Improving Quality, Reproducibility, and Usability of FRET‐Based Tension Sensors

Abstract

Mechanobiology, the study of how mechanical forces affect cellular behavior, is an emerging field of study that has garnered broad and significant interest. Researchers are currently seeking to better understand how mechanical signals are transmitted, detected, and integrated at a subcellular level. One tool for addressing these questions is a Förster resonance energy transfer (FRET)‐based tension sensor, which enables the measurement of molecular‐scale forces across proteins based on changes in emitted light. However, the reliability and reproducibility of measurements made with these sensors has not been thoroughly examined. To address these concerns, we developed numerical methods that improve the accuracy of measurements made using sensitized emission‐based imaging. To establish that FRET‐based tension sensors are versatile tools that provide consistent measurements, we used these methods, and demonstrated that a vinculin tension sensor is unperturbed by cell fixation, permeabilization, and immunolabeling. This suggests FRET‐based tension sensors could be coupled with a variety of immuno‐fluorescent labeling techniques. Additionally, as tension sensors are frequently employed in complex biological samples where large experimental repeats may be challenging, we examined how sample size affects the uncertainty of FRET measurements. In total, this work establishes guidelines to improve FRET‐based tension sensor measurements, validate novel implementations of these sensors, and ensure that results are precise and reproducible. © 2018 International Society for Advancement of Cytometry

Förster Resonance Energy Transfer Pioneers Biomechanics: Molecular‐Scale Force Meters for Visualizing Intracellular Stress Fields

“…As a relatively new branch of science, biomechanics, is dealing with measurements of forces at different levels of organization of the living state and studying transmission of force through the cell membrane toward the cell interior (mechano-transduction), as well as the conversion of mechanical signals to electric and chemical ones, that is, signal transductions evoked by mechanical forces (1-3). As a general rule, while increasing the characteristic Förster-distance R 0 , FRET efficiency is increased, increasing the length and stiffness ("spring constant") of the linker protein, FRET efficiency is reduced.A specific example for high tuning a tension sensor is shown by the group of Hoffman et al (8,9). In order to monitor these types of signaling, an important step forward is the development of biosensors capable for mechanical force measurement at the molecular level, even inside the living cell.…”

“…A specific example for high tuning a tension sensor is shown by the group of Hoffman et al (8,9). They developed and further improved a vinculin-based tension sensor (VinTSMod) to measure force transmission of vinculin, which bridges adhesion receptors in the cell membrane and the actin filaments of cytoskeleton.…”

“…This problem was solved here by constructing "VinTS internals," where either mTFP1 or Venus has been inserted into a vinculin molecule (8). The FRET signal of a sensing module, as the primary intramolecular FRET, can be contaminated with FRET signal cross talks from the neighboring sensing modules, serving as the secondary, inter-molecular FRET.…”

“…The authors used the short 2x(GGSGGS) and the long TRAF (TNFα receptor derivative) linkers for high (48%) and low (4%) FRET between mTFP1 and Venus (8). They used the "sensitized emission method" of FRET.…”

“…The main parameters of a sensor-the lower and upper limits of detectable forces, that is, the dynamic range and the magnitude of FRET response to unit stress, that is, the sensitivity-are determined by the stiffness (spring constant) and length of the linker, as well as the FRET efficiency, quantified by the characteristic Förster-distance R 0 . As a general rule, while increasing the characteristic Förster-distance R 0 , FRET efficiency is increased, increasing the length and stiffness ("spring constant") of the linker protein, FRET efficiency is reduced.A specific example for high tuning a tension sensor is shown by the group of Hoffman et al (8,9). They developed and further improved a vinculin-based tension sensor (VinTSMod) to measure force transmission of vinculin, which bridges adhesion receptors in the cell membrane and the actin filaments of cytoskeleton.…”