Membrane fusion protein annexin A7 and lamellar body protein ABCA3 co‐localize at the cell surface in secretagogue‐stimulated alveolar type II cells

Abstract

Lung surfactant secretion requires fusion of lamellar bodies (LB) with the plasma membrane in type II (T2) cells. Annexin A7 (A7) contributes to such membrane fusion since 1) it promotes surfactant secretion in permeabilized T2 cells and 2) secretagogues increase its association with the LB and plasma membrane in T2 cells. In this study, we hypothesized that secretagogues would increase membrane insertion of A7 to facilitate its fusion promoting activity. As a result, A7 may be expressed at the cell surface. Isolated rat T2 cells were cultured for 20–22h on glass cover slips and stimulated for 2h without or with 100nM phorbol myristate acetate (PMA). The cells were fixed in 2% paraformaldehyde, (PFA, non‐permeabilized) or in ice‐cold methanol (permeabilized) and immuno‐stained for A7 and LB specific protein, ABCA3 (P180). With methanol fixation, control cells showed P180 and A7 staining at the cell surface as well as in the cell interior without significant co‐localization of two proteins. The PMA‐stimulated cells showed increased co‐localization of two proteins. With PFA fixation, control cells showed that A7 and some P180 staining at the cell surface without significant co‐localization of the two proteins. Very little staining was seen inside the cells. In PMA‐stimulated cells, increased staining and co‐localization of the two proteins were observed at the cell surface. Our results suggest that increased secretion is associated with increased insertion of A7 and P180 into the plasma membrane leading to exposure of these two proteins at the cells surface, possibly at the secretion site.