Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA078

Poster Communications

Knockout of caveolin-1 delays calcium signalling in human alveolar type I epithelial cells

K. A. Diem1, G. Fois1, P. Dietl1, M. Frick1

1. Institute of General Physiology, Ulm University, Ulm, Germany.

Secretion of pulmonary surfactant from alveolar type II epithelial cells is essential to maintain alveolar homeostasis and gas exchange within the lung. Earlier studies showed that mechanical forces are important for alveolar homeostasis and stretching of the epithelial cells during inspiration is believed to be the main stimulus for surfactant secretion. Yet the precise mechanisms how mechanical forces result in surfactant secretion are still elusive. The alveolar epithelium consists of alveolar type I (ATI) and type II (ATII) epithelial cells and both have been shown to respond to stretching. Moreover, paracrine signals from ATI cells can stimulate a rise in intracellular Ca2+ (Ca2+i) in ATII cells, the main stimulus for surfactant secretion. Caveolae, small plasma membrane invaginations present in ATI but absent in ATII cells, function as membrane buffers and may play a role as mechanosensors. To investigate the impact of caveolae on cell stiffness and response to mechanical stress we established a CRISPR/Cas9-induced human caveolin-1 knockout (CAV1 KO) ATI cell line. The cells were cultured on flexible substrates (PDMS membranes) in mono- or co-culture with primary ATII cells. The cells were stretched intermittently with increasing amplitudes to a maximum of 75 % longitudinal distension. FURA-2 assays to measure changes in Ca2+i indicated that ATI WT cells respond more frequently (20.72 % responders) and at lower thresholds (20 % stretch) to mechanical stress than KO cells (4.98 % responders, 40 % stretch) suggesting that caveolae serve as a hub for downstream Ca-signalling in response to mechanical deformation of ATI cells. Moreover, using confocal microscopy, we quantitatively investigated the colocalization and amount of CAV1 and PTRF, both caveolar proteins, in the plasma membrane of stretched (15, 25, 50, 75 % stretch) versus unstretched ATI WT cells. The colocalization of both proteins was significantly reduced when membranes were stretched above 50 %. This confirms the idea that caveolae disassemble and proteins are released from the plasma membrane when mechanical forces appear. These data further add to the idea that caveolae within ATI cells are potential mechanosensors in the alveolar epithelium.

Where applicable, experiments conform with Society ethical requirements