Substrate elasticity regulates cytoskeletal remodeling and mechanical behavior of U2OS osteosarcoma cells

Abstract

Substrate elasticity plays a pivotal role in regulating the morphology, mechanical properties, and cytoskeletal organization of cancer cells. In this study, we examined the response of U2OS osteosarcoma cells to substrates of varying stiffness, with a particular focus on cytoskeletal remodeling, cell elasticity, and microparticle internalization. To simulate environments of moderate and high stiffness, cells were cultured on polyacrylamide (PA) hydrogels with a stiffness of 40 kPa and on rigid glass substrates, respectively. Changes in cell morphology and cytoskeletal organization were assessed using fluorescence microscopy, while cell mechanical properties were measured using atomic force microscopy (AFM). To investigate the relationship between substrate mechanics and endocytic activity, carboxylated fluorescent 2 {micro}m latex microspheres were introduced to the cell culture system. Our study showed that cell spreading increased with substrate stiffness. U2OS cells cultured on glass exhibited a significantly larger surface area, more actin stress fibers, and a more organized, stretched cytoskeletal architecture compared to cells grown on 40 kPa PA gels. AFM measurements further demonstrated that cells on glass were mechanically stiffer than those on PA substrates. Microparticle uptake was also strongly influenced by substrate stiffness. Cells cultured in the 40 kPa PA gels internalized a significantly greater number of fluorescent microspheres. Notably, these cells frequently formed distinct, cup-like structures composed of microtubules around the beads. Three-dimensional image reconstructions revealed that these structures encapsulate the particles in an asymmetrical manner, indicative of active cytoskeletal remodeling. To better understand the molecular composition of these microtubule-based structures, we analyzed the localization of selected microtubule-associated proteins (MAPs), including IQGAP1, CLIP1, and MARK2 (Conboy J.P. et al. 2024). Interestingly, only IQGAP1 was localized prominently to the microtubule cups on 40 kPa gels, often forming ring-like structures surrounding the beads. In some cases, these rings were observed independently of detectable microtubules, suggesting the involvement of an active, possibly microtubule-initiated, endocytic process. In conclusion, our findings demonstrate that substrate stiffness modulates multiple aspects of U2OS cell behavior, including morphology, cytoskeletal arrangement, mechanical properties, and microparticle uptake. These results underscore the mechanosensitive nature of osteosarcoma cells and highlight novel roles for microtubule structures and MAPs, particularly IQGAP1 in stiffness-dependent cellular uptake mechanisms.