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 involvedin 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 newline newline