Abstract
Caveolae 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 Gαq/phospholipase Cβ 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 Gαq and one of its receptors, bradykinin type 2 receptor (B2R), as well as a significant population of Gαi and its coupled β2-adrenergic receptor (βAR), 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 Gαq-B2R, but does not affect cAMP signals mediated through Gαi and βAR. Structurally, we find that mild osmotic stress corresponding roughly to a pressure of 3.82 newtons/m2 increases the domain diameter by ∼30% 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.
Highlights
Many cells undergo mechanical deformation in their normal function
Caveolae are structurally composed of ϳ140 copies of either caveolin-1 or caveolin-3 (Cav1 or Cav3), which form a thimble-like structure in the inner leaflet of the plasma membrane promoting membrane curvature and leading to the invaginations seen by electron microscopy [2]
We found that when rat aortic smooth muscle cells are subjected to relatively mild osmotic stress, Ca2ϩ signals mediated through G␣q/PLC are diminished suggesting a disruption of the interactions between Cav-1 and G␣q that stabilize the activated state
Summary
PLC, phospholipase C; B2R, bradykinin type 2 receptor; AR, 2-adrenergic receptor; GPCR, G protein-coupled receptor; eGFP, enhanced GFP; eCFP, enhanced cyan fluorescent protein; eYFP, enhanced YFP; FCS, fluorescence correlation spectroscopy; N, newton; STORM, stochastic optical reconstruction microscopy; NND, Nearest Neighbor Distance; HBSS, Hanks’ balanced salt solution. We found that when rat aortic smooth muscle cells are subjected to relatively mild osmotic stress, Ca2ϩ signals mediated through G␣q/PLC are diminished suggesting a disruption of the interactions between Cav-1 and G␣q that stabilize the activated state. Under these mild stress conditions, we could not detect dissociation of caveola domains [11]. Our studies support a model in which mild deformation of caveolae, such as those encountered during normal cell function, provides a mechanism for cells to control calcium signals and maintain membrane integrity These structural measurements allow us to estimate the stability of caveolae and, in turn, the stability that caveola domains provide to cells
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