No abstract

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.

No abstract

Integrin receptors transduce the mechanical properties of the extracellular matrix. Past studies using DNA probes showed that integrins sense the magnitude of ligand forces with pN resolution. An open question is whether integrin receptors also sense the force-extension trajectory of their ligands. The challenge in addressing this question pertains to the lack of molecular probes that can control force-extension trajectories independently of force magnitude. To address this limitation, we synthesized two reversible DNA probes that fold with identical self-complementary domains but with different topologies. Thus, these probes unfold at the same steady-state force magnitude but following different kinetic pathways to reach the fully extended ssDNA state. Hairpin-like probes unzip with a low barrier of 14 pN while the pseudo-knot-like probes shear at 59 pN. Confirming that we had created probes with different barriers of unfolding, we quantified platelet integrin forces and measured 50-fold more tension signal with the unzipping probes over the shearing probes. In contrast, fibroblasts opened both probes to similar levels indicating more static forces. Surprisingly, fibroblast mechanotransduction markers, such as YAP levels, fibronectin production, actin organization, and integrin activation were significantly elevated on unzipping probes. This demonstrates that integrin receptors within focal adhesions sense the molecular force-extension profile of their ligands and not only the magnitude of equilibrium mechanical resistance.

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.