Molecular Force Sensors Research Articles

In-Silico Analyses of Molecular Force Sensors for Mechanical Characterization of Biological Systems.

Mechanical forces play key roles in biological processes such as cell migration and sensory perception. In recent years molecular force sensors have been developed as tools for in situ force measurements. Here we use all-atom steered molecular dynamics simulations to predict and study the relationship between design parameters and mechanical properties for three types of molecular force sensors commonly used in cellular biological research: two peptide- and one DNA-based. The peptide-based sensors consist of a pair of fluorescent proteins, which can undergo Förster resonance energy transfer (FRET), linked by spider silk (GPGGA)n or synthetic (GGSGGS)n disordered regions. The DNA-based sensor consists of two fluorophore-labeled strands of DNA that can be unzipped or sheared upon force application with a FRET signal as readout of dissociation. We simulated nine sensors, three of each kind. After equilibration, flexible peptide linkers of three different lengths were stretched by applying forces to their N- and C-terminal Cα atoms in opposite directions. Similarly, we equilibrated a DNA-based sensor and pulled on the phosphate atom of the terminal guanine of one strand and a selected phosphate atom on the other strand in the opposite direction. These simulations were performed at constant velocity (0.01 nm/ns - 10 nm/ns) and constant force (10 pN - 500 pN) for all versions of the sensors. Our results show how the force response of these sensors depends on their length, sequence, configuration and loading rate. Mechanistic insights gained from simulations analyses indicate that interpretation of experimental results should consider the influence of transient formation of secondary structure in peptide-based sensors and of overstretching in DNA-based sensors. These predictions can guide optimal fluorophore choice and facilitate the rational design of new sensors for use in protein, DNA, hybrid systems, and molecular devices.

Molecular Force Sensors for Biological Application.

The mechanical forces exerted by cells on their surrounding microenvironment are known as cellular traction forces. These forces play crucial roles in various biological processes, such as tissue development, wound healing and cell functions. However, it is hard for traditional techniques to measure cellular traction forces accurately because their magnitude (from pN to nN) and the length scales over which they occur (from nm to μm) are extremely small. In order to fully understand mechanotransduction, highly sensitive tools for measuring cellular forces are needed. Current powerful techniques for measuring traction forces include traction force microscopy (TFM) and fluorescent molecular force sensors (FMFS). In this review, we elucidate the force imaging principles of TFM and FMFS. Then we highlight the application of FMFS in a variety of biological processes and offer our perspectives and insights into the potential applications of FMFS.

In silico analyses of molecular force sensors for mechanical characterization of biological systems

In silico analyses of molecular force sensors for mechanical characterization of biological systems

Measurement of Forces Acting on Single T-Cell Receptors.

Molecular forces are increasingly recognized as an important parameter to understand cellular signaling processes. In the recent years, evidence accumulated that also T-cells exert tensile forces via their T-cell receptor during the antigen recognition process. To measure such intercellular pulling forces, one can make use of the elastic properties of spider silk peptides, which act similar to Hookean springs: increased strain corresponds to increased stress applied to the peptide. Combined with Förster resonance energy transfer (FRET) to read out the strain, such peptides represent powerful and versatile nanoscopic force sensing tools. In this paper, we provide a detailed protocol how to synthesize a molecular force sensor for application in T-cell antigen recognition and hands-on guidelines on experiments and analysis of obtained single molecule FRET data.

A mechanical-coupling mechanism in OSCA/TMEM63 channel mechanosensitivity

Mechanosensitive (MS) ion channels are a ubiquitous type of molecular force sensor sensing forces from the surrounding bilayer. The profound structural diversity in these channels suggests that the molecular mechanisms of force sensing follow unique structural blueprints. Here we determine the structures of plant and mammalian OSCA/TMEM63 proteins, allowing us to identify essential elements for mechanotransduction and propose roles for putative bound lipids in OSCA/TMEM63 mechanosensation. Briefly, the central cavity created by the dimer interface couples each subunit and modulates dimeric OSCA/TMEM63 channel mechanosensitivity through the modulating lipids while the cytosolic side of the pore is gated by a plug lipid that prevents the ion permeation. Our results suggest that the gating mechanism of OSCA/TMEM63 channels may combine structural aspects of the ‘lipid-gated’ mechanism of MscS and TRAAK channels and the calcium-induced gating mechanism of the TMEM16 family, which may provide insights into the structural rearrangements of TMEM16/TMC superfamilies.

Development of mechanosensitive synthetic cells for biomedical applications

The ability of cells to sense and respond to their physical environment plays a fundamental role in a broad spectrum of biological processes. As one of the most essential molecular force sensors and transducers found in cell membranes, mechanosensitive (MS) ion channels can convert mechanical inputs into biochemical or electrical signals to mediate a variety of sensations. The bottom-up construction of cell-sized compartments displaying cell-like organization, behaviors, and complexity, also known as synthetic cells, has gained popularity as an experimental platform to characterize biological functions in isolation. By reconstituting MS channels in the synthetic lipid bilayers, we envision using mechanosensitive synthetic cells for several medical applications. Here, we describe three different concepts for using ultrasound, shear stress, and compressive stress as mechanical stimuli to activate drug release from mechanosensitive synthetic cells for disease treatments.

Novel imaging methods and force probes for molecular mechanobiology of cytoskeleton and adhesion.

Detection and conversion of mechanical forces into biochemical signals is known as mechanotransduction. From cells to tissues, mechanotransduction regulates migration, proliferation, and differentiation in processes such as immune responses, development, and cancer progression. Mechanosensitive structures such as integrin adhesions, the actin cortex, ion channels, caveolae, and the nucleus sense and transmit forces. In vitro approaches showed that mechanosensing is based on force-dependent protein deformations and reorganizations. However, the mechanisms in cells remained unclear since cell imaging techniques lacked molecular resolution. Thanks to recent developments in super-resolution microscopy (SRM) and molecular force sensors, it is possible to obtain molecular insight of mechanosensing in live cells. We discuss how understanding of molecular mechanotransduction was revolutionized by these innovative approaches, focusing on integrin adhesions, actin structures, and the plasma membrane.

Visualizing Neurons Under Tension In Vivo with Optogenetic Molecular Force Sensors.

The visualization of mechanical stress distribution in specific molecular networks within a living and physiologically active cell or animal remains a formidable challenge in mechanobiology. The advent of fluorescence-resonance energy transfer (FRET)-based molecular tension sensors overcame a significant hurdle that now enables us to address previously technically limited questions. Here, we describe a method that uses genetically encoded FRET tension sensors to visualize the mechanics of cytoskeletal networks in neurons of living animals with sensitized emission FRET and confocal scanning light microscopy. This method uses noninvasive immobilization of living animals to image neuronal β-spectrin cytoskeleton at the diffraction limit, and leverages multiple imaging controls to verify and underline the quality of the measurements. In combination with a semiautomated machine-vision algorithm to identify and trace individual neurites, our analysis performs simultaneous calculation of FRET efficiencies and visualizes statistical uncertainty on a pixel by pixel basis. Our approach is not limited to genetically encoded spectrin tension sensors, but can also be used for any kind of ratiometric imaging in neuronal cells both in vivo and in vitro.

Cellular force-sensing through actin filaments.

The actin cytoskeleton orchestrates cell mechanics and facilitates the physical integration of cells into tissues, while tissue-scale forces and extracellular rigidity in turn govern cell behaviour. Here, we discuss recent evidence that actin filaments (F-actin), the core building blocks of the actin cytoskeleton, also serve as molecular force sensors. We delineate two classes of proteins, which interpret forces applied to F-actin through enhanced binding interactions: 'mechanically tuned' canonical actin-binding proteins, whose constitutive F-actin affinity is increased by force, and 'mechanically switched' proteins, which bind F-actin only in the presence of force. We speculate mechanically tuned and mechanically switched actin-binding proteins are biophysically suitable for coordinating cytoskeletal force-feedback and mechanical signalling processes, respectively. Finally, we discuss potential mechanisms mediating force-activated actin binding, which likely occurs both through the structural remodelling of F-actin itself and geometric rearrangements of higher-order actin networks. Understanding the interplay of these mechanisms will enable the dissection of force-activated actin binding's specific biological functions.

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate multiple functions, including adhesion, proliferation, migration, and differentiation. Mechanical forces can be transmitted from the cell via the adhesion receptor integrin to ligands in the ECM. The amount and spatial organization of these cell-generated forces can be modulated by growth factor receptors, including epidermal growth factor receptor (EGFR). The tools currently available to quantify crosstalk-mediated changes in cell mechanics and relate them to focal adhesions, cellular morphology, and signaling are limited. DNA-based molecular force sensors known as tension gauge tethers (TGTs) have been employed to quantify these changes. TGT probes are unique in their ability to both modulate the underlying force threshold and report piconewton scale receptor forces across the entire adherent cell surface at diffraction-limited spatial resolution. The TGT probes used here rely on the irreversible dissociation of a DNA duplex by receptor-ligand forces that generate a fluorescent signal. This allows quantification of the cumulative integrin tension (force history) of the cell. This article describes a protocol employing TGTs to study the impact of EGFR on integrin mechanics and adhesion formation. The assembly of the TGT mechanical sensing platform is systematically detailed and the procedure to image forces, focal adhesions, and cell spreading is outlined. Overall, the ability to modulate the underlying force threshold of the probe, the adhesion ligand, and the type and concentration of growth factor employed for stimulation make this a robust platform for studying the interplay of diverse membrane receptors in regulating integrin-mediated forces.

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion.

Multicellular organisms rely on interactions between membrane receptors and cognate ligands in the surrounding extracellular matrix (ECM) to orchestrate multiple functions, including adhesion, proliferation, migration, and differentiation. Mechanical forces can be transmitted from the cell via the adhesion receptor integrin to ligands in the ECM. The amount and spatial organization of these cell-generated forces can be modulated by growth factor receptors, including epidermal growth factor receptor (EGFR). The tools currently available to quantify crosstalk-mediated changes in cell mechanics and relate them tofocal adhesions, cellular morphology, and signaling are limited. DNA-based molecular force sensors known as tension gauge tethers (TGTs) have been employed to quantify these changes. TGT probes are unique in their ability to both modulate the underlying force threshold and report piconewton scale receptor forces across the entire adherent cell surface at diffraction-limited spatial resolution. The TGT probes used here rely on the irreversible dissociation of a DNA duplex by receptor-ligand forces that generate a fluorescent signal. This allows quantification of the cumulative integrin tension (force history) of the cell. This article describes a protocol employing TGTs to study the impact of EGFR on integrin mechanics and adhesion formation. The assembly ofthe TGT mechanical sensing platform is systematically detailed and the procedure to image forces, focal adhesions, and cell spreading is outlined. Overall, the ability to modulate the underlying force threshold of the probe, the adhesion ligand, and the type and concentration of growth factor employed for stimulation make this a robust platform for studying the interplay of diverse membrane receptors in regulating integrin-mediated forces.

Engineering the extracellular matrix with DNA-based molecular force sensors

Mechanical forces due to fluid shear stress can promote the metastatic capabilities of cancer cells. However, measuring shear flow in native 3-D tissue environments can be challenging due to the high inhomogeneity of the composition and microarchitecture of the tumor extracellular matrix (ECM). DNA-based molecular sensors have been of particular interest for studying cell interactions due to their high tunability, sensitivity and easy integrability into various biological systems. However, measuring cell-ECM interactions due to fluid flow using these sensors is yet to be achieved.

Quantification of fast molecular adhesion by fluorescence footprinting.

Rolling adhesion is a unique process in which the adhesion events are short-lived and operate under highly nonequilibrium conditions. These characteristics pose a challenge in molecular force quantification, where in situ measurement of these forces cannot be achieved with molecular force sensors that probe near equilibrium. Here, we demonstrated a quantitative adhesion footprint assay combining DNA-based nonequilibrium force probes and modeling to measure the molecular force involved in fast rolling adhesion. We were able to directly profile the ensemble molecular force distribution in our system during rolling adhesion with a dynamic range between 0 and 18 pN. Our results showed that the shear stress driving bead rolling motility directly controls the molecular tension on the probe-conjugated adhesion complex. Furthermore, the shear stress can steer the dissociation bias of components within the molecular force probe complex, favoring either DNA probe dissociation or receptor-ligand dissociation.

Quantifying Molecular Forces with Serially Connected Force Sensors

To understand the mechanical forces involved in cell adhesion, molecular force sensors have been developed to study tension through adhesion proteins. Recently, a class of molecular force sensors called tension gauge tethers (TGTs) have been developed that rely on irreversible force-dependent dissociation of a DNA duplex to study cell adhesion forces. Although the TGT offers a high signal-to-noise ratio and is ideal for studying fast/single-molecular adhesion processes, quantitative interpretation of experimental results has been challenging. Here, we use a computational approach to investigate how TGT fluorescence readout can be quantitatively interpreted. In particular, we studied force sensors made of a single TGT, multiplexed single TGTs, and two TGTs connected in series. Our results showed that fluorescence readout using a single TGT can result from drastically different combinations of force history and adhesion event density that span orders of magnitude. In addition, the apparent behavior of the TGT is influenced by the tethered receptor-ligand, making it necessary to calibrate the TGT with every new receptor-ligand. To solve this problem, we proposed a system of two serially connected TGTs. Our result shows that not only is the ratiometric readout of serial TGT independent of the choice of receptor-ligand, it is able to reconstruct force history with sub-pN force resolution. This is also not possible by simply multiplexing different types of TGTs together. Last, we systematically investigated how the sequence composition of the two serially connected TGTs can be tuned to achieve different dynamic range. This computational study demonstrated how serially connected irreversible molecular dissociation processes can accurately quantify molecular force and laid the foundation for subsequent experimental studies.

Extending the Capabilities of Molecular Force Sensors via DNA Nanotechnology.

At the nanoscale, pushing, pulling, and shearing forces drive biochemical processes in development and remodeling as well as in wound healing and disease progression. Research in the field of mechanobiology investigates not only how these loads affect biochemical signaling pathways but also how signaling pathways respond to local loading by triggering mechanical changes such as regional stiffening of a tissue. This feedback between mechanical and biochemical signaling is increasingly recognized as fundamental in embryonic development, tissue morphogenesis, cell signaling, and disease pathogenesis. Historically, the interdisciplinary field of mechanobiology has been driven by the development of technologies for measuring and manipulating cellular and molecular forces, with each new tool enabling vast new lines of inquiry. In this review, we discuss recent advances in the manufacturing and capabilities of molecular-scale force and strain sensors. We also demonstrate how DNA nanotechnology has been critical to the enhancement of existing techniques and to the development of unique capabilities for future mechanosensor assembly. DNA is a responsive and programmable building material for sensor fabrication. It enables the systematic interrogation of molecular biomechanics with forces at the 1- to 200-pN scale that are needed to elucidate the fundamental means by which cells and proteins transduce mechanical signals.

Quantifying Molecular Forces with Serially Connected Force Sensors

To understand the mechanical forces involved in cell adhesion, molecular force sensors have been developed to study tension through adhesion proteins. Recently, a class of molecular force sensors called tension gauge tethers (TGTs) have been developed that rely on irreversible force-dependent dissociation of a DNA duplex to study cell adhesion forces. Although the TGT offers a high signal-to-noise ratio and is ideal for studying fast/single-molecular adhesion processes, quantitative interpretation of experimental results has been challenging. Here, we use a computational approach to investigate how TGT fluorescence readout can be quantitatively interpreted. In particular, we studied force sensors made of a single TGT, multiplexed single TGTs, and two TGTs connected in series. Our results showed that fluorescence readout using a single TGT can result from drastically different combinations of force history and adhesion event density that span orders of magnitude. In addition, the apparent behavior of the TGT is influenced by the tethered receptor-ligand, making it necessary to calibrate the TGT with every new receptor-ligand. To solve this problem, we proposed a system of two serially connected TGTs. Our result shows that not only is the ratiometric readout of serial TGT independent of the choice of receptor-ligand, it is able to reconstruct force history with sub-pN force resolution. This is also not possible by simply multiplexing different types of TGTs together. Last, we systematically investigated how the sequence composition of the two serially connected TGTs can be tuned to achieve different dynamic range. This computational study demonstrated how serially connected irreversible molecular dissociation processes can accurately quantify molecular force and laid the foundation for subsequent experimental studies.

Real-Time Measurement of Molecular Tension during Cell Adhesion and Migration Using Multiplexed Differential Analysis of Tension Gauge Tethers.

Cells must respond specifically and dynamically to mechanical cues from the extracellular environment and dysregulation of extracellular force sensing leads to a variety of diseases. Therefore, it is important to deconvolve the many inputs that transduce mechanical signals and understand how these signals are interpreted and responded to. DNA and peptide-based molecular force sensors have been previously developed to measure forces applied through single membrane receptors including integrins and Notch receptors. The tension gauge tether (TGT) exploits the physical rupture force of double-stranded DNA to measure and modulate the force applied through single receptor-ligand bonds and can cover a wide range of tension (10-60 pN). By exploiting a fluorescent dye-quencher pair and collecting differential fluorescence signals over time, we characterized the quenched tension gauge tether (qTGT) system and developed an image analysis protocol to measure molecular tension in quasi-real time. We show that this differential qTGT analysis method can simultaneously measure multiple levels of integrin-mediated molecular tension over a wide time scale during the onset of adhesion and cell migration.

Mechanical Switching of Aromaticity and Homoaromaticity in Molecular Optical Force Sensors for Polymers.

The sensing of mechanical stress in polymers is indispensable for investigating the origin and propagation of cracks that lead to material failure and for designing mechanically responsive polymers. Here the unique approaches of using the force-induced switching of aromaticity and homoaromaticity in molecular optical force sensors for the real-time measurement of mechanical forces acting in stretched polymers are suggested. The mechanical switching of aromaticity in Dewar benzene is an irreversible event, whereas the degree of π-orbital overlap in homoaromatic compounds like homotropylium can be adjusted progressively over a wide range of forces. Using computational methods, it is demonstrated that both approaches lead to significant changes in the visible part of the UV/Vis spectra of the force sensors upon application of weak forces (pN-nN). Polymers that incorporate such molecular force sensors therefore change their color well before material failure occurs.

Robust mechanobiological behavior emerges in heterogeneous myosin systems

Biological complexity presents challenges for understanding natural phenomenon and engineering new technologies, particularly in systems with molecular heterogeneity. Such complexity is present in myosin motor protein systems, and computational modeling is essential for determining how collective myosin interactions produce emergent system behavior. We develop a computational approach for altering myosin isoform parameters and their collective organization, and support predictions with in vitro experiments of motility assays with α-actinins as molecular force sensors. The computational approach models variations in single myosin molecular structure, system organization, and force stimuli to predict system behavior for filament velocity, energy consumption, and robustness. Robustness is the range of forces where a filament is expected to have continuous velocity and depends on used myosin system energy. Myosin systems are shown to have highly nonlinear behavior across force conditions that may be exploited at a systems level by combining slow and fast myosin isoforms heterogeneously. Results suggest some heterogeneous systems have lower energy use near stall conditions and greater energy consumption when unloaded, therefore promoting robustness. These heterogeneous system capabilities are unique in comparison with homogenous systems and potentially advantageous for high performance bionanotechnologies. Findings open doors at the intersections of mechanics and biology, particularly for understanding and treating myosin-related diseases and developing approaches for motor molecule-based technologies.

Simulating Force Response at Cellular Junctions: Desmoplakin as a Molecular Force Sensor?

Desmosomes are cell-cell junctions with a key role in mechano-sensation. However, it is currently unknown how the desmosomal complex and its proteins respond to mechanical force, transmitting chemical signals. We study desmoplakin, one of the obligate desmosomal proteins, identifying its possible role in mechanosensing. The most striking feature of this protein is the presence of an SH3 domain embedded in one of its six spectrin repeat domains. Intriguingly, the spectrin repeats hide the typical SH3 binding site, suggesting that either (i) the SH3 domain serves as a stabilizer of the protein through its interaction with one of the spectrin repeats, or (ii) it is activated only under force, initiating a signaling cascade for mechanosensing. To explore these alternatives, we performed equilibrium as well as force-probe Molecular Dynamics simulations on the central region of desmoplakin. To bridge towards physiologically relevant time scales, we repeated our simulations through a wide range of pulling velocities with a total simulation time of several tens of microseconds. We find desmoplakin to feature a clear sawtooth-like force profile, with each peak corresponding to modular spectrin repeat unfolding events followed by the final SH3 unfolding. The SH3 domain not only stabilizes desmoplakin, but also remains intact until the very last part of the trajectory, allowing interactions with downstream partners. Interestingly, we find the same behavior in the homologous plectin protein, suggesting this dual role of the SH3 domain to be conserved throughout the plakin family of proteins. However, plectin unfolds at significantly lower forces, involving an early or forceless loss of the SH3 domain-spectrin repeat interface. We identify specific mutations that promote this unfolding pathway, explaining the evolutionary adaptation of the cytosolic compared to the desmosomal mechano-sensing molecule. Our results suggest a direct role of unique SH3-insertion in spectrin repeat proteins in cellular mechanotransduction, and will be validated by direct comparison to single-molecule force spectroscopy experiments carried out by our collaborators.