Poster abstracts
Poster number 22 submitted by Jon Fritz
Visualizing AnxA2-driven active membrane repair dynamics in alveolar type I cells
Jonathan R. Fritz (Biophysics Program, The Ohio State University), Noah Weisleder (Department of Physiology & Cell Biology, the Ohio State University), Joseph Bednash (Division of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, the Ohio State University), Joshua A. Englert (Division of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, the Ohio State University)
Abstract:
Physical forces generated during mechanical ventilation can damage the plasma membranes of alveolar epithelial cells and trigger active plasma membrane (PM) repair to reseal membrane disruptions and prevent cell death. In many mammalian cell types, calcium influx through injury-induced membrane disruptions initiates active PM repair mechanisms. One such repair mechanism involves Annexin A2 (AnxA2) translocation to PM injury sites to mediate vesicle delivery, patch formation, and membrane bending. Recently, we have shown that global AnxA2-knockout mice have increased injury following ventilator-induced lung injury (VILI) compared to wild-type mice. However, the mechanisms by which AnxA2 mediates PM repair in lung cells are not well understood. In this study, we used multiphoton microscopy and a dye-entry assay to demonstrate that extracellular calcium is essential for PM repair in primary human alveolar (i.e. lung) type I (ATI) cells following laser injury. To visualize AnxA2 organization and translocation post-injury, we transiently transfected ATI cells with GFP-tagged AnxA2. As expected, absent extracellular calcium and more intense injury dampens AnxA2-GFP fluorescence at the site of PM injury. Furthermore, by using rapamycin to inhibit mTOR complex 1 (mTORC1), a multiprotein kinase known to phosphorylate AnxA2, we show a dose-dependent decrease in AnxA2-mediated PM repair. By establishing an AnxA2-dependent active PM repair post-injury in ATI cells, we possess a dynamic, cell-scale platform upon which to investigate additional targets of active PM repair in human lung cells. A better understanding of PM repair in lung cells would provide potential therapeutic targets for treatment of lung diseases.
Keywords: lung, membrane repair, microscopy