Measuring integrin force loading rates using a two-step DNA tension sensor.

Cells apply forces to extracellular matrix (ECM) ligands through transmembrane integrin receptors: an interaction which is intimately involved in cell motility, wound healing, cancer invasion and metastasis. These small (pN) forces exerted by cells have been studied by molecular tension fluorescence microscopy (MTFM), which utilizes a force-induced conformational change of a probe to detect mechanical events. MTFM has revealed the force magnitude for integrins receptors in a variety of cell models including primary cells. However, force dynamics and specifically the force loading rate (LR) have important implications in receptor signaling and adhesion formation and remain poorly characterized. Here, we develop a LR probe which is comprised of an engineered DNA structures that undergoes two mechanical transitions at distinct force thresholds: a low force threshold at 4.7 pN corresponding to hairpin unfolding and a high force threshold at 56 pN triggered through duplex shearing. These transitions yield distinct fluorescence signatures observed through single-molecule fluorescence microscopy in live-cells. Automated analysis of tens of thousands of events from 8 cells showed that the bond lifetime of integrins that engage their ligands and transmit a force >4.7 pN decays exponentially with a τ of 45.6 sec. A small subset of these events (<10%) mature in magnitude to >56pN with a median loading rate of 1.3 pNs-1 with these mechanical ramp events localizing at the periphery of the cell-substrate junction. Importantly, the LR probe design is modular and can be adapted to measure force ramp rates for a broad range of mechanoreceptors and cell models, thus aiding in the study of mechanotransduction.

DNA Tension Probes Show that Cardiomyocyte Maturation Is Sensitive to the Piconewton Traction Forces Transmitted by Integrins.

Cardiac muscle cells (CMCs) are the unit cells that comprise the heart. CMCs go through different stages of differentiation and maturation pathways to fully mature into beating cells. These cells can sense and respond to mechanical cues through receptors such as integrins which influence maturation pathways. For example, cell traction forces are important for the differentiation and development of functional CMCs, as CMCs cultured on varying substrate stiffness function differently. Most work in this area has focused on understanding the role of bulk extracellular matrix stiffness in mediating the functional fate of CMCs. Given that stiffness sensing mechanisms are mediated by individual integrin receptors, an important question in this area pertains to the specific magnitude of integrin piconewton (pN) forces that can trigger CMC functional maturation. To address this knowledge gap, we used DNA adhesion tethers that rupture at specific thresholds of force (∼12, ∼56, and ∼160 pN) to test whether capping peak integrin tension to specific magnitudes affects CMC function. We show that adhesion tethers with greater force tolerance lead to functionally mature CMCs as determined by morphology, twitching frequency, transient calcium flux measurements, and protein expression (F-actin, vinculin, α-actinin, YAP, and SERCA2a). Additionally, sarcomeric actinin alignment and multinucleation were significantly enhanced as the mechanical tolerance of integrin tethers was increased. Taken together, the results show that CMCs harness defined pN integrin forces to influence early stage development. This study represents an important step toward biophysical characterization of the contribution of pN forces in early stage cardiac differentiation.

Supramolecular DNA Photonic Hydrogels for On-Demand Control of Coloration with High Spatial and Temporal Resolution.

Hydrogels embedded with periodic arrays of nanoparticles display a striking photonic crystal coloration that may be useful for applications such as camouflage, anticounterfeiting, and chemical sensing. Dynamically generating color patterns requires control of nanoparticle organization within a polymer network on-demand, which is challenging. We solve this problem by creating a DNA hydrogel system that shows a 50 000-fold decrease in modulus upon heating by ∼10 °C. Magnetic nanoparticles entrapped within these DNA gels generate a structural color only when the gel is heated and a magnetic field is applied. A spatially controlled photonic crystal coloration was achieved by photopatterning with a near-infrared illumination. Color was "erased" by illuminating or heating the gel in the absence of an external magnetic field. The on-demand assembly technology demonstrated here may be beneficial for the development of a new generation of smart materials with potential applications in erasable lithography, encryption, and sensing.

An Outside-In Switch in Integrin Signaling Caused by Chemical and Mechanical Signals in Reactive Astrocytes.

Astrocyte reactivity is associated with poor repair capacity after injury to the brain, where chemical and physical changes occur in the damaged zone. Astrocyte surface proteins, such as integrins, are upregulated, and the release of pro-inflammatory molecules and extracellular matrix (ECM) proteins upon damage generate a stiffer matrix. Integrins play an important role in triggering a reactive phenotype in astrocytes, and we have reported that α V ß3 Integrin binds to the Thy-1 (CD90) neuronal glycoprotein, increasing astrocyte contractility and motility. Alternatively, α V ß3 Integrin senses mechanical forces generated by the increased ECM stiffness. Until now, the association between the α V ß3 Integrin mechanoreceptor response in astrocytes and changes in their reactive phenotype is unclear. To study the response to combined chemical and mechanical stress, astrocytes were stimulated with Thy-1-Protein A-coated magnetic beads and exposed to a magnetic field to generate mechanical tension. We evaluated the effect of such stimulation on cell adhesion and contraction. We also assessed traction forces and their effect on cell morphology, and integrin surface expression. Mechanical stress accelerated the response of astrocytes to Thy-1 engagement of integrin receptors, resulting in cell adhesion and contraction. Astrocyte contraction then exerted traction forces onto the ECM, inducing faster cell contractility and higher traction forces than Thy-1 alone. Therefore, cell-extrinsic chemical and mechanical signals regulate in an outside-in manner, astrocyte reactivity by inducing integrin upregulation, ligation, and signaling events that promote cell contraction. These changes in turn generate cell-intrinsic signals that increase traction forces exerted onto the ECM (inside-out). This study reveals α V ß3 Integrin mechanoreceptor as a novel target to regulate the harmful effects of reactive astrocytes in neuronal healing.

Turn-key mapping of cell receptor force orientation and magnitude using a commercial structured illumination microscope.

Many cellular processes, including cell division, development, and cell migration require spatially and temporally coordinated forces transduced by cell-surface receptors. Nucleic acid-based molecular tension probes allow one to visualize the piconewton (pN) forces applied by these receptors. Building on this technology, we recently developed molecular force microscopy (MFM) which uses fluorescence polarization to map receptor force orientation with diffraction-limited resolution (~250 nm). Here, we show that structured illumination microscopy (SIM), a super-resolution technique, can be used to perform super-resolution MFM. Using SIM-MFM, we generate the highest resolution maps of both the magnitude and orientation of the pN traction forces applied by cells. We apply SIM-MFM to map platelet and fibroblast integrin forces, as well as T cell receptor forces. Using SIM-MFM, we show that platelet traction force alignment occurs on a longer timescale than adhesion. Importantly, SIM-MFM can be implemented on any standard SIM microscope without hardware modifications.

Chameleon-Inspired Strain-Accommodating Smart Skin.

Stimuli-responsive color-changing hydrogels, commonly colored using embedded photonic crystals (PCs), have potential applications ranging from chemical sensing to camouflage and anti-counterfeiting. A major limitation in these PC hydrogels is that they require significant deformation (>20%) in order to change the PC lattice constant and generate an observable chromatic shift (∼100 nm). By analyzing the mechanism of how chameleon skin changes color, we developed a strain-accommodating smart skin (SASS), which maintains near-constant size during chromatic shifting. SASS is composed of two types of hydrogels: a stimuli-responsive, PC-containing hydrogel that is patterned within a second hydrogel with robust mechanical properties, which permits strain accommodation. In contrast to conventional "accordion"-type PC responsive hydrogels, SASS maintains near-constant volume during chromatic shifting. Importantly, SASS materials are stretchable (strain ∼150%), amenable to patterning, spectrally tunable, and responsive to both heat and natural sunlight. We demonstrate examples of using SASS for biomimicry. Our strategy, to embed responsive materials within a mechanically matched scaffolding polymer, provides a general framework to guide the future design of artificial smart skins.