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

• Published in: Cytometry. Part A Vol. 95; no. 2; pp. 201 - 213
• Main Authors: Gates, Evan M., LaCroix, Andrew S., Rothenberg, Katheryn E., Hoffman, Brenton D.
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01.02.2019; Wiley Subscription Services, Inc
• Subjects: Animals; Biological properties; Biological samples; Biosensing Techniques - methods; Cell Line; Cytometry; Emission measurements; Energy transfer; Fluorescence; Fluorescence resonance energy transfer; Fluorescence Resonance Energy Transfer - methods; FRET efficiency; FRET‐based biosensor; Mathematical models; Mechanical properties; Mechanical stimuli; mechanotransduction; Mice; Numerical methods; Reproducibility; Reproducibility of Results; sensitized emission; Sensors; Tension; Vinculin; Vinculin - metabolism; FRET efficiency; sensitized emission; FRET-based biosensor; mechanotransduction
• ISSN: 1552-4922; 1552-4930; 1552-4930
• DOI: 10.1002/cyto.a.23688

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