title: Spatiotemporal dynamics of actin remodeling and endomembrane trafficking in alveolar epithelial type I cell wound healing.
abstract: 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.
authors.affiliation: Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Lindsay M
authors.initials: LM
authors.lastname: Godin
authors.forename: Jorge
authors.initials: J
authors.lastname: Vergen
authors.forename: Y S
authors.initials: YS
authors.lastname: Prakash
authors.forename: Richard E
authors.initials: RE
authors.lastname: Pagano
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Hubmayr
mesh_terms: D000199:Actins / Q000378:metabolism*; D056809:Alveolar Epithelial Cells / Q000378:metabolism* / Q000473:pathology*; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D051356:Cortactin / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D050356:Lipid Metabolism; D008954:Models, Biological; D010714:Phosphatidylethanolamines / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012235:Rhodamines / Q000378:metabolism; D013997:Time Factors; D014945:Wound Healing
chemical_list: D000199:Actins; D051356:Cortactin; D005452:Fluoresceins; D010714:Phosphatidylethanolamines; D012235:Rhodamines; C085925:calcein AM; C483858:phosphatidylethanolamine