Improving Quality, Reproducibility, and Usability of FRET-Based Tension Sensors.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Gates, EM; LaCroix, AS; Rothenberg, KE; Hoffman, BD

Published Date

  • February 2019

Published In

Volume / Issue

  • 95 / 2

Start / End Page

  • 201 - 213

PubMed ID

  • 30523675

Pubmed Central ID

  • 30523675

Electronic International Standard Serial Number (EISSN)

  • 1552-4930

International Standard Serial Number (ISSN)

  • 1552-4922

Digital Object Identifier (DOI)

  • 10.1002/cyto.a.23688

Language

  • eng