Search

Mechanotransduction, the interplay between physical and chemical signaling, plays vital roles in many biological processes ranging from cell differentiation to metastasis. The state-of-the-art techniques to quantify cell forces employ deformable polymer films or molecular probes tethered to glass substrates. These types of flat substrates limit applications in investigating mechanotransduction on non-planar geometries where physiological activities such as phagocytosis and immunological synapse formation mostly occur. A second challenge is the low throughput of microscopy readout which limits the application of current assays in fundamental and clinical research. We address these challenges by developing a DNA-based microparticle tension sensor (μTS), which features a spherical surface and thus allows for investigation of mechanical events at curved interfaces or within groups of cells in suspension. Importantly, the micron-scale of μTS enables flow cytometry readout, which is rapid and high throughput. To demonstrate the scope of μTS, we applied the method to map and measure T-cell receptor (TCR) 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.

Cells transmit piconewton forces to receptors to mediate processes such as migration and immune recognition. A major challenge in quantifying such forces is the sparsity of cell mechanical events. Accordingly, molecular tension is typically quantified with high resolution fluorescence microscopy, which hinders widespread adoption and application. Here, we report a mechanically-triggered hybridization chain reaction (mechano-HCR) that allows chemical amplification of mechanical events. The amplification is triggered when a cell receptor mechanically denatures a duplex revealing a cryptic initiator to activate the HCR reaction in situ. Importantly, mechano-HCR enables direct readout of pN forces using a plate reader. We leverage this capability and measured mechano-IC(50) for aspirin, Y-27632, and eptifibatide. Given that cell mechanical phenotypes are of clinical importance, mechano-HCR may offer a convenient route for drug discovery, personalized medicine, and disease diagnosis.

Oligonucleotide-based probes offer the highest spatial resolution, force sensitivity, and molecular specificity for cellular tension sensing and have been developed to measure a variety of molecular forces mediated by individual receptors in T cells, platelets, fibroblasts, B-cells, and immortalized cancer cell lines. These fluorophore-oligonucleotide conjugate probes are designed with a stem-loop structure that engages cell receptors and reversibly unfolds due to mechanical strain. With the growth of recent work bridging molecular mechanobiology and biomaterials, there is a need for a detailed spectroscopic analysis of DNA tension probes that are used for cellular imaging. In this manuscript, we conducted an analysis of 19 DNA hairpin-based tension probe variants using molecular dynamics simulations, absorption spectroscopy, and fluorescence imaging (epifluorescence and fluorescence lifetime imaging microscopy). We find that tension probes are highly sensitive to their molecular design, including donor and acceptor proximity and pairing, DNA stem-loop structure, and conjugation chemistry. We demonstrate the impact of these design features using a supported lipid bilayer model of podosome-like adhesions. Finally, we discuss the requirements for tension imaging in various biophysical contexts and offer a series of experimental recommendations, thus providing a guide for the design and application of DNA hairpin-based molecular tension probes.

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 quantify and visualize the piconewton (pN) forces applied by these receptors. Building on this technology, we recently imaged DNA tension probes using fluorescence polarization imaging to map the magnitude and 3D orientation of receptor forces with diffraction limited resolution (~ 250 nm). Further improvements in spatial resolution are desirable as many force-sensing receptors are organized at the nano-scale in supramolecular complexes such as focal adhesions. Here, we show that structured illumination microscopy (SIM), a super-resolution technique, can be used to perform super-resolution molecular force microscopy (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 integrins forces, as well as T cell receptor forces. The method reveals that platelets dynamically re-arrange the orientation of their integrin forces during activation. Monte Carlo simulations validated the results and provided analysis of the sources of noise. Importantly, we envision that SIM-MFM will be broadly adopted by the cell biology and mechanobiology communities because it can be implemented on any standard SIM microscope without hardware modifications.

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. Tension-PAINT integrates molecular tension probes with DNA-PAINT to enable ~25-nm-resolution mapping of piconewton mechanical events. Tension-PAINT can be used to study dynamic forces, and an irreversible variant integrates force history over time.

Podosomes are ubiquitous cellular structures important to diverse processes including cell invasion, migration, bone resorption, and immune surveillance. Structurally, podosomes consist of a protrusive actin core surrounded by adhesion proteins. Although podosome protrusion forces have been quantified, the magnitude, spatial distribution, and orientation of the opposing tensile forces remain poorly characterized. Here we use DNA nanotechnology to create probes that measure and manipulate podosome tensile forces with molecular piconewton (pN) resolution. Specifically, Molecular Tension-Fluorescence Lifetime Imaging Microscopy (MT-FLIM) produces maps of the cellular adhesive landscape, revealing ring-like tensile forces surrounding podosome cores. Photocleavable adhesion ligands, breakable DNA force probes, and pharmacological inhibition demonstrate local mechanical coupling between integrin tension and actin protrusion. Thus, podosomes use pN integrin forces to sense and respond to substrate mechanics. This work deepens our understanding of podosome mechanotransduction and contributes tools that are widely applicable for studying receptor mechanics at dynamic interfaces., Podosomes are protrusive structures that coordinate diverse functions related to cell invasion, migration, bone resorption and immune surveillance. Here the authors integrate DNA nanotechnology with FLIM-FRET to demonstrate that podosomes apply pN integrin tensile forces to sense and respond to substrate mechanics.

Modifying RNA through either splicing or editing is a fundamental biological process for creating protein diversity from the same genetic code. Developing novel chemical biology tools for RNA editing has potential to transiently edit genes and to provide a better understanding of RNA biochemistry. Current techniques used to modify RNA include the use of ribozymes, adenosine deaminase and tRNA endonucleases. Herein, we report a nanozyme that is capable of splicing of virtually any RNA stem-loop. This nanozyme is comprised of a gold nanoparticle functionalized with three enzymes: two catalytic DNA strands with ribonuclease function and an RNA ligase. The nanozyme cleaves and then ligates RNA targets, performing a splicing reaction that is akin to the function of the spliceosome. Our results show that the three-enzyme reaction can remove a 19 nt segment from a 67 nt RNA loop with up to 66% efficiency. The complete nanozyme can perform the same splice reaction at 10% efficiency. These splicing nanozymes represent a new promising approach for gene manipulation that has potential for applications in living cells.

Mammalian and bacterial cells sense and exert mechanical forces through the process of mechanotransduction, which interconverts biochemical and physical signals. This is especially important in contact-dependent signaling, where ligand-receptor binding occurs at cell-cell or cell-ECM junctions. By virtue of occurring within these specialized junctions, receptors engaged in contact-dependent signaling undergo oligomerization and coupling with the cytoskeleton as part of their signaling mechanisms. While our ability to measure and map biochemical signaling within cell junctions has advanced over the past decades, physical cues remain difficult to map in space and time. Recently, supported lipid bilayer (SLB) technologies have emerged as a flexible platform to mimic and perturb cell-cell and cell-ECM junctions, allowing one to study membrane receptor mechanotransduction. Changing the lipid composition and underlying substrate tunes bilayer fluidity, and lipid and ligand micro- and nano-patterning spatially control positioning and clustering of receptors. Patterning metal gridlines within SLBs confines lipid mobility and introduces mechanical resistance. Here we review fundamental SLB mechanics and how SLBs can be engineered as tunable cell substrates for mechanotransduction studies. Finally, we highlight the impact of this work in understanding the biophysical mechanisms of cell adhesion. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.