Osmotic Stress Alters the Intracellular Distribution of Non-erythroidal Spectrin (Fodrin) in Bovine Aortic Endothelial Cells

Abstract

Cell swelling is known to result in unfolding of membrane invaginations and restructuring of F-actin. The effect of cell swelling on the intracellular distributions of other cytoskeletal proteins that constitute the submembrane cortical cytoskeleton is virtually unknown. This study focuses on the effects of cell swelling on non-erythroidal spectrin (fodrin, also known as spectrin II), a predominant component of the membrane cytoskeleton. The intracellular distribution of spectrin in vascular endothelial cells was studied by optical sectioning using a 3-D deconvolution microscopy system. Our results show that once bovine aortic endothelial cells (BAECs) reach confluency, the non-erythroidal spectrin is localized in the submembrane regions of the cells. Analysis of the intensity profiles of the non-erythroidal spectrin under isotonic and hypotonic conditions show that: (a) the width of the submembrane spectrin staining increases gradually with time within the first 5 minutes after the osmotic shock; (b) significant recovery is observed after 10 minutes even if the cells are maintained in hypotonic medium, and (c) spectrin distribution is altered by disrupting F-actin with latrunculin A but not by stabilizing F-actin with jasplakinolide. We suggest that cell swelling results in partial translocation of the submembrane spectrin to the cytosol and that it may play a major role in initiation of swelling-induced cellular events.

Stimulation of Gαq-coupled M1 muscarinic receptor causes reversible spectrin redistribution mediated by PLC, PKC and ROCK

“…We next examined whether changes in osmotic pressure can induce membrane blebbing associated with ␣II spectrin distribution in M1-CHO cells. Switching the perfusion buffer between the isotonic and hypotonic solution caused the global expansion and/or shrinkage of the spectrin lattice, as reported previously (Sun and Levitan, 2003), but lengthy oscillatory Effects of inhibitory compounds on ␣II spectrin remodeling and membrane blebbing. M1-CHO cells expressing YFP-␣II spectrin were pre-incubated in Hepes buffer containing inhibitors at the indicated concentration.…”

Hypotonic Challenge of Endothelial Cells Increases Membrane Stiffness with No Effect on Tether Force

“…The general expectation is that, as cells swell, membrane tension should increase; however, there is little direct evidence to support this notion. It is also expected that upon swelling, the elastic modulus of the cells should decrease, representing cell softening, because earlier studies showed that cell swelling results in the disruption and reorganization of the cortical cytoskeleton (14)(15)(16)(17)(18)(19)(20)(21). In this study, we analyze the impact of cell swelling on the biomechanical properties of human aortic endothelial cells by simultaneous measurements of endothelial elastic moduli and the force required for the formation of membrane tethers.…”

“…Indeed, Beyder and Sachs (44) showed that the application of hydrostatic pressure through a micropipette directly to the cell interior in a whole-cell patch configuration results in a decrease in membrane deformability, which indicates membrane stiffening. To discriminate between these possibilities, we tested whether depolymerization of F-actin, which causes a collapse of the cytoskeleton (18,32), abrogates the observed stiffening effect. As expected, we observed a dramatic decrease in the elastic modulus for cells treated with latrunculin A, indicating the collapse of the cytoskeleton and possibly the transfer of stress from deep cytoskeleton to the cortex.…”

“…Multiple studies have demonstrated that the membrane elastic modulus of living cells depends primarily on the submembrane cytoskeleton, which represents the mechanical scaffold of the cells (reviewed by (12,13)). Because cell swelling is expected to induce disruption of the cytoskeleton (14)(15)(16)(17)(18)(19)(20)(21) and possibly its detachment from the membrane, cell swelling could be expected to result in cell softening as well. It is not clear, however, how the two biomechanical parameters (membrane tension and elastic modulus) are interrelated during cell swelling.…”

Membrane Stiffening in Osmotic Swelling

“…First, the cellular cytoskeleton may be thought of as a cross-linked filamentous gel having a poroelastic structure, which has been likened to the structure of a sponge, and has therefore been termed the “sponge-effect” by Sachs et al (Chao, Sivaselvan, & Sachs, 2018; Charras, Mitchison, & Mahadevan, 2009; Sachs & Sivaselvan, 2015). Another possibility is that the softening could be explained by mild disruption and reorganization of the cytoskeleton as a result of osmotic challenge (Hoffmann, 2000; Hoffmann, Lambert, & Pedersen, 2009; Jorgensen et al, 2003; Lambert, Hoffmann, & Pedersen, 2008; Levitan, Almonte, Mollard, & Garber, 1995; Pasantes-Morales, Cardin, & Tuz, 2000; Pedersen et al, 1999, 2001; Spagnoli et al, 2008; Sun & Levitan, 2003). It is not clear which of these possibilities would account for the observed mild softening of cells as a response to osmotic challenge.…”