Construction, imaging, and analysis of FRET-based tension sensors in living cells.
Journal Article (Journal Article)
Due to an increased appreciation for the importance of mechanical stimuli in many biological contexts, an interest in measuring the forces experienced by specific proteins in living cells has recently emerged. The development and use of Förster resonance energy transfer (FRET)-based molecular tension sensors has enabled these types of studies and led to important insights into the mechanisms those cells utilize to probe and respond to the mechanical nature of their surrounding environment. The process for creating and utilizing FRET-based tension sensors can be divided into three main parts: construction, imaging, and analysis. First we review several methods for the construction of genetically encoded FRET-based tension sensors, including restriction enzyme-based methods as well as the more recently developed overlap extension or Gibson Assembly protocols. Next, we discuss the intricacies associated with imaging tension sensors, including optimizing imaging parameters as well as common techniques for estimating artifacts within standard imaging systems. Then, we detail the analysis of such data and describe how to extract useful information from a FRET experiment. Finally, we provide a discussion on identifying and correcting common artifacts in the imaging of FRET-based tension sensors.
Full Text
Duke Authors
Cited Authors
- LaCroix, AS; Rothenberg, KE; Berginski, ME; Urs, AN; Hoffman, BD
Published Date
- January 2015
Published In
Volume / Issue
- 125 /
Start / End Page
- 161 - 186
PubMed ID
- 25640429
Pubmed Central ID
- PMC4511473
International Standard Serial Number (ISSN)
- 0091-679X
Digital Object Identifier (DOI)
- 10.1016/bs.mcb.2014.10.033
Language
- eng