Super-resolved Measurement of Piconewton Receptor Forces via Tension-PAINT

Abstract

Piconewton receptor forces exerted by cellular receptors regulate critical cellular processes, including development, migration, immune recognition, and hemostasis. Despite the vital role of mechanical forces in biology, no current technique can image cellular force organization with sub-100 nm resolution. Here, we present tension-PAINT, which leverages DNA-based molecular tension probes in combination with the stochastic super-resolution microscopy technique DNA-Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT), to enable super-resolved receptor tension measurements. DNA-PAINT relies on the transient binding of fluorophore-labeled oligonucleotide “imager” sequences to complementary “docking” sequences to produce the single-molecule fluorescence required for stochastic super-resolution imaging. We reasoned that encoding a cryptic docking sequence within DNA-based molecular tension probes would allow these probes to function like receptor force-triggered switches. These DNA based molecular force probes reveal a single-stranded DNA docking site only under the influence of pN receptor forces. The resulting technique, which we have named tension-PAINT, provides maps of receptor tension with up to 25 nm spatial resolution and pN force sensitivity. Here, we present variants of tension-PAINT capable of real-time super-resolved tension measurement in living cells, and of integrating cellular force history over time. We apply tension-PAINT to map the integrin forces of living platelets, revealing that platelets exert integrin mediated forces in a 100-200 nm wide spreading “ring” pattern at the lamellipodial edge. Additionally, we reveal the dynamics of fibroblast focal-adhesion and filopodial retraction forces. Due to its compatibility with widely available, unmodified fluorescence microscopes, we anticipate that tension-PAINT will provide a valuable, widely accessible tool to quantify the role of the nanoscale distribution of receptor forces in biological function.