Role of caveolin-1 in regulating membrane properties in cells in a 3D microenvironment

Abstract

Caveolae are 60-80 nm omega shaped structures on the plasma membrane which comprise of Caveolin-1 (Cav1), the major structural protein and are rich in cholesterol and sphingolipids. Caveolae play an important role in cellular signaling, endocytosis and mechanosensing. Apart from these functions, caveolae have been recently emerged as plasma membrane organizers and protectors. Their presence or absence changes the membrane composition, membrane order and membrane tension, further regulating signal transduction in cells. All these properties of plasma membrane are either known or expected to be different in cells which are cultured in a three-dimensional (3D) microenvironment as compared to conventional rigid two-dimensional (2D) tissue culture plate. In this project, we have tried to elucidate the role of Cav1 in cells in a 3D microenvironment. We focused on two aspects - 1) how Cav1 modulate mobility of various membrane associated proteins and 2) whether and how Cav1 affect endocytosis in cells in a 3D microenvironment. pTyr14-cav1 is one of the important modification on Cav1 and we have studied its relevance in both these aspects. We compared mobility of various markers (K-Ras-CAAX-GFP, H-Ras-CAAX-GFP and GPI-GFP) in WT Cav1-KO MEFs (Mouse Embryonic Fibroblasts) and found it to be differentially regulated by Cav1 in 2D versus 3D. Mobility of K-Ras-CAAX-GFP on the plasma membrane by FRAP was found to be reduced in WT MEFs as compared to Cav1-KO MEFs. Interestingly, reconstitution of Cav1-KO MEFs with WT Cav1 or Y14FCav1 (phosphodefecient version) both reduced this mobility, suggesting pTyr14-Cav1 not being involved in regulating the mobility. H-Ras-CAAX-GFP and GPI-GFP did not have any difference in their mobility in WT MEFs versus Cav1-KO MEFs in 3D collagen. This altered mobility of K-Ras-CAAX-GFP could be because of altered plasma membrane properties (like fluidity or tension) of WT MEFs versus Cav1- KO MEFs. Our studies looking at endocytosis in WT-MEFs in 3D collagen showed that, at concentrations 1.5 mg/ml and above, GM1-CTxB endocytosis was blocked. However, at lower collagen concentrations (0.5 mg/ml and 1 mg/ml) the endocytosis was supported. Cav1-KO MEFs did not show such differential uptake of GM1-CTxB at different collagen concentrations. Also, the mobility of K-Ras-CAAX-GFP was higher in WT MEFs embedded in 1 mg/ml than in 1.5 mg/ml collagen. Reconstitution of Cav1-KO MEFs with WT-Cav1 or Y14F-Cav1 both blocked the endocytosis at 1.5 mg/ml, again suggesting pTyr14-Cav1 not being involved in regulating GM1-CTxB endocytosis in 3D. Preliminary laser ablation and actin disruption experiments indicate to a possible mechanism for this differential regulation, which is, the membrane tension and / or cortical actin network could be different in WT MEFs embedded in different concentrations of collagen gels.