Abstract
Substrate mechanical properties have emerged as potent determinants of cell functions and fate. We here tested the hypothesis that different forms of endocytosis are regulated by the elasticity of the synthetic hydrogels cells are cultured on. Towards this objective, we quantified cell-associated fluorescence of the established endocytosis markers transferrin (Tf) and cholera toxin subunit B (CTb) using a flow-cytometry based protocol, and imaged marker internalization using microscopy techniques. Our results demonstrated that clathrin-mediated endocytosis of Tf following a 10-minute incubation with a fibroblast cell line was lower on the softer substrates studied (5 kPa) compared to those with elasticities of 40 and 85 kPa. This effect was cancelled after 1-hour incubation revealing that intracellular accumulation of Tf at this time point did not depend on substrate elasticity. Lipid-raft mediated endocytosis of CTb, on the other hand, was not affected by substrate elasticity in the studied range of time and substrate elasticity. The use of pharmacologic contractility inhibitors revealed inhibition of endocytosis for both Tf and CTb after a 10-minute incubation and a dissimilar effect after 1 hour depending on the inhibitor type. Further, the internalization of fluorescent NPs, used as model drug delivery systems, showed a dependence on substrate elasticity, while transfection efficiency was unaffected by it. Finally, an independence on substrate elasticity of Tf and CTb association with HeLa cells indicated that there are cell-type differences in this respect. Overall, our results suggest that clathrin-mediated but not lipid-raft mediated endocytosis is potentially influenced by substrate mechanics at the cellular level, while intracellular trafficking and accumulation show a more complex dependence. Our findings are discussed in the context of previous work on how substrate mechanics affect the fundamental process of endocytosis and highlight important considerations for future studies.
Highlights
Cells respond to the mechanics of their microenvironment by transducing mechanical cues to biochemical information that contributes to the regulation of their adhesion, migration and differentiation [1,2]
Association of cholera toxin subunit B (CTb) or high MW dextran molecules was unaffected by substrate elasticity in the examined range, indicating that different forms of endocytosis are not regulated in the same manner by the substrate
Pharmacological inhibition of actomyosin contractility had significant effects on uptake of Tf, depending on inhibitor type; treatment with blebbistatin initially inhibited its uptake, while at longer time points an accumulation of Tf inside blebbistatin-treated cells was observed compared to control cells
Summary
Cells respond to the mechanics of their microenvironment by transducing mechanical cues to biochemical information that contributes to the regulation of their adhesion, migration and differentiation [1,2]. How this is mechanistically achieved is a subject of intense study, given the significance of mechanotransduction in development and disease progression [3,4]. Endocytosis is the process by which cells internalize extracellular material along with surrounding fluid by engulfing part of their plasma membrane. Many molecules and viruses utilize clathrin-independent pathways to enter cells after binding to the plasma membrane [8]
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