The magnitude of LFA-1/ICAM-1 forces fine-tune TCR-triggered T cell activation.

T cells defend against cancer and viral infections by rapidly scanning the surface of target cells seeking specific peptide antigens. This key process in adaptive immunity is sparked upon T cell receptor (TCR) binding of antigens within cell-cell junctions stabilized by integrin (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) complexes. A long-standing question in this area is whether the forces transmitted through the LFA-1/ICAM-1 complex tune T cell signaling. Here, we use spectrally encoded DNA tension probes to reveal the first maps of LFA-1 and TCR forces generated by the T cell cytoskeleton upon antigen recognition. DNA probes that control the magnitude of LFA-1 force show that F>12 pN potentiates antigen-dependent T cell activation by enhancing T cell-substrate engagement. LFA-1/ICAM-1 mechanical events with F>12 pN also enhance the discriminatory power of the TCR when presented with near cognate antigens. Overall, our results show that T cells integrate multiple channels of mechanical information through different ligand-receptor pairs to tune function.

ST6Gal-I-mediated sialylation of the epidermal growth factor receptor modulates cell mechanics and enhances invasion.

Heterogeneity within the glycocalyx influences cell adhesion mechanics and signaling. However, the role of specific glycosylation subtypes in influencing cell mechanics via alterations of receptor function remains unexplored. It has been shown that the addition of sialic acid to terminal glycans impacts growth, development, and cancer progression. In addition, the sialyltransferase ST6Gal-I promotes epidermal growth factor receptor (EGFR) activity, and we have shown EGFR is an 'allosteric mechano-organizer' of integrin tension. Here, we investigated the impact of ST6Gal-I on cell mechanics. Using DNA-based tension gauge tether probes of variable thresholds, we found that high ST6Gal-I activity promotes increased integrin forces and spreading in Cos-7 and OVCAR3, OVCAR5, and OV4 cancer cells. Further, employing inhibitors and function-blocking antibodies against ß1, ß3, and ß5 integrins and ST6Gal-I targets EGFR, tumor necrosis factor receptor, and Fas cell surface death receptor, we validated that the observed phenotypes are EGFR-specific. We found that while tension, contractility, and adhesion are extracellular-signal-regulated kinase pathway-dependent, spreading, proliferation, and invasion are phosphoinositide 3-kinase-Akt serine/threonine kinase dependent. Using total internal reflection fluorescence microscopy and flow cytometry, we also show that high ST6Gal-I activity leads to sustained EGFR membrane retention, making it a key regulator of cell mechanics. Our findings suggest a novel sialylation-dependent mechanism orchestrating cellular mechanics and enhancing cell motility via EGFR signaling.

Gene Regulation Using Nanodiscs Modified with HIF-1-α Antisense Oligonucleotides.

Delivery of nucleic acids can be hindered by multiple factors including nuclease susceptibility, endosome trapping, and clearance. Multiple nanotechnology scaffolds have offered promising solutions, and among these, lipid-based systems are advantageous because of their high biocompatibility and low toxicity. However, many lipid nanoparticle systems still have issues regarding stability, rapid clearance, and cargo leakage. Herein, we demonstrate the use of a synthetic nanodisc (ND) scaffold functionalized with an anti-HIF-1-α antisense oligonucleotide (ASO) to reduce HIF-1-α mRNA transcript levels. We prepared ND conjugates by using a mixture of phosphoglycerolipids with phosphocholine and phosphothioethanol headgroups that self-assemble into a ∼13 × 5 nm discoidal structure upon addition of a 22-amino-acid ApoA1 mimetic peptide. Optimized reaction conditions yield 15 copies of the anti-HIF-1-α ASO DNA covalently conjugated to the thiolated phospholipids using maleimide-thiol chemistry. We show that DNA-ND conjugates are active, nuclease resistant, and rapidly internalized into cells to regulate HIF-1-α mRNA levels without the use of transfection agents. DNA-ND uptake is partially mediated through Scavenger Receptor B1 and the ND conjugates show enhanced knockdown of HIF-1-α compared to that of the soluble ASOs in multiple cell lines. Our results demonstrate that covalently functionalized NDs may offer an improved platform for ASO therapeutics.

DNA-Based Microparticle Tension Sensors (µTS) for Measuring Cell Mechanics in Non-planar Geometries and for High-Throughput Quantification.

Mechanotransduction, the interplay between physical and chemical signaling, plays vital roles in many biological processes. The state-of-the-art techniques to quantify cell forces employ deformable polymer films or molecular probes tethered to glass substrates. However, the applications of these assays in fundamental and clinical research are restricted by the planar geometry and low throughput of microscopy readout. Herein, we develop a DNA-based microparticle tension sensor, which features a spherical surface and thus allows for investigation of mechanotransduction at curved interfaces. The micron-scale of µTS enables flow cytometry readout, which is rapid and high throughput. We applied the method to map and measure T-cell receptor forces and platelet integrin forces at 12 and 56 pN thresholds. Furthermore, we quantified the inhibition efficiency of two anti-platelet drugs providing a proof-of-concept demonstration of µTS to screen drugs that modulate cellular mechanics.

EGFR activation attenuates the mechanical threshold for integrin tension and focal adhesion formation.

Mechanical forces, growth factors and the extracellular matrix all play crucial roles in cell adhesion. To understand how epidermal growth factor receptor (EGFR) impacts the mechanics of adhesion, we employed tension gauge tether (TGT) probes displaying the integrin ligand cRGDfK and quantified integrin tension. EGF exposure significantly increased spread area, cell circularity, integrated integrin tension, mechanical rupture density, radial organization and size of focal adhesions in Cos-7 cells on TGT surfaces. These findings suggest that EGFR regulates integrin tension and the spatial organization of focal adhesions. Additionally, we found that the mechanical tension threshold for outside-in integrin activation is tunable by EGFR. Parallel genetic and pharmacologic strategies demonstrated that these phenotypes are driven by ligand-dependent EGFR signaling. Our results establish a novel mechanism whereby EGFR regulates integrin activation and cell adhesion, providing control over cellular responses to the environment.This article has an associated First Person interview with the first author of the paper.

Engineering DNA-Functionalized Nanostructures to Bind Nucleic Acid Targets Heteromultivalently with Enhanced Avidity.

Improving the affinity of nucleic acids to their complements is an important goal for many fields spanning from genomics to antisense therapy and diagnostics. One potential approach to achieving this goal is to use multivalent binding, which often boosts the affinity between ligands and receptors, as exemplified by virus-cell binding and antibody-antigen interactions. Herein, we investigate the binding of heteromultivalent DNA-nanoparticle conjugates, where multiple unique oligonucleotides displayed on a nanoparticle form a multivalent complex with a long DNA target containing the complementary sequences. By developing a strategy to spatially pattern oligonucleotides on a nanoparticle, we demonstrate that the molecular organization of heteromultivalent nanostructures is critical for effective binding; patterned particles have a ∼23 order-of-magnitude improvement in affinity compared to chemically identical particles patterned incorrectly. We envision that nanostructures presenting spatially patterned heteromultivalent DNA will offer important biomedical applications given the utility of DNA-functionalized nanostructures in diagnostics and therapeutics.

DNA probes that store mechanical information reveal transient piconewton forces applied by T cells.

The advent of molecular tension probes for real-time mapping of piconewton forces in living systems has had a major impact on mechanobiology. For example, DNA-based tension probes have revealed roles for mechanics in platelet, B cell, T cell, and fibroblast function. Nonetheless, imaging short-lived forces transmitted by low-abundance receptors remains a challenge. This is a particular problem for mechanoimmunology where ligand-receptor bindings are short lived, and a few antigens are sufficient for cell triggering. Herein, we present a mechanoselection strategy that uses locking oligonucleotides to preferentially and irreversibly bind DNA probes that are mechanically strained over probes at rest. Thus, infrequent and short-lived mechanical events are tagged. This strategy allows for integration and storage of mechanical information into a map of molecular tension history. Upon addition of unlocking oligonucleotides that drive toehold-mediated strand displacement, the probes reset to the real-time state, thereby erasing stored mechanical information. As a proof of concept, we applied this strategy to study OT-1 T cells, revealing that the T cell receptor (TCR) mechanically samples antigens carrying single amino acid mutations. Such events are not detectable using conventional tension probes. Each mutant peptide ligand displayed a different level of mechanical sampling and spatial scanning by the TCR that strongly correlated with its functional potency. Finally, we show evidence that T cells transmit pN forces through the programmed cell death receptor-1 (PD1), a major target in cancer immunotherapy. We anticipate that mechanical information storage will be broadly useful in studying the mechanobiology of the immune system.

DNA Nanotechnology as an Emerging Tool to Study Mechanotransduction in Living Systems.

The ease of tailoring DNA nanostructures with sub-nanometer precision has enabled new and exciting in vivo applications in the areas of chemical sensing, imaging, and gene regulation. A new emerging paradigm in the field is that DNA nanostructures can be engineered to study molecular mechanics. This new development has transformed the repertoire of capabilities enabled by DNA to include detection of molecular forces in living cells and elucidating the fundamental mechanisms of mechanotransduction. This Review first describes fundamental aspects of force-induced melting of DNA hairpins and duplexes. This is then followed by a survey of the currently available force sensing DNA probes and different fluorescence-based force readout modes. Throughout the Review, applications of these probes in studying immune receptor signaling, including the T cell receptor and B cell receptor, as well as Notch and integrin signaling, are discussed.

A brighter force gauge for cells.

An improved biosensor sheds new light on tension within proteins.

Molecular Tension Probes to Investigate the Mechanopharmacology of Single Cells: A Step toward Personalized Mechanomedicine.

Given that dysregulation of mechanics contributes to diseases ranging from cancer metastasis to lung disease, it is important to develop methods for screening the efficacy of drugs that target cellular forces. Here, nanoparticle-based tension sensors are used to quantify the mechanical response of individual cells upon drug treatment. As a proof-of-concept, the activity of bronchodilators is tested on human airway smooth muscle cells derived from seven donors, four of which are asthmatic. It is revealed that airway smooth muscle cells isolated from asthmatic donors exhibit greater traction forces compared to the control donors. Additionally, the mechanical signal is abolished using myosin inhibitors or further enhanced in the presence of inflammatory inducers, such as nicotine. Using the signal generated by the probes, single-cell dose-response measurements are performed to determine the "mechano" effective concentration (mechano-EC50 ) of albuterol, a bronchodilator, which reduces integrin forces by 50%. Mechano-EC50 values for each donor present discrete readings that are differentially enhanced as a function of nicotine treatment. Importantly, donor mechano-EC50 values varied by orders of magnitude, suggesting significant variability in their sensitivity to nicotine and albuterol treatment. To the best of the authors' knowledge, this is the first study harnessing a piconewton tension sensor platform for mechanopharmacology.

Light-Responsive Polymer Particles as Force Clamps for the Mechanical Unfolding of Target Molecules.

Single-molecule force spectroscopy techniques are powerful tools for investigating the mechanical unfolding of biomolecules. However, they are limited in throughput and require dedicated instrumentation. Here, we report a force-generating particle that can unfold target molecules on-demand. The particle consists of a plasmonic nanorod core encapsulated with a thermoresponsive polymer shell. Optical heating of the nanorod leads to rapid collapse of the polymer, thus transducing light into mechanical work to unfold target molecules. The illumination tunes the duration and degree of particle collapse, thus controlling the lifetime and magnitude of applied forces. Single-molecule fluorescence imaging showed reproducible mechanical unfolding of DNA hairpins. We also demonstrate the triggering of 50 different particles in <1 min, exceeding the speed of conventional atomic force microscopy. The polymer force clamp represents a facile and bottom-up approach to force manipulation.

Platelet integrins exhibit anisotropic mechanosensing and harness piconewton forces to mediate platelet aggregation.

Platelet aggregation at the site of vascular injury is essential in clotting. During this process, platelets are bridged by soluble fibrinogen that binds surface integrin receptors. One mystery in the mechanism of platelet aggregation pertains to how resting platelets ignore soluble fibrinogen, the third most abundant protein in the bloodstream, and yet avidly bind immobile fibrinogen on the surface of other platelets at the primary injury site. We speculate that platelet integrins are mechanosensors that test their ligands across the platelet-platelet synapse. To investigate this model, we interrogate human platelets using approaches that include the supported lipid bilayer platform as well as DNA tension sensor technologies. Experiments suggest that platelet integrins require lateral forces to mediate platelet-platelet interactions. Mechanically labile ligands dampen platelet activation, and the onset of piconewton integrin tension coincides with calcium flux. Activated platelets display immobilized fibrinogen on their surface, thus mediating further recruitment of resting platelets. The distribution of integrin tension was shown to be spatially regulated through two myosin-signaling pathways, myosin light chain kinase and Rho-associated kinase. Finally, we discovered that the termination of integrin tension is coupled with the exposure of phosphatidylserine. Our work reveals the highest spatial and temporal resolution maps of platelet integrin mechanics and its role in platelet aggregation, suggesting that platelets are physical substrates for one another that establish mechanical feedback loops of activation. The results are reminiscent of mechanical regulation of the T-cell receptor, E-cadherin, and Notch pathways, suggesting a common feature for signaling at cell junctions.

Molecular Tension Probes for Imaging Forces at the Cell Surface.

Mechanical forces are essential for a variety of biological processes ranging from transcription and translation to cell adhesion, migration, and differentiation. Through the activation of mechanosensitive signaling pathways, cells sense and respond to physical stimuli from the surrounding environment, a process widely known as mechanotransduction. At the cell membrane, many signaling receptors, such as integrins, cadherins and T- or B-cell receptors, bind to their ligands on the surface of adjacent cells or the extracellular matrix (ECM) to mediate mechanotransduction. Upon ligation, these receptor-ligand bonds transmit piconewton (pN) mechanical forces that are generated, in part, by the cytoskeleton. Importantly, these forces expose cryptic sites within mechanosensitive proteins and modulate the binding kinetics (on/off rate) of receptor-ligand complexes to further fine-tune mechanotransduction and the corresponding cell behavior. Over the past three decades, two categories of methods have been developed to measure cell receptor forces. The first class is traction force microscopy (TFM) and micropost array detectors (mPADs). In these methods, cells are cultured on elastic polymers or microstructures that deform under mechanical forces. The second category of techniques is single molecule force spectroscopy (SMFS) including atomic force microscopy (AFM), optical or magnetic tweezers, and biomembrane force probe (BFP). In SMFS, the experimenter applies external forces to probe the mechanics of individual cells or single receptor-ligand complexes, serially, one bond at a time. Although these techniques are powerful, the limited throughput of SMFS and the nN force sensitivity of TFM have hindered further elucidation of the molecular mechanisms of mechanotransduction. In this Account, we introduce the recent advent of molecular tension fluorescence microscopy (MTFM) as an emerging tool for molecular imaging of receptor mechanics in living cells. MTFM probes are composed of an extendable linker, such as polymer, oligonucleotide, or protein, and flanked by a fluorophore and quencher. By measuring the fluorescence emission of immobilized MTFM probes, one can infer the extension of the linker and the externally applied force. Thus, MTFM combines aspects of TFM and SMFS to optically report receptor forces across the entire cell surface with pN sensitivity. Specifically, we provide an in-depth review of MTFM probe design, which includes the extendable "spring", spectroscopic ruler, surface immobilization chemistry, and ligand design strategies. We also demonstrate the strengths and weaknesses of different versions of MTFM probes by discussing case studies involving the pN forces involved in epidermal growth factor receptor, integrin, and T-cell receptor signaling pathways. Lastly, we present a brief future outlook, primarily from a chemists' perspective, on the challenges and opportunities for the design of next generation MTFM probes.

DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity.

T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12-19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically "filtered" to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.

Ratiometric Tension Probes for Mapping Receptor Forces and Clustering at Intermembrane Junctions.

Short-range communication between cells is required for the survival of multicellular organisms. One mechanism of chemical signaling between adjacent cells employs surface displayed ligands and receptors that only bind when two cells make physical contact. Ligand-receptor complexes that form at the cell-cell junction and physically bridge two cells likely experience mechanical forces. A fundamental challenge in this area pertains to mapping the mechanical forces experienced by ligand-receptor complexes within such a fluid intermembrane junction. Herein, we describe the development of ratiometric tension probes for direct imaging of receptor tension, clustering, and lateral transport within a model cell-cell junction. These probes employ two fluorescent reporters that quantify both the ligand density and the ligand tension and thus generate a tension signal independent of clustering. As a proof-of-concept, we applied the ratiometric tension probes to map the forces experienced by the T-cell receptor (TCR) during activation and showed the first direct evidence that the TCR-ligand complex experiences sustained pN forces within a fluid membrane junction. We envision that the ratiometric tension probes will be broadly useful for investigating mechanotransduction in juxtacrine signaling pathways.

Mechanically Induced Catalytic Amplification Reaction for Readout of Receptor-Mediated Cellular Forces.

Mechanics play a fundamental role in cell biology, but detecting piconewton (pN) forces is challenging because of a lack of accessible and high throughput assays. A mechanically induced catalytic amplification reaction (MCR) for readout of receptor-mediated forces in cells is described. Mechanically labile DNA duplexes presenting ligands are surface immobilized such that specific receptor forces denature the duplex and thus expose a blocked primer. Amplification of primers is achieved using an isothermal polymerization reaction and quantified by fluorescence readout. As a proof of concept, the assay was used to test the activity of a mechanomodulatory drug and integrin adhesion receptor antibodies. To the best of our knowledge, this is the first example of a catalytic reaction triggered in response to molecular piconewton forces. The MCR may transform the field of mechanobiology by providing a new facile tool to detect receptor specific mechanics with the convenience of the polymerase chain reaction (PCR).

Protection against ß-amyloid-induced synaptic and memory impairments via altering ß-amyloid assembly by bis(heptyl)-cognitin.

ß-amyloid (Aß) oligomers have been closely implicated in the pathogenesis of Alzheimer's disease (AD). We found, for the first time, that bis(heptyl)-cognitin, a novel dimeric acetylcholinesterase (AChE) inhibitor derived from tacrine, prevented Aß oligomers-induced inhibition of long-term potentiation (LTP) at concentrations that did not interfere with normal LTP. Bis(heptyl)-cognitin also prevented Aß oligomers-induced synaptotoxicity in primary hippocampal neurons. In contrast, tacrine and donepezil, typical AChE inhibitors, could not prevent synaptic impairments in these models, indicating that the modification of Aß oligomers toxicity by bis(heptyl)-cognitin might be attributed to a mechanism other than AChE inhibition. Studies by using dot blotting, immunoblotting, circular dichroism spectroscopy, and transmission electron microscopy have shown that bis(heptyl)-cognitin altered Aß assembly via directly inhibiting Aß oligomers formation and reducing the amount of preformed Aß oligomers. Molecular docking analysis further suggested that bis(heptyl)-cognitin presumably interacted with the hydrophobic pockets of Aß, which confers stabilizing powers and assembly alteration effects on Aß. Most importantly, bis(heptyl)-cognitin significantly reduced cognitive impairments induced by intra-hippocampal infusion of Aß oligomers in mice. These results clearly demonstrated how dimeric agents prevent Aß oligomers-induced synaptic and memory impairments, and offered a strong support for the beneficial therapeutic effects of bis(heptyl)-cognitin in the treatment of AD.

Detection of base excision repair enzyme activity using a luminescent G-quadruplex selective switch-on probe.

We report herein a simple and convenient luminescent assay for detection of base excision repair enzyme activity using an Ir(III) complex as a G-quadruplex selective probe. Using uracil-DNA glycosylase (UDG) as a model enzyme, the assay achieved high sensitivity and selectivity for UDG over other tested enzymes. The utility of the assay for screening potential UDG inhibitors was also demonstrated.

Structure-based design of flavone derivatives as c-myc oncogene down-regulators.

Based on molecular docking analysis of complexes between flavone and the c-myc G-quadruplex, we designed and screened 30 flavone derivatives containing various side chains that could potentially form interactions with the G-quadruplex grooves. As a proof-of-concept, the highest-scoring flavone derivatives containing cationic pyridinium side chains were synthesized and their interactions with the c-myc G-quadruplex were examined using a PCR-stop assay. The stabilizing effects of the flavone derivatives were found to be selective towards the c-myc G-quadruplex over other biologically relevant G-quadruplex structures, such as the human telomeric sequence (HTS). The interaction between the most potent compound of the series and the c-myc G-quadruplex was examined in depth using UV-Vis titration, molecular modeling and CD spectroscopy. Our results suggest that in addition to stabilizing the c-myc G-quadruplex, the flavone derivatives were capable of inducing the formation of the G-quadruplex structure even in the absence of monovalent cations. The flavone derivatives were found to be potent inhibitors of c-myc promoters within the cellular environment and displayed promising cytotoxic behavior against human cancer cell lines.

A luminescent G-quadruplex switch-on probe for the highly selective and tunable detection of cysteine and glutathione.

A G-quadruplex-selective luminescent iridium(III) switch-on probe has been developed for the detection of cysteine (Cys) in aqueous solution. The system is highly sensitive and selective towards Cys with a tunable range of detection. The detection of glutathione (GSH) is also examined.