Many essential cellular processes are regulated by mechanical properties of their microenvironment. Here, we introduce stimuli-responsive composite scaffolds fabricated by three-dimensional (3D) laser lithography to simultaneously stretch large numbers of single cells in tailored 3D microenvironments. The key material is a stimuli-responsive photoresist containing cross-links formed by noncovalent, directional interactions between β-cyclodextrin (host) and adamantane (guest). This allows reversible actuation under physiological conditions by application of soluble competitive guests. Cells adhering in these scaffolds build up initial traction forces of ~80 nN. After application of an equibiaxial stretch of up to 25%, cells remodel their actin cytoskeleton, double their traction forces, and equilibrate at a new dynamic set point within 30 min. When the stretch is released, traction forces gradually decrease until the initial set point is retrieved. Pharmacological inhibition or knockout of nonmuscle myosin 2A prevents these adjustments, suggesting that cellular tensional homeostasis strongly depends on functional myosin motors.

αVβ3 integrin can bind to multiple extracellular matrix proteins, including vitronectin (Vn) and fibronectin (Fn), which are often presented to cells in culture as homogenous substrates. However, in tissues, cells experience highly complex and changing environments. To better understand integrin ligand selection in such complex environments, we employed binary-choice substrates of Fn and Vn to dissect αVβ3 integrin-mediated binding to different ligands on the subcellular scale. Super-resolution imaging revealed that αVβ3 integrin preferred binding to Vn under various conditions. In contrast, binding to Fn required higher mechanical load on αVβ3 integrin. Integrin mutations, structural analysis and chemical inhibition experiments indicated that the degree of hybrid domain swing-out is relevant for the selection between Fn and Vn; only a force-mediated, full hybrid domain swing-out facilitated αVβ3-Fn binding. Thus, force-dependent conformational changes in αVβ3 integrin increased the diversity of available ligands for binding and therefore enhanced the ligand promiscuity of this integrin. This article has an associated First Person interview with the first author of the paper.

The shape of animal cells is an important regulator for many essential processes such as cell migration or division. It is strongly determined by the organization of the actin cytoskeleton, which is also the main regulator of cell forces. Quantitative analysis of cell shape helps to reveal the physical processes underlying cell shape and forces, but it is notoriously difficult to conduct it in three dimensions. Here we use direct laser writing to create 3D open scaffolds for adhesion of connective tissue cells through well-defined adhesion platforms. Due to actomyosin contractility in the cell contour, characteristic invaginations lined by actin bundles form between adjacent adhesion sites. Using quantitative image processing and mathematical modeling, we demonstrate that the resulting shapes are determined not only by contractility, but also by elastic stress in the peripheral actin bundles. In this way, cells can generate higher forces than through contractility alone.

Nonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration. Mammalian cells express up to three isoforms termed NM IIA, B, and C. We used U2OS cells to create CRISPR/Cas9-based knockouts of all three isoforms and analyzed the phenotypes on homogenously-coated surfaces, in collagen gels, and on micropatterned substrates. In contrast to homogenously-coated surfaces, a structured environment supports a cellular phenotype with invaginated actin arcs even in the absence of NM IIA-induced contractility. A quantitative shape analysis of cells on micropatterns combined with a scale-bridging mathematical model reveals that NM IIA is essential to build up cellular tension during initial stages of force generation, while NM IIB is necessary to elastically stabilize NM IIA-generated tension. A dynamic cell stretch/release experiment in a three-dimensional scaffold confirms these conclusions and in addition reveals a novel role for NM IIC, namely the ability to establish tensional homeostasis.

Integrin-mediated adhesions are convergence points of multiple signaling pathways. Their inner structure and their diverse functions can be studied with super-resolution microscopy. Here, we examined the spatial organization within focal adhesion by analyzing several adhesion proteins with structured illumination microscopy (SIM). We found that phosphorylated paxillin (pPax) and phosphorylated focal adhesion kinase (pFAK) form spot-like, spatially defined clusters within adhesions in several cell lines and confirmed these findings with additional super-resolution techniques. These clusters showed a more regular separation from each other compared to more randomly distributed labels of general FAK or paxillin. Mutational analysis indicated that the active (open) FAK conformation is a prerequisite for the pattern formation of pFAK. Live-cell super-resolution imaging revealed that organization in clusters is preserved over time for FAK constructs; however, distance between clusters is dynamic for FAK, while paxillin is more stable. Combined, these data introduce spatial clusters of pPax and pFAK as substructures in adhesions and highlight the relevance of paxillin-FAK binding for establishing a regular substructure in focal adhesions.