Role of Caveolin-1 in cellular mechanosensing in 3D and 2D microenvironment

Abstract

Cell–extracellular matrix (ECM) interactions are fundamental to maintaining tissue architecture, function, and homeostasis. Alterations in the biochemical or mechanical properties of the ECM can disrupt these interactions, contributing to pathological conditions such as fibrosis and cancer. While in 2D microenvironments, matrix stiffness serves as a dominant ECM medicated mechanical cue influencing cellular behaviour, in 3D microenvironments, the regulation is more complex, involving additional physical parameters such as matrix porosity, rigidity and fibre architecture. In this study, we investigated the bidirectional interactions between fibroblasts and collagen under varying mechanochemical conditions in 3D (on different collagen concentration and time) and in 2D (on differential stiffnesses) microenvironments focusing on the role of Caveolin-1 (Cav-1) in the regulation. We quantitatively measured changes in collagen branch number and junctions in 3D hydrogels using confocal reflectance microscopy and existing analysis protocols. This reveals the impact small changes in collagen concertation (1.0 vs 1.5 mg/ml) over time (15 minutes to 4 hours) have on 3D gels. Embedded in 3D hydrogels, wild-type mouse fibroblasts (WT MEFs) differentially affect collagen organisation in their immediate proximity with changing concentration and time which is interestingly lost in Cav-1 null fibroblasts (Cav-1 Null MEFs) with altered stiffness, mechanosensing and cytoskeletal regulation. Inhibition of the Rho- ROCK pathway (known to be changed in Cav-1 null fibroblasts) drives cellular protrusions and concentration-dependent 3D collagen organisation in wildtype fibroblasts, but surprisingly not in Caveolin-1 null fibroblasts. This interestingly depends on dynamin, which when inhibited, disrupts ROCK-dependent protrusions and alters collagen organisation in 3D collagen. Together, these observations quantitatively demonstrate how cells respond at the cell- matrix interphase to subtle changes in collagen concentration and organisation in 3D hydrogels, regulated by the presence of Caveolin-1. On the other hand, cells in 2D microenvironments of varying stiffnesses respond differently, which is also dependent on Caveolin-1. In this study, we standardized conditions and investigated how the presence or absence of Caveolin-1 regulate stiffness dependent endocytosis in cell.