Godin, Lindsay M.

Stress failure of alveolar epithelial cells is a common feature of ventilator‐injured lungs. While some injured cells undergo necrosis, others repair their plasma membrane (PM) wounds through non‐secretory exocytosis of intracellular vesicles (e.g. lysosomes) to and cytoskeleton remodeling at and around the wound. We measured the timing of lysosomal trafficking and F‐actin reorganization following micropuncture injury in primary type I alveolar epithelial cells (ATI). ATI were isolated from female rats and loaded with Calcein AM, N‐rhodamine phosphatidylethanolamine (lysosome membrane label) or transfected with LifeAct (GFP tagged actin label). Changes in fluorescence intensity were indicators of time to wound repair (cessation of calcein fluorescence decay following injury), lysosomal trafficking to the wound site, and actin remodeling. ATI repaired wounds within 27.9±3.9s. Recruitment of lysosomes to the wound site was detected at 2.9±1.5s following injury and preceded actin polymerization by 23.7±5.4s. Failure to repair PM wounds was associated with a weak or absent actin polymerization response. We conclude that local actin polymerization is a critical step in PM wound repair and postulate that adhesive interactions between PM lipids and the remodeled actin cytoskeleton serve as a driving force for wound closure by promoting lateral PM lipid bilayer flow. Support: HL63178

We present plasma membrane (PM) internalization responses of type I alveolar epithelial cells to a 50 mosmol/l increase in tonicity. Our research is motivated by interest in ATI repair, for which endocytic retrieval of PM appears to be critical. We validated pharmacological and molecular tools to dissect the endocytic machinery of these cells and used these tools to test the hypothesis that osmotic stress triggers a pathway-specific internalization of PM domains. Validation experiments confirmed the fluorescent analogs of lactosyl-ceramide, transferrin, and dextran as pathway-specific cargo of caveolar, clathrin, and fluid-phase uptake, respectively. Pulse-chase experiments indicate that hypertonic exposure causes a downregulation of clathrin and fluid-phase endocytosis while stimulating caveolar endocytosis. The tonicity-mediated increase in caveolar endocytosis was associated with the translocation of caveolin-1 from the PM and was absent in cells that had been transfected with dominant-negative dynamin constructs. In separate experiments we show that hypertonic exposure increases the probability of PM wound repair following micropuncture from 82 ± 4 to 94 ± 2% ( P < 0.01) and that this effect depends on Src pathway activation-mediated caveolar endocytosis. The therapeutic and biological implications of our findings are discussed.

Alveolar epithelial type I cell (ATI) wounding is prevalent in ventilator-injured lungs and likely contributes to pathogenesis of “barotrauma” and “biotrauma.” In experimental models most wounded alveolar cells repair plasma membrane (PM) defects and survive insults. Considering the force balance between edge energy at the PM wound margins and adhesive interactions of the lipid bilayer with the underlying cytoskeleton (CSK), we tested the hypothesis that subcortical actin depolymerization is a key facilitator of PM repair. Using real-time fluorescence imaging of primary rat ATI transfected with a live cell actin-green fluorescent protein construct (Lifeact-GFP) and loaded with N-rhodamine phosphatidylethanolamine (PE), we examined the spatial and temporal coordination between cytoskeletal remodeling and PM repair following micropuncture. Membrane integrity was inferred from the fluorescence intensity profiles of the cytosolic label calcein AM. Wounding led to rapid depolymerization of the actin CSK near the wound site, concurrent with accumulation of endomembrane-derived N-rhodamine PE. Both responses were sustained until PM integrity was reestablished, which typically occurs between ∼10 and 40 s after micropuncture. Only thereafter did the actin CSK near the wound begin to repolymerize, while the rate of endomembrane lipid accumulation decreased. Between 60 and 90 s after successful PM repair, after translocation of the actin nucleation factor cortactin, a dense actin fiber network formed. In cells that did not survive micropuncture injury, actin remodeling did not occur. These novel results highlight the importance of actin remodeling in ATI cell repair and suggest molecular targets for modulating the repair process.