Caveolin-1 dependent regulation of cell-matrix interphase in 3D collagen gels.

Cell and extracellular matrix (ECM) interactions are essential for maintaining tissue function and homeostasis. Changes in the biochemical or mechanical properties of the ECM can lead to diseases such as fibrosis or cancer. In a 3D microenvironment, cell-matrix interaction is vital to how cells sense and respond to biochemical and biophysical cues. This study examines the reciprocal interactions between fibroblasts and collagen in 3D hydrogels. 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 differentially affect collagen organisation in their immediate proximity with changing concentration and time. This regulation is interestingly lost in Caveolin-1 null fibroblasts with altered stiffness, mechanosensing and cytoskeletal regulation. Inhibition of the Rho-ROCK pathway (known to be changed in Caveolin-1 null fibroblasts) drives cellular protrusions and concentration-dependent 3D collagen organisation in wildtype fibroblasts, but surprisingly not in Caveolin-1 null fibroblasts. This depends on dynamin-dependent endocytosis, 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. Statement of SignificanceThis study highlights the critical role of Caveolin-1 in cell-mediated regulation of the extracellular matrix (ECM) in a 3D microenvironment. Using confocal reflectance microscopy, we demonstrate that subtle changes in collagen concentration significantly influence collagen organisation over time. While wild-type fibroblasts exhibit dynamic, concentration-dependent regulation of the cell-matrix interphase, Caveolin-1 null fibroblasts lose this regulation. Furthermore, Rho-ROCK pathway inhibition promotes protrusions and collagen reorganisation in wild-type cells but not Caveolin-1 null fibroblasts. Dynamin-dependent endocytosis also emerges as a key modulator of this process. These findings provide quantitative insights into how mechanosensing and cytoskeletal regulation, dependent on endocytosis can control the cell-matrix interphase, with possible implications for understanding disease-associated matrix changes.