PT  - JOURNAL ARTICLE
AU  - Eric J. Aird
AU  - Kassidy J. Tompkins
AU  - Wendy R. Gordon
TI  - An enhanced molecular tension sensor based on bioluminescence resonance energy transfer (BRET)
AID  - 10.1101/617696
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 617696
4099  - http://biorxiv.org/content/early/2019/04/24/617696.short
4100  - http://biorxiv.org/content/early/2019/04/24/617696.full
AB  - Molecular tension sensors measure piconewton forces experienced by individual proteins in the context of the cellular microenvironment. Current genetically-encoded tension sensors use FRET to report on extension of an elastic peptide encoded in a cellular protein of interest. Here we present the development and characterization of a new type of molecular tension sensor based on bioluminescence resonance energy transfer (BRET) which exhibits more desirable spectral properties and an enhanced dynamic range compared to other molecular tension sensors. Moreover, it avoids many disadvantages of FRET measurements in cells, including heating of the sample, autofluorescence, photobleaching, and corrections of direct acceptor excitation. We benchmark the sensor by inserting it into the canonical mechanosensing focal adhesion protein vinculin, observing highly resolved gradients of tensional changes across focal adhesions. We anticipate that the BRET-TS will expand the toolkit available to study mechanotransduction at a molecular level and allow potential extension to an in vivo context.BRETBioluminescence resonance energy transferFRETFörster resonance energy transferFPfluorescent proteinTStension sensorRETresonance energy transferHLhelical linkerFAfocal adhesionRLUrelative light unitsNlucNanoLuc