Abstract
Focal adhesion (FA) assembly, mediated by integrin activation, responds to matrix stiffness; however, the underlying mechanisms are unclear. Here, we showed that β1 integrin and caveolin-1 (Cav1) levels were decreased with declining matrix stiffness. Soft matrix selectively downregulated β1 integrin by endocytosis and subsequent lysosomal degradation. Disruption of lipid rafts with methyl-β-cyclodextrin or nystatin, or knockdown of Cav1 by siRNA decreased cell spreading, FA assembly, and β1 integrin protein levels in cells cultured on stiff matrix. Overexpression of Cav1, particularly the phospho-mimetic mutant Cav1-Y14D, averted soft matrix-induced decreases in β1 integrin protein levels, cell spreading, and FA assembly in NMuMG cells. Interestingly, overexpression of an auto-clustering β1 integrin hindered soft matrix-induced reduction of Cav1 and cell spreading, which suggests a reciprocal regulation between β1 integrin and Cav1. Finally, co-expression of this auto-clustering β1 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 β1 integrin and Cav1, which reciprocally control each other, and subsequently determine FA assembly and turnover.
Highlights
Caveolin-1 (Cav1), a structural protein of caveolae/lipid rafts that conducts and coordinates multiple signals at the cell surface[12,13]
We show a reciprocal regulation between Cav[1] and β1 integrin that is orchestrated by matrix stiffness, and highlighted their functions in mechanical sensing machinery and delineated their role in generating platforms at the cell surface for the initiation of focal adhesion (FA) assembly
To correctly determine the effect of matrix stiffness on cell spreading and focal adhesion (FA) assembly, cells were cultured on collagen-coated dishes (>1 GPa) or less stiff polyacrylamide (PA) gels
Summary
Caveolin-1 (Cav1), a structural protein of caveolae/lipid rafts that conducts and coordinates multiple signals at the cell surface[12,13]. It is well accepted that Cav[1] is involved in integrin-dependent signaling[11,14,15] and FA assembly/turnover[16,17], and acts as a mechanosensor in sensing flow and stretch-induced mechanotransduction[18,19]. The underlying mechanism by which Cav[1] regulates mechanosensation and matrix stiffness-dependent integrin www.nature.com/scientificreports/. We show a reciprocal regulation between Cav[1] and β1 integrin that is orchestrated by matrix stiffness, and highlighted their functions in mechanical sensing machinery and delineated their role in generating platforms at the cell surface for the initiation of FA assembly
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