Detection of Fluorescent Protein Mechanical Switching in Cellulo.

The ability of cells to sense and respond to mechanical forces is critical in many physiological and pathological processes. However, the mechanisms by which forces affect protein function inside cells remain unclear. Motivated by in vitro demonstrations of fluorescent proteins (FPs) undergoing reversible mechanical switching of fluorescence, we investigated if force-sensitive changes in FP function could be visualized in cells. Guided by a computational model of FP mechanical switching, we develop a formalism for its detection in Förster resonance energy transfer (FRET)-based biosensors and demonstrate its occurrence in cellulo in a synthetic actin-crosslinker and the mechanical linker protein vinculin. We find that in cellulo mechanical switching is reversible and altered by manipulation of cellular force generation as well as force-sensitive bond dynamics of the biosensor. Together, this work describes a new framework for assessing FP mechanical stability and provides a means of probing force-sensitive protein function inside cells. MOTIVATION: The ability of cells to sense mechanical forces is critical in developmental, physiological, and pathological processes. Cells sense mechanical cues via force-induced alterations in protein structure and function, but elucidation of the molecular mechanisms is hindered by the lack of approaches to directly probe the effect of forces on protein structure and function inside cells. Motivated by in vitro observations of reversible fluorescent protein mechanical switching, we developed an approach for detecting fluorescent protein mechanical switching in cellulo . This enables the visualization of force-sensitive protein function inside living cells.

Can a bulky glycocalyx promote catch bonding in early integrin adhesion? Perhaps a bit.

Many types of cancer cells overexpress bulky glycoproteins to form a thick glycocalyx layer. The glycocalyx physically separates the cell from its surroundings, but recent work has shown that the glycocalyx can paradoxically increase adhesion to soft tissues and therefore promote the metastasis of cancer cells. This surprising phenomenon occurs because the glycocalyx forces adhesion molecules (called integrins) on the cell's surface into clusters. These integrin clusters have cooperative effects that allow them to form stronger adhesions to surrounding tissues than would be possible with equivalent numbers of un-clustered integrins. These cooperative mechanisms have been intensely scrutinized in recent years. A more nuanced understanding of the biophysical underpinnings of glycocalyx-mediated adhesion could uncover therapeutic targets, deepen our general understanding of cancer metastasis, and elucidate general biophysical processes that extend far beyond the realm of cancer research. This work examines the hypothesis that the glycocalyx has the additional effect of increasing mechanical tension experienced by clustered integrins. Integrins function as mechanosensors that undergo catch bonding-meaning the application of moderate tension increases integrin bond lifetime relative to the lifetime of integrins experiencing low tension. In this work, a three-state chemomechanical catch bond model of integrin tension is used to investigate catch bonding in the presence of a bulky glycocalyx. A pseudo-steady-state approximation is applied, which relies on the assumption that integrin bond dynamics occur on a much faster timescale than the evolution of the full adhesion between the plasma membrane and the substrate. Force-dependent kinetic rate constants are used to calculate a steady-state distribution of integrin-ligand bonds for Gaussian-shaped adhesion geometries. The relationship between the energy of the system and adhesion geometry is then analyzed in the presence and absence of catch bonding in order to evaluate the extent to which catch bonding alters the energetics of adhesion formation. This modeling suggests that a bulky glycocalyx can lightly trigger catch bonding, increasing the bond lifetime of integrins at adhesion edges by up to 100%. The total number of integrin-ligand bonds within an adhesion is predicted to increase by up to ~ 60% for certain adhesion geometries. Catch bonding is predicted to decrease the activation energy of adhesion formation by ~ 1-4 kBT, which translates to a ~ 3-50 × increase in the kinetic rate of adhesion nucleation. This work reveals that integrin mechanics and clustering likely both contribute to glycocalyx-mediated metastasis.

Can a bulky glycocalyx promote catch bonding in early integrin adhesion? Perhaps a bit.

Many types of cancer overexpress bulky glycoproteins to form a thick glycocalyx layer. The glycocalyx physically separates the cell from its surroundings, but recent work has shown that the glycocalyx can paradoxically increase adhesion to soft tissues and therefore promote the metastasis of cancer cells. This surprising phenomenon occurs because the glycocalyx forces adhesion molecules (called integrins) on the cell's surface into clusters. These integrin clusters have cooperative effects that allow them to form stronger adhesions to surrounding tissues than would be possible with equivalent numbers of un-clustered integrins. These cooperative mechanisms have been intensely scrutinized in recent years; a more nuanced understanding of the biophysical underpinnings of glycocalyx-mediated adhesion could uncover therapeutic targets, deepen our general understanding of cancer metastasis, and elucidate general biophysical processes that extend far beyond the realm of cancer research. This work examines the hypothesis that the glycocalyx has the additional effect of increasing mechanical tension experienced by clustered integrins. Integrins function as mechanosensors that undergo catch bonding - meaning the application of moderate tension increases integrin bond lifetime relative to the lifetime of integrins experiencing low tension. In this work, a three-state chemomechanical catch bond model of integrin tension is used to investigate catch bonding in the presence of a bulky glycocalyx. This modeling suggests that a bulky glycocalyx can lightly trigger catch bonding, increasing the bond lifetime of integrins at adhesion edges by up to 100%. The total number of integrin-ligand bonds within an adhesion is predicted to increase by up to ~60% for certain adhesion geometries. Catch bonding is predicted to decrease the activation energy of adhesion formation by ~1-4 k B T, which translates to a ~3-50× increase in the kinetic rate of adhesion nucleation. This work reveals that integrin mechanic and clustering likely both contribute to glycocalyx-mediated metastasis.

Adhesive Dynamics Simulations of Highly Polyvalent DNA Motors.

Molecular motors, such as myosin and kinesin, perform diverse tasks ranging from vesical transport to bulk muscle contraction. Synthetic molecular motors may eventually be harnessed to perform similar tasks in versatile synthetic systems. The most promising type of synthetic molecular motor, the DNA walker, can undergo processive motion but generally exhibits low speeds and virtually no capacity for force generation. However, we recently showed that highly polyvalent DNA motors (HPDMs) can rival biological motors by translocating at micrometer per minute speeds and generating 100+ pN of force. Accordingly, DNA nanotechnology-based designs may hold promise for the creation of synthetic, force-generating nanomotors. However, the dependencies of HPDM speed and force on tunable design parameters are poorly understood and difficult to characterize experimentally. To overcome this challenge, we present RoloSim, an adhesive dynamics software package for fine-grained simulations of HPDM translocation. RoloSim uses biophysical models for DNA duplex formation and dissociation kinetics to explicitly model tens of thousands of molecular scale interactions. These molecular interactions are then used to calculate the nano- and microscale motions of the motor. We use RoloSim to uncover how motor force and speed scale with several tunable motor properties such as motor size and DNA duplex length. Our results support our previously defined hypothesis that force scales linearly with polyvalency. We also demonstrate that HPDMs can be steered with external force, and we provide design parameters for novel HPDM-based molecular sensor and nanomachine designs.

Ultra-photostable DNA FluoroCubes: Mechanism of Photostability and Compatibility with FRET and Dark Quenching.

DNA-based FluoroCubes were recently developed as a solution to photobleaching, a ubiquitous limitation of fluorescence microscopy (Niekamp; ; Stuurman; ; Vale Nature Methods, 2020). FluoroCubes, that is, compact ∼4 × 4 × 5.4 nm3 four-helix bundles coupled to ≤6 fluorescent dyes, remain fluorescent up to ∼50× longer than single dyes and emit up to ∼40× as many photons. The current work answers two important questions about the FluoroCubes. First, what is the mechanism by which photostability is enhanced? Second, are FluoroCubes compatible with Förster resonance energy transfer (FRET) and similar techniques? We use single particle photobleaching studies to show that photostability arises through interactions between the fluorophores and the four-helix DNA bundle. Supporting this, we discover that smaller ∼4 × 4 × 2.7 nm3 FluoroCubes also confer ultraphotostability. However, we find that certain dye-dye interactions negatively impact FluoroCube performance. Accordingly, 4-dye FluoroCubes lacking these interactions perform better than 6-dye FluoroCubes. We also demonstrate that FluoroCubes are compatible with FRET and dark quenching applications.

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.

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.

Massively Parallelized Molecular Force Manipulation with On-Demand Thermal and Optical Control.

In single-molecule force spectroscopy (SMFS), a tethered molecule is stretched using a specialized instrument to study how macromolecules extend under force. One problem in SMFS is the serial and slow nature of the measurements, performed one molecule at a time. To address this long-standing challenge, we report on the origami polymer force clamp (OPFC) which enables parallelized manipulation of the mechanical forces experienced by molecules without the need for dedicated SMFS instruments or surface tethering. The OPFC positions target molecules between a rigid nanoscale DNA origami beam and a responsive polymer particle that shrinks on demand. As a proof-of-concept, we record the steady state and time-resolved mechanical unfolding dynamics of DNA hairpins using the fluorescence signal from ensembles of molecules and confirm our conclusion using modeling.

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.

Rapid kinetic fingerprinting of single nucleic acid molecules by a FRET-based dynamic nanosensor.

Biofluid-derived cell-free nucleic acids such as microRNAs (miRNAs) and circulating tumor-derived DNAs (ctDNAs) have emerged as promising disease biomarkers. Conventional detection of these biomarkers by digital PCR and next generation sequencing, although highly sensitive, requires time-consuming extraction and amplification steps that also increase the risk of sample loss and cross-contamination. To achieve the direct, rapid, and amplification-free detection of miRNAs and ctDNAs with near-perfect specificity and single-molecule level sensitivity, we herein designed a single-molecule kinetic fingerprinting assay, termed intramolecular single-molecule recognition through equilibrium Poisson sampling (iSiMREPS). iSiMREPS exploits a dynamic DNA nanosensor comprising a surface anchor and a pair of fluorescent detection probes: one probe captures a target molecule onto the surface, while the other transiently interrogates the target to generate kinetic fingerprints by intramolecular single-molecule Förster resonance energy transfer (smFRET) that are recorded by single-molecule fluorescence microscopy and identify the target after kinetic filtering and data analysis. We optimize the sensor design, use formamide to further accelerate the fingerprinting kinetics, and maximize sensitivity by removing non-target-bound probes using toehold-mediated strand displacement to reduce background. We show that iSiMREPS can detect, in as little as 10 s, two distinct, promising cancer biomarkers-miR-141 and a common EGFR exon 19 deletion-reaching a limit of detection (LOD) of ~3 fM and a mutant allele fraction among excess wild-type as low as 1 in 1 million, or 0.0001%. We anticipate that iSiMREPS will find utility in research and clinical diagnostics based on its features of rapid detection, high specificity, sensitivity, and generalizability.

Burnt bridge ratchet motor force scales linearly with polyvalency: a computational study.

Nano- and micro-scale burnt bridge ratchet motors, which translocate via "guide" molecules that bind to and degrade a field of "fuel" molecules, have recently emerged in several biological and engineering contexts. The capacity of these motors to generate mechanical forces remains an open question. Here, chemomechanical modeling suggests that BBR force scales linearly with the steady-state number of guide-fuel bonds.

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells.

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions.

Multivalent molecular tension probes as anisotropic mechanosensors: concept and simulation.

Cells use protein-based mechanosensors to measure the physical properties of their surroundings. Synthetic tension sensors made of proteins, DNA, and other molecular building blocks have recently emerged as tools to visualize and perturb the mechanics of these mechanosensors. While almost all synthetic tension sensors are designed to exhibit orientation-independent force responses, recent work has shown that biological mechanosensors often function in a manner that is highly dependent on force orientation. Accordingly, the design of synthetic mechanosensors with orientation-dependent force responses can provide a means to study the role of orientation in mechanosensation. Furthermore, the process of designing anisotropic force responses may yield insight into the physical basis for orientation-dependence in biological mechanosensors. Here, we propose a DNA-based molecular tension sensor design wherein multivalency is used to create an orientation-dependent force response. We apply chemomechanical modeling to show that multivalency can be used to create synthetic mechanosensors with force response thresholds that vary by tens of pN with respect to force orientation.

Live-cell super-resolved PAINT imaging of piconewton cellular traction forces.

Despite the vital role of mechanical forces in biology, it still remains a challenge to image cellular force with sub-100-nm resolution. Here, we present tension points accumulation for imaging in nanoscale topography (tPAINT), integrating molecular tension probes with the DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique to map piconewton mechanical events with ~25-nm resolution. To perform live-cell dynamic tension imaging, we engineered reversible probes with a cryptic docking site revealed only when the probe experiences forces exceeding a defined mechanical threshold (~7-21 pN). Additionally, we report a second type of irreversible tPAINT probe that exposes its cryptic docking site permanently and thus integrates force history over time, offering improved spatial resolution in exchange for temporal dynamics. We applied both types of tPAINT probes to map integrin receptor forces in live human platelets and mouse embryonic fibroblasts. Importantly, tPAINT revealed a link between platelet forces at the leading edge of cells and the dynamic actin-rich ring nucleated by the Arp2/3 complex.

Conditional Deoxyribozyme-Nanoparticle Conjugates for miRNA-Triggered Gene Regulation.

DNA-nanoparticle (NP) conjugates have been used to knockdown gene expression transiently and effectively, making them desirable tools for gene regulation therapy. Because DNA-NPs are constitutively active and are rapidly taken up by most cell types, they offer limited control in terms of tissue or cell type specificity. To take a step toward solving this issue, we incorporate toehold-mediated strand exchange, a versatile molecular programming modality, to switch the DNA-NPs from an inactive state to an active state in the presence of a specific RNA input. Because many transcripts are unique to cell subtype or disease state, this approach could one day lead to responsive nucleic acid therapeutics with enhanced specificity. As a proof of concept, we designed conditional deoxyribozyme-nanoparticles (conditional DzNPs) that knockdown tumor necrosis factor α (TNFα) mRNA upon miR-33 triggering. We demonstrate toehold-mediated strand exchange and restoration of TNFα DNAzyme activity in the presence of miR-33 trigger, with optimization of the preparation, configuration, and toehold length of conditional DzNPs. Our results indicate specific and strong ON/OFF response of conditional DzNPs to the miR-33 trigger in buffer. Furthermore, we demonstrate endogenous miR-33-triggered knockdown of TNFα mRNA in mouse macrophages, implying the potential of conditional gene regulation applications using these DzNPs.

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.

Variable incidence angle linear dichroism (VALiD): a technique for unique 3D orientation measurement of fluorescent ensembles.

A fundamental challenge with fluorophore orientation measurement is degeneracy, which is the inability to distinguish between multiple unique fluorophore orientations. Techniques exist for the non-degenerate measurement of the orientations of single, static fluorophores. However, such techniques are unsuitable for densely labeled and/or dynamic samples common to biological research. Accordingly, a rapid, widefield microscopy technique that can measure orientation parameters for ensembles of fluorophores in a non-degenerate manner is desirable. We propose that exciting samples with polarized light and multiple incidence angles could enable such a technique. We use Monte Carlo simulations to validate this approach for specific axially symmetric ensembles of fluorophores and obtain optimal experimental parameters for its future implementation.

Tunable DNA Origami Motors Translocate Ballistically Over µm Distances at nm/s Speeds.

Inspired by biological motor proteins, that efficiently convert chemical fuel to unidirectional motion, there has been considerable interest in developing synthetic analogues. Among the synthetic motors created thus far, DNA motors that undertake discrete steps on RNA tracks have shown the greatest promise. Nonetheless, DNA nanomotors lack intrinsic directionality, are low speed and take a limited number of steps prior to stalling or dissociation. Herein, we report the first example of a highly tunable DNA origami motor that moves linearly over micron distances at an average speed of 40 nm/min. Importantly, nanomotors move unidirectionally without intervention through an external force field or a patterned track. Because DNA origami enables precise testing of nanoscale structure-function relationships, we were able to experimentally study the role of motor shape, chassis flexibility, leg distribution, and total number of legs in tuning performance. An anisotropic rigid chassis coupled with a high density of legs maximizes nanomotor speed and endurance.