Mechanotransduction of matrix stiffness in regulation of focal adhesion size and number: reciprocal regulation of caveolin-1 and β1 integrin

Focal adhesion (FA) assembly, mediated by integrin activation, responds to matrix stiffness; however, the underlying mechanisms are unclear. Here, we showed that beta 1 integrin and caveolin-1 (Cav1) levels were decreased with declining matrix stiffness. Soft matrix selectively downregulated beta 1 integrin by endocytosis and subsequent lysosomal degradation. Disruption of lipid rafts with methyl-beta cyclodextrin or nystatin, or knockdown of Cav1 by siRNA decreased cell spreading, FA assembly, and beta 1 integrin protein levels in cells cultured on stiff matrix. Overexpression of Cav1, particularly the phosphomimetic mutant Cav1-Y14D, averted soft matrix-induced decreases in beta 1 integrin protein levels, cell spreading, and FA assembly in NMuMG cells. Interestingly, overexpression of an auto-clustering beta 1 integrin hindered soft matrix-induced reduction of Cav1 and cell spreading, which suggests a reciprocal regulation between beta 1 integrin and Cav1. Finally, co-expression of this auto-clustering beta 1 integrin and Cav1-Y14D synergistically enhanced cell spreading, and FA assembly in HEK293T cells cultured on either stiff (>G Pa) or soft (0.2 kPa) matrices. Collectively, these results suggest that matrix stiffness governs the expression of beta 1 integrin and Cav1, which reciprocally control each other, and subsequently determine FA assembly and turnover.