that the fusion pore was closed and luminal pH was reacidified. Scale bar: 5 μm. (J and K) Transmission electron microscopy images and cross-sectional areas (CSAs) of LBs from Ctrl-63ab and Sftpc-63ab mice. n = 41 and 94 LBs, respectively. Scale bars: 1 μm.
Fig 3: Lethal pulmonary phenotypes of Tmem63a/b-KO mice.(A) Survival curves for constitutive Tmem63a/b-KO mice of different genotypes. The numbers of mice are shown in parentheses. (B) Failure of alveolar expansion in Tmem63b-KO mice after birth (P0). Original magnification, ×2. (C) Survival curves of AEC-specific Tmem63a/b conditional-KO mice with different genotypes. Aqp5-Cre was expressed in AT1 cells and approximately 50% of AT2 cells SPC-Cre was expressed in all AT2 cells. (D) Survival curves for tamoxifen-inducible, AEC-specific Tmem63a/b-cDKO mice. Ager-CreERT2 was expressed in AT1 cells and approximately 10% of AT2 cells; Nkx2.1-CreERT2 was expressed in AT1 cells and approximately 80% of AT2 cells; and Sftpc-CreERT2 was expressed in all AT2 cells. Ctrl-63ab represents 63afl/fl 63bfl/fl mice without Cre, and others are with the corresponding CreERT2. (E) Micro-CT images of mouse lungs after tamoxifen induction. Arrows indicate regions of atelectasis. (F) Mean lung volume intensities for mice before and after tamoxifen induction, measured from the micro-CT images. n = 3 mice. (G) Atelectasis in cDKO mice illustrated by freshly dissected lungs and H&E-stained sections. Arrows indicate collapsed lobes. Scale bars: 200 μm. (H) A dramatic decline of SpO2 before respiratory failure was observed in cDKO mice. n = 3 mice. (I) Characteristics of pulmonary edema in cDKO mice at post-tamoxifen day 12. n = 5, 9, and 7 mice for lung weight from left to right; n = 3 mice for wet/dry ratio. (J) Body weights in cDKO mice at post-tamoxifen day 12. n = 5, 9, and 7 mice from left to right. (K) Deficiency of secreted SPC, surfactant phospholipid DPPC, and ATP in BALF collected from cDKO mice at post-tamoxifen day 10. n = 3, 5, and 3 mice from left to right in each graph. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA with Tukey’s test.
Fig 4: Human TMEM63A/B rescues respiratory failure in Tmem63a/b-cDKO mice.(A) Survival curves for Ager-Sftpc-63ab-cDKO mice that received intratracheally delivered AAVs with empty vector (Ctrl), human TMEM63A (h63A), human TMEM63B (h63B), or the h63B-Y572A mutant after tamoxifen induction. The numbers of mice are shown in parentheses. (B) Images show the relatively normal appearance and structure of h63A- and h63B-rescued lungs compared with the lungs transduced with control or h63B-Y572A AAV. Scale bars: 200 μm. d11, day 11; d12, day 12; d60, day 60. (C) Expression of h63A, h63B, and h63B-Y572A in mouse lungs and localization in AT2 cells. Images show immunofluorescence staining for Pro-SPC (AT2), PDPN (AT1), Flag (h63A/B), and LAMP1 (LBs). Scale bars: 100 μm and 10 μm.
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