Turn-key mapping of cell receptor force orientation and magnitude using a commercial structured illumination microscope

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.

Unbreakable DNA tension probes show that cell adhesion receptors detect the molecular force-extension curve of their ligands

“…Platelets are a useful model because of their small size and their ability to generate force. 34, 36, 37, 38, 39, 40 Understanding platelet force is also biomedically relevant in the study of clotting disorders, wound healing, and drug interactions for common cardiovascular diseases. 41, 42 Platelets were cultured on RS and RU probe for 1 hour before activation with 10 µM ADP for 10 minutes ( Fig.…”

Integrin molecular tension required for focal adhesion maturation and YAP nuclear translocation

“…This design strategy provides a way to monitor the real-time molecular tension during cell spreading. The combination of RGD-ligand-DNA tension sensors with super-resolution imaging technology takes the study of cell mechanics to another ultrastructure level [ [52] , [53] , [54] , [55] ]. In our study, we observed that the requirement of molecular tension on integrins is positively correlated with FA size.…”

Super‐Resolution Tension PAINT Imaging with a Molecular Beacon

“…DNA‐based hairpin probe offers the flexibility to fine‐tune the force regime for measuring integrin dynamics, making it ideally suited for live‐cell tension imaging [19, 20] . Tension imaging using structured illumination microscopy (SIM) enabled super‐resolution force mapping with orientation but the resolution was lower than DNA‐PAINT [21, 22] . An improved DNA‐PAINT‐based super‐resolution method for fast, live‐cell tension imaging is crucial for understanding integrin‐mediated cellular mechanics.…”

“…[19,20] Tension imaging using structured illumination microscopy (SIM) enabled super-resolution force mapping with orientation but the resolution was lower than DNA-PAINT. [21,22] An improved DNA-PAINT-based super-resolution method for fast, live-cell tension imaging is crucial for understanding integrin-mediated cellular mechanics. Here we developed a new strategy using a molecular beacon (MB) as the imager molecule that only becomes fluorescent upon binding to target sequences (Figure 1), eliminating the drawbacks of traditional DNA-PAINT while retaining all its advantages.…”