Mechanical strain of alveolar type II cells in culture: changes in the transcellular cytokeratin network and adaptations

Abstract

Mechanical forces exert multiple effects in cells, ranging from altered protein expression patterns to cell damage and death. Despite undisputable biological importance, little is known about structural changes in cells subjected to strain ex vivo. Here, we undertake the first transmission electron microscopy investigation combined with fluorescence imaging on pulmonary alveolar type II cells that are subjected to equibiaxial strain. When cells are investigated immediately after stretch, we demonstrate that curved cytokeratin (CK) fibers are straightened out at 10% increase in cell surface area (CSA) and that this is accompanied by a widened extracellular gap of desmosomesthe insertion points of CK fibers. Surprisingly, a CSA increase by 20% led to higher fiber curvatures of CK fibers and a concurrent return of the desmosomal gap to normal values. Since 20% CSA increase also induced a significant phosphorylation of CK8-ser431, we suggest CK phosphorylation might lower the tensile force of the transcellular CK network, which could explain the morphological observations. Stretch durations of 5 min caused membrane injury in up to 24% of the cells stretched by 30%, but the CK network remained surprisingly intact even in dead cells. We conclude that CK and desmosomes constitute a strong transcellular scaffold that survives cell death and hypothesize that phosphorylation of CK fibers is a mechano-induced adaptive mechanism to maintain epithelial overall integrity. mechanical stress; cytoskeleton; desmosome; injury MANY CELL TYPES are subjected to deformation or other mechanical forces. An increasing number of studies has addressed cellular and molecular mechanisms that lead to coordinated responses of cells to such stimuli (for review, see Refs. 1, 23). Besides physiological responses occurring within a tolerable range of mechanical stress, a certain level of deformation inevitably threatens the structural integrity of the cell and leads to cell death. Little is known about structural cell changes in this life-threatening state, and even less is known about possible defense mechanisms a cell might employ to resist potentially fatal amplitudes of tensile stress. The goal of this study was to gather basic information regarding these questions on a cellular level by application of controlled strain to cells in culture, allowing direct comparison of stretched cells with unstretched controls.We used primary cultures of alveolar type II (ATII) cells, the most abundant cell type in the lung, because in vivo this cell type is exposed to life-long mechanical strain during breathing. Although different lung volumes (as % of total lung capacity) were suggested (2, 36, and for a review, see Ref. 5) to induce single cell stretch on a cellular level, it has been shown that within a certain range, these cells respond to mechanical distension with release of surfactant into the alveolus in vivo (25, 27) as well as in vitro (13, 43). Repetitive stretch induces a variety of cellular responses associated with gene regulation and protein...

Lamellar Body Exocytosis by Cell Stretch or Purinergic Stimulation: Possible Physiological Roles, Messengers and Mechanisms

“…Indeed, Gajic et al found signs of reversible cell membrane stress failure in rats ventilated with high tidal volumes using propidium iodide as a cell-impermeable marker of cell damage [72]. In addition, we found ultrastructural evidence for membrane damage in type II pneumocytes stretched on silastic membranes [71]. In summary, in pathophysiological conditions, ATP leakage from wounded cells could influence type II cells and surfactant secretion in an autocrine/paracrine way.…”

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