Combined Atomic Force Microscopy–Fluorescence Microscopy: Analyzing Exocytosis in Alveolar Type II Cells

Abstract

Hybrid atomic force microscopy (AFM)-fluorescence microscopy (FM) investigation of exocytosis in lung epithelial cells (ATII cells) allows the detection of individual exocytic events by FM, which can be simultaneously correlated to structural changes in individual cells by AFM. Exocytosis of lamellar bodies (LBs) represents a slow form of exocytosis found in many non-neuronal cells. Exocytosis of LBs, following stimulation with adenosine-5'-triphosphate (ATP) and phorbol 12-myristate 13-acetate (PMA), results in a cation influx via P2X(4) receptors at the site of LB fusion with the plasma membrane (PM), which should induce a temporary increase in cell height/volume. AFM measurements were performed in single-line scans across the cell surface. Five minutes after stimulation, ATII cells revealed a cell height and volume increase of 13.7% ± 4.1% and 15.9 ± 4.8% (N = 9), respectively. These transient changes depend on exocytic LB-PM fusion. Nonstimulated cells and cells lacking LB fusions did not show a significant change in cell height/volume (N = 8). In addition, a cell height decrease was observed in ATII cells stimulated by uridine-5'-triphosphate (UTP) and PMA, agonists inducing LB fusion with the PM, but not activation of P2X(4) receptors. The cell height and volume decreased by -8.6 ± 3.6% and -11.2 ± 3.9% (N = 5), respectively. Additionally, low force contact and dynamic mode AFM imaging of cell areas around the nucleus after stimulation with ATP/PMA was performed. Fused LBs are more pronounced in AFM topography images compared to nonfused LBs, concluding that different "dynamic states" of LBs or locations from the PM are captured during imaging.