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.

Force-Induced Site-Specific Enzymatic Cleavage Probes Reveal That Serial Mechanical Engagement Boosts T Cell Activation.

The T cell membrane is studded with >104 T cell receptors (TCRs) that are used to scan target cells to identify short peptide fragments associated with viral infection or cancerous mutation. These peptides are presented as peptide-major-histocompatibility complexes (pMHCs) on the surface of virtually all nucleated cells. The TCR-pMHC complex forms at cell-cell junctions, is highly transient, and experiences mechanical forces. An important question in this area pertains to the role of the force duration in immune activation. Herein, we report the development of force probes that autonomously terminate tension within a time window following mechanical triggering. Force-induced site-specific enzymatic cleavage (FUSE) probes tune the tension duration by controlling the rate of a force-triggered endonuclease hydrolysis reaction. This new capability provides a method to study how the accumulated force duration contributes to T cell activation. We screened DNA sequences and identified FUSE probes that disrupt mechanical interactions with F > 7.1 piconewtons (pN) between TCRs and pMHCs. This rate of disruption, or force lifetime (τF), is tunable from tens of minutes down to 1.9 min. T cells challenged with FUSE probes with F > 7.1 pN presenting cognate antigens showed up to a 23% decrease in markers of early activation. FUSE probes with F > 17.0 pN showed weaker influence on T cell triggering further showing that TCR-pMHC with F > 17.0 pN are less frequent compared to F > 7.1 pN. Taken together, FUSE probes allow a new strategy to investigate the role of force dynamics in mechanotransduction broadly and specifically suggest a model of serial mechanical engagement boosting TCR activation.

Molecular Compressive Force Sensor for Mapping Forces at the Cell-Substrate Interface.

Mechanical forces are crucial for biological processes such as T cell antigen recognition. A suite of molecular tension probes to measure pulling forces have been reported over the past decade; however, there are no reports of molecular probes for measuring compressive forces, representing a gap in the current mechanobiology toolbox. To address this gap, we report a molecular compression reporter using pseudostable hairpins (M-CRUSH). The design principle was based on a pseudostable DNA structure that folds in response to an external compressive force. We created a library of DNA stem-loop hairpins with varying thermodynamic stability, and then used Förster Resonance Energy Transfer (FRET) to quantify hairpin folding stability as a function of temperature and crowding. We identified an optimal pseudostable DNA hairpin highly sensitive to molecular crowding that displayed a shift in melting temperature (Tm) of 7 °C in response to a PEG crowding agent. When immobilized on surfaces, this optimized DNA hairpin showed a 29 ± 6% increase in FRET index in response to 25% w/w PEG 8K. As a proof-of-concept demonstration, we employed M-CRUSH to map the compressive forces generated by primary naïve T cells. We noted dynamic compressive forces that were highly sensitive to antigen presentation and coreceptor engagement. Importantly, mechanical forces are generated by cytoskeletal protrusions caused by acto-myosin activity. This was confirmed by treating cells with cytoskeletal inhibitors, which resulted in a lower FRET response when compared to untreated cells. Furthermore, we showed that M-CRUSH signal is dependent on probe density with greater density probes showing enhanced signal. Finally, we demonstrated that M-CRUSH probes are modular and can be applied to different cell types by displaying a compressive signal observed under human platelets. M-CRUSH offers a powerful tool to complement tension sensors and map out compressive forces in living systems.

Cbl-b mitigates the responsiveness of naive CD8+ T cells that experience extensive tonic T cell receptor signaling.

Naive T cells experience tonic T cell receptor (TCR) signaling in response to self-antigens presented by major histocompatibility complex (MHC) in secondary lymphoid organs. We investigated how relatively weak or strong tonic TCR signals influence naive CD8+ T cell responses to stimulation with foreign antigens. The heterogeneous expression of Nur77-GFP, a transgenic reporter of tonic TCR signaling, in naive CD8+ T cells suggests variable intensities or durations of tonic TCR signaling. Although the expression of genes associated with acutely stimulated T cells was increased in Nur77-GFPHI cells, these cells were hyporesponsive to agonist TCR stimulation compared with Nur77-GFPLO cells. This hyporesponsiveness manifested as diminished activation marker expression and decreased secretion of IFN-γ and IL-2. The protein abundance of the ubiquitin ligase Cbl-b, a negative regulator of TCR signaling, was greater in Nur77-GFPHI cells than in Nur77-GFPLO cells, and Cbl-b deficiency partially restored the responsiveness of Nur77-GFPHI cells. Our data suggest that the cumulative effects of previously experienced tonic TCR signaling recalibrate naive CD8+ T cell responsiveness. These changes include gene expression changes and negative regulation partially dependent on Cbl-b. This cell-intrinsic negative feedback loop may enable the immune system to restrain naive CD8+ T cells with higher self-reactivity.

The T-cell niche tunes immune function through modulation of the cytoskeleton and TCR-antigen forces.

Obesity is a major public health crisis given its rampant growth and association with an increased risk for cancer. Interestingly, patients with obesity tend to have an increased tumor burden and decreased T-cell function. It remains unclear how obesity compromises T-cell mediated immunity. To address this question, we modeled the adipocyte niche using the secretome released from adipocytes as well as the niche of stromal cells and investigated how these factors modulated T-cell function. We found that the secretomes altered antigen-specific T-cell receptor (TCR) triggering and activation. RNA-sequencing analysis identified thousands of gene targets modulated by the secretome including those associated with cytoskeletal regulation and actin polymerization. We next used molecular force probes to show that T-cells exposed to the adipocyte niche display dampened force transmission to the TCR-antigen complex and conversely, stromal cell secreted factors lead to significantly enhanced TCR forces. These results were then validated in diet-induced obese mice. Importantly, secretome-mediated TCR force modulation mirrored the changes in T-cell functional responses in human T-cells using the FDA-approved immunotherapy, blinatumomab. Thus, this work shows that the adipocyte niche contributes to T-cell dysfunction through cytoskeletal modulation and reduces TCR triggering by dampening TCR forces consistent with the mechanosensor model of T-cell activation.

An Endosomal Escape Trojan Horse Platform to Improve Cytosolic Delivery of Nucleic Acids.

Endocytosis is a major bottleneck toward cytosolic delivery of nucleic acids, as the vast majority of nucleic acid drugs remain trapped within endosomes. Current trends to overcome endosomal entrapment and subsequent degradation provide varied success; however, active delivery agents such as cell-penetrating peptides have emerged as a prominent strategy to improve cytosolic delivery. Yet, these membrane-active agents have poor selectivity for endosomal membranes, leading to toxicity. A hallmark of endosomes is their acidic environment, which aids in degradation of foreign materials. Here, we develop a pH-triggered spherical nucleic acid that provides smart antisense oligonucleotide (ASO) release upon endosomal acidification and selective membrane disruption, termed DNA EndosomaL Escape Vehicle Response (DELVR). We anchor i-Motif DNA to a nanoparticle (AuNP), where the complement strand contains both an ASO sequence and a functionalized endosomal escape peptide (EEP). By orienting the EEP toward the AuNP core, the EEP is inactive until it is released through acidification-induced i-Motif folding. In this study, we characterize a small library of i-Motif duplexes to develop a structure-switching nucleic acid sequence triggered by endosomal acidification. We evaluate antisense efficacy using HIF1a, a hypoxic indicator upregulated in many cancers, and demonstrate dose-dependent activity through RT-qPCR. We show that DELVR significantly improves ASO efficacy in vitro. Finally, we use fluorescence lifetime imaging and activity measurement to show that DELVR benefits synergistically from nuclease- and pH-driven release strategies with increased ASO endosomal escape efficiency. Overall, this study develops a modular platform that improves the cytosolic delivery of nucleic acid therapeutics and offers key insights for overcoming intracellular barriers.

"Turbo-Charged" DNA Motors with Optimized Sequence Enable Single-Molecule Nucleic Acid Sensing.

DNA motors that consume chemical energy to generate processive mechanical motion mimic natural motor proteins and have garnered interest due to their potential applications in dynamic nanotechnology, biosensing, and drug delivery. Such motors translocate by a catalytic cycle of binding, cleavage, and rebinding between DNA "legs" on the motor body and RNA "footholds" on a track. Herein, we address the well-documented trade-off between motor speed and processivity and investigate how these parameters are controlled by the affinity between DNA legs and their complementary footholds. Specifically, we explore the role of DNA leg length and GC content in tuning motor performance by dictating the rate of leg-foothold dissociation. Our investigations reveal that motors with 0 % GC content exhibit increased instantaneous velocities of up to 150 nm/sec, three-fold greater than previously reported DNA motors and comparable to the speeds of biological motor proteins. We also demonstrate that the faster speed and weaker forces generated by 0 % GC motors can be leveraged for enhanced capabilities in sensing. We observe single-molecule sensitivity when programming the motors to stall in response to the binding of nucleic acid targets. These findings offer insights for the design of high-performance DNA motors with promising real-world biosensing applications.

Simulated microgravity improves maturation of cardiomyocytes derived from human induced pluripotent stem cells.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) possess tremendous potential for basic research and translational application. However, these cells structurally and functionally resemble fetal cardiomyocytes, which is a major limitation of these cells. Microgravity can significantly alter cell behavior and function. Here we investigated the effect of simulated microgravity on hiPSC-CM maturation. Following culture under simulated microgravity in a random positioning machine for 7 days, 3D hiPSC-CMs had increased mitochondrial content as detected by a mitochondrial protein and mitochondrial DNA to nuclear DNA ratio. The cells also had increased mitochondrial membrane potential. Consistently, simulated microgravity increased mitochondrial respiration in 3D hiPSC-CMs, as indicated by higher levels of maximal respiration and ATP content, suggesting improved metabolic maturation in simulated microgravity cultures compared with cultures under normal gravity. Cells from simulated microgravity cultures also had improved Ca2+ transient parameters, a functional characteristic of more mature cardiomyocytes. In addition, these cells had improved structural properties associated with more mature cardiomyocytes, including increased sarcomere length, z-disc length, nuclear diameter, and nuclear eccentricity. These findings indicate that microgravity enhances the maturation of hiPSC-CMs at the structural, metabolic, and functional levels.

DNA mechanocapsules for programmable piconewton responsive drug delivery.

The mechanical dysregulation of cells is associated with a number of disease states, that spans from fibrosis to tumorigenesis. Hence, it is highly desirable to develop strategies to deliver drugs based on the "mechanical phenotype" of a cell. To achieve this goal, we report the development of DNA mechanocapsules (DMC) comprised of DNA tetrahedrons that are force responsive. Modeling shows the trajectory of force-induced DMC rupture and predicts how applied force spatial position and orientation tunes the force-response threshold. DMCs functionalized with adhesion ligands mechanically denature in vitro as a result of cell receptor forces. DMCs are designed to encapsulate macromolecular cargos such as dextran and oligonucleotide drugs with minimal cargo leakage and high nuclease resistance. Force-induced release and uptake of DMC cargo is validated using flow cytometry. Finally, we demonstrate force-induced mRNA knockdown of HIF-1α in a manner that is dependent on the magnitude of cellular traction forces. These results show that DMCs can be effectively used to target biophysical phenotypes which may find useful applications in immunology and cancer biology.

Heteromultivalency enables enhanced detection of nucleic acid mutations.

Detecting genetic mutations such as single nucleotide polymorphisms (SNPs) is necessary to prescribe effective cancer therapies, perform genetic analyses and distinguish similar viral strains. Traditionally, SNP sensing uses short oligonucleotide probes that differentially bind the SNP and wild-type targets. However, DNA hybridization-based techniques require precise tuning of the probe's binding affinity to manage the inherent trade-off between specificity and sensitivity. As conventional hybridization offers limited control over binding affinity, here we generate heteromultivalent DNA-functionalized particles and demonstrate optimized hybridization specificity for targets containing one or two mutations. By investigating the role of oligo lengths, spacer lengths and binding orientation, we reveal that heteromultivalent hybridization enables fine-tuned specificity for a single SNP and dramatic enhancements in specificity for two non-proximal SNPs empowered by highly cooperative binding. Capitalizing on these abilities, we demonstrate straightforward discrimination between heterozygous cis and trans mutations and between different strains of the SARS-CoV-2 virus. Our findings indicate that heteromultivalent hybridization offers substantial improvements over conventional monovalent hybridization-based methods.

Nanodiscoidal Nucleic Acids for Gene Regulation.

Therapeutic nucleic acids represent a powerful class of drug molecules to control gene expression and protein synthesis. A major challenge in this field is that soluble oligonucleotides have limited serum stability, and the majority of nucleic acids that enter the cells are trapped within endosomes. Delivery efficiency can be improved using lipid scaffolds. One such example is the nanodisc (ND), a self-assembled nanostructure composed of phospholipids and peptides and modeled after high density lipoproteins (HDLs). Herein, we describe the development of the nanodiscoidal nucleic acid (NNA) which is a ND covalently modified with nucleic acids on the top and bottom lipid faces as well as the lateral peptide belt. The 13 nm ND was doped with thiolated phospholipids and thiol-containing peptides and coupled in a one-pot reaction with oligonucleotides to achieve ∼30 DNA/NNA nucleic acid density. NNAs showed superior nuclease resistance and enhanced cellular uptake that was mediated through the scavenger receptor B1. Time-dependent Förster resonance energy transfer (FRET) analysis of internalized NNA confirmed that NNAs display increased stability. NNAs modified with clinically validated antisense oligonucleotides (ASOs) that target hypoxia inducible factor 1-α (HIF-1-α) mRNA showed enhanced activity compared with that of the soluble DNA across multiple cell lines as well as a 3D cancer spheroid model. Lastly, in vivo experiments show that ASO-modified NNAs are primarily localized into livers and kidneys, and NNAs were potent in downregulating HIF-1-α using 5-fold lower doses than previously reported. Collectively, our results highlight the therapeutic potential for NNAs.

Detection of cellular traction forces via the force-triggered Cas12a-mediated catalytic cleavage of a fluorogenic reporter strand.

Molecular forces generated by cell receptors are infrequent and transient, and hence difficult to detect. Here we report an assay that leverages the CRISPR-associated protein 12a (Cas12a) to amplify the detection of cellular traction forces generated by as few as 50 adherent cells. The assay involves the immobilization of a DNA duplex modified with a ligand specific for a cell receptor. Traction forces of tens of piconewtons trigger the dehybridization of the duplex, exposing a cryptic Cas12-activating strand that sets off the indiscriminate Cas12-mediated cleavage of a fluorogenic reporter strand. We used the assay to perform hundreds of force measurements using human platelets from a single blood draw to extract individualized dose-response curves and half-maximal inhibitory concentrations for a panel of antiplatelet drugs. For seven patients who had undergone cardiopulmonary bypass, platelet dysfunction strongly correlated with the need for platelet transfusion to limit bleeding. The Cas12a-mediated detection of cellular traction forces may be used to assess cell state, and to screen for genes, cell-adhesion ligands, drugs or metabolites that modulate cell mechanics.

Molecular mechanocytometry using tension-activated cell tagging.

Flow cytometry is used routinely to measure single-cell gene expression by staining cells with fluorescent antibodies and nucleic acids. Here, we present tension-activated cell tagging (TaCT) to label cells fluorescently based on the magnitude of molecular force transmitted through cell adhesion receptors. As a proof-of-concept, we analyzed fibroblasts and mouse platelets after TaCT using conventional flow cytometry.

Force-Triggered Self-Destructive Hydrogels.

Self-destructive polymers (SDPs) are defined as a class of smart polymers that autonomously degrade upon experiencing an external trigger, such as a chemical cue or optical excitation. Because SDPs release the materials trapped inside the network upon degradation, they have potential applications in drug delivery and analytical sensing. However, no known SDPs that respond to external mechanical forces have been reported, as it is fundamentally challenging to create mechano-sensitivity in general and especially so for force levels below those required for classical force-induced bond scission. To address this challenge, the development of force-triggered SDPs composed of DNA crosslinked hydrogels doped with nucleases is described here. Externally applied piconewton forces selectively expose enzymatic cleavage sites within the DNA crosslinks, resulting in rapid polymer self-degradation. The synthesis and the chemical and mechanical characterization of DNA crosslinked hydrogels, as well as the kinetics of force-triggered hydrolysis, are described. As a proof-of-concept, force-triggered and time-dependent rheological changes in the polymer as well as encapsulated nanoparticle release are demonstrated. Finally, that the kinetics of self-destruction are shown to be tuned as a function of nuclease concentration, incubation time, and thermodynamic stability of DNA crosslinkers.

Mechanical force regulates ligand binding and function of PD-1.

Immune checkpoint blockade targeting PD-1 shows great success in cancer therapy. However, the mechanism of how ligand binding initiates PD-1 signaling remains unclear. As prognosis markers of multiple cancers, soluble PD-L1 is found in patient sera and can bind PD-1, but fails to suppress T cell function. This and our previous observations that T cells exert endogenous forces on PD-1-PD-L2 bonds prompt the hypothesis that mechanical force might be critical to PD-1 triggering, which is missing in the soluble ligand case due to the lack of mechanical support afforded by surface-anchored ligand. Here we show that PD-1 function is eliminated or reduced when mechanical support on ligand is removed or dampened, respectively. Force spectroscopic analysis reveals that PD-1 forms catch bonds with both PD-Ligands <7 pN where force prolongs bond lifetime, but slip bonds >8 pN where force accelerates dissociation. Steered molecular dynamics finds PD-1-PD-L2 complex very sensitive to force due to the two molecules' "side-to-side" binding via ß sheets. Pulling causes relative rotation and translation between the two molecules by stretching and aligning the complex along the force direction, yielding new atomic contacts not observed in the crystal structure. Compared to wild-type, PD-1 mutants targeting the force-induced new interactions maintain the same binding affinity but display lower rupture force, shorter bond lifetime, reduced tension, and most importantly, impaired capacity to suppress T cell activation. Our results uncover a mechanism for cells to probe the mechanical support of PD-1-PD-Ligand bonds using endogenous forces to regulate PD-1 triggering.

Force-induced site-specific enzymatic cleavage probes reveal that serial mechanical engagement boosts T cell activation.

The surface of T cells is studded with T cell receptors (TCRs) that are used to scan target cells to identify peptide-major histocompatibility complexes (pMHCs) signatures of viral infection or cancerous mutation. It is now established that the TCR-pMHC complex is highly transient and experiences mechanical forces that augment the fidelity of T cell activation. An important question in this area pertains to the role of force duration in immune activation. Herein, we report the development of force probes that autonomously terminate tension within a time window following mechanical triggering. Force-induced site-specific enzymatic cleavage (FUSE) probes tune tension duration by controlling the rate of a force-triggered endonuclease hydrolysis reaction. This new capability provides a method to study how accumulated force duration contributes to T cell activation. We screened DNA sequences and identified FUSE probes that disrupt mechanical interactions with F >7.1 piconewtons (pN) between TCRs and pMHCs. Force lifetimes (τF) are tunable from tens of min down to 1.9 min. T cells challenged with FUSE probes presenting cognate antigens with τF of 1.9 min demonstrated dampened markers of early activation, thus demonstrating that repeated mechanical sampling boosts TCR activation. Repeated mechanical sampling F >7.1 pN was found to be particularly critical at lower pMHC antigen densities, wherein the T cell activation declined by 23% with τF of 1.9 min. FUSE probes with F >17.0 pN response showed weaker influence on T cell triggering further showing that TCR-pMHC with F >17.0 pN are less frequent compared to F >7.1 pN. Taken together, FUSE probes allow a new strategy to investigate the role of force dynamics in mechanotransduction broadly and specifically suggest a model of serial mechanical engagement in antigen recognition.

Optomechanically Actuated Hydrogel Platform for Cell Stimulation with Spatial and Temporal Resolution.

Cells exist in the body in mechanically dynamic environments, yet the vast majority of in vitro cell culture is conducted on static materials such as plastic dishes and gels. To address this limitation, we report an approach to transition widely used hydrogels into mechanically active substrates by doping optomechanical actuator (OMA) nanoparticles within the polymer matrix. OMAs are composed of gold nanorods surrounded by a thermoresponsive polymer shell that rapidly collapses upon near-infrared (NIR) illumination. As a proof of concept, we crosslinked OMAs into laminin-gelatin hydrogels, generating up to 5 µm deformations triggered by NIR pulsing. This response was tunable by NIR intensity and OMA density within the gel and is generalizable to other hydrogel materials. Hydrogel mechanical stimulation enhanced myogenesis in C2C12 myoblasts as evidenced by ERK signaling, myocyte fusion, and sarcomeric myosin expression. We also demonstrate rescued differentiation in a chronic inflammation model as a result of mechanical stimulation. This work establishes OMA-actuated biomaterials as a powerful tool for in vitro mechanical manipulation with broad applications in the field of mechanobiology.

DNA Origami Tension Sensors (DOTS) to study T cell receptor mechanics at membrane junctions.

The T cell receptor (TCR) is thought to be a mechanosensor, meaning that it transmits mechanical force to its antigen and leverages the force to amplify the specificity and magnitude of TCR signaling. The past decade has witnessed the development of molecular probes which have revealed many aspects of receptor mechanotransduction. However, most force probes are immobilized on hard substrates, thus failing to reveal mechanics in the physiological context of cell membranes. In this report, we developed DNA origami tension sensors (DOTS) which bear force sensors on a DNA origami breadboard and allow mapping of TCR mechanotransduction at dynamic intermembrane junctions. We demonstrate that TCR-antigen bonds experience 5-10 pN forces, and the mechanical events are dependent on cell state, antigen mobility, antigen potency, antigen height and F-actin activity. We tethered DOTS onto a microparticle to mechanically screen antigen in high throughput using flow cytometry. Finally, DOTS were anchored onto live B cell membranes thus producing the first quantification of TCR mechanics at authentic immune cell-cell junctions.

All-Covalent Nuclease-Resistant and Hydrogel-Tethered DNA Hairpin Probes Map pN Cell Traction Forces.

Cells sense and respond to the physical properties of their environment through receptor-mediated signaling, a process known as mechanotransduction, which can modulate critical cellular functions such as proliferation, differentiation, and survival. At the molecular level, cell adhesion receptors, such as integrins, transmit piconewton (pN)-scale forces to the extracellular matrix, and the magnitude of the force plays a critical role in cell signaling. The most sensitive approach to measuring integrin forces involves DNA hairpin-based sensors, which are used to quantify and map forces in living cells. Despite the broad use of DNA hairpin sensors to study a variety of mechanotransduction processes, these sensors are typically anchored to rigid glass slides, which are orders of magnitude stiffer than the extracellular matrix and hence modulate native biological responses. Here, we have developed nuclease-resistant DNA hairpin probes that are all covalently tethered to PEG hydrogels to image cell traction forces on physiologically relevant substrate stiffness. Using HeLa cells as a model cell line, we show that the molecular forces transmitted by integrins are highly sensitive to the bulk modulus of the substrate, and cells cultured on the 6 and 13 kPa gels produced a greater number of hairpin unfolding events compared to the 2 kPa substrates. Tension signals are spatially colocalized with pY118-paxillin, confirming focal adhesion-mediated probe opening. Additionally, we found that integrin forces are greater than 5.8 pN but less than 19 pN on 13 kPa gels. This work provides a general strategy to integrate molecular tension probes into hydrogels, which can better mimic in vivo mechanotransduction.