Super-resolution Visualization of Caveola Deformation in Response to Osmotic Stress

Abstract

Edited by George M. CarmanCaveolae are protein-dense plasma membrane domains structurally composed of caveolin-1 or -3 along with other proteins. Our previous studies have shown that caveolae enhance calcium signals generated through the G␣ q /phospholipase C␤ signaling pathway and that subjecting cells to hypo-osmotic stress reverses this enhancement. In this study, we have used super-resolution fluorescence microscopy supplemented by fluorescence correlation studies to determine the structural factors that underlie this behavior. We find similar and significant population of G␣ q and one of its receptors, bradykinin type 2 receptor (B2R), as well as a significant population of G␣ i and its coupled ␤2-adrenergic receptor (␤AR), are localized to caveola domains. Although mild osmotic stress deforms caveolae and alters interactions between the caveolae and these proteins, the general structure and the localization of caveola components remain largely unchanged. This deformation eliminates the ability of caveolae to stabilize calcium signals mediated through G␣ q -B2R, but does not affect cAMP signals mediated through G␣ i and ␤AR. Structurally, we find that mild osmotic stress corresponding roughly to a pressure of 3.82 newtons/m 2 increases the domain diameter by ϳ30% and increases the fluorescence intensity in the center of the domain mouth suggesting a flattening of the invagination. Approximate calculations show that caveolae in muscle tissue have the strength to handle the stress of muscle movement.

Regulation of bifunctional proteins in cells: Lessons from the phospholipase Cβ/G protein pathway

“…Caveolae are prominent in smooth muscle cells and provide mechanical strength by flattening during stretch to provide more membrane area . We have found that the flattening of these domains by mechanical stretch or hypo‐osmotic conditions eliminates stabilization of activated Gαq by caveolin molecules reducing Ca 2+ signals .…”

Stimulation of phospholipase Cβ1 by Gαq promotes the assembly of stress granule proteins

“…Fluorescence correlation (FCS) and N&B measurements-FCS measurements were performed on the dual-channel confocal fluorescence correlation spectrometer (Alba version 5, ISS Inc.) equipped with avalanche photodiodes and a Nikon Eclipse Ti-U inverted microscope as previously described (27). For N&B (see (57)), we collected ~100 PC12 cell images viewing either free eGFP (control) or eGFP-Ago2, at a 66nm/pixel resolution and at a rate of 4 µs/pixel.…”

“…To determine whether PLCβ1 can impact stress granule formation, we started with mild, hypo-osmotic stress (300 to 150 mOsm) that some cells may experience under normal physiological conditions and where we have found has reversible effects on the Gαq/PLCβ signaling pathway in muscle cells (26,27). It is probable that osmotic stress would produce stress granules that are different in size, number and composition from the well-studied structures produced by NaAsO2.…”

The Development of Compartmentation of cAMP Signaling in Cardiomyocytes: The Role of T-Tubules and Caveolae Microdomains

“…Furthermore, Sinha et al and others have shown that acute mechanical stress induced by osmotic swelling or stretching, reduced the interaction of caveolin with cavin-1. It increased the concentration of free caveolin proteins at the plasma membrane, thus resulting in a rapid disappearance of caveolae [ 38 , 41 ]. Mohan et al further elucidated that Cavin-1 regulates caveolae dynamics, by targeting Cavin-3 to caveolae where it interacts with the scaffolding domain of Cav1 [ 42 ].…”