Measuring Exocytosis Rate Using Corrected Fluorescence Recovery After Photoconversion
- Creators
- Luo, Nan
- Yan, An
- Yang, Zhenbiao
Abstract
Exocytosis plays crucial roles in regulating the distribution and function of plasma membrane (PM) and extracellular matrix proteins. However, measuring the exocytosis rate of a specific protein by conventional methods is very difficult because of exocytosis-independent trafficking such as endocytosis, which also affects membrane protein distribution. Here, we describe a novel method, corrected fluorescence recovery after photoconversion, in which exocytosis-dependent and -independent trafficking events are measured simultaneously to accurately determine exocytosis rate. In this method, the protein-of-interest is tagged with Dendra2, a green-to-red photoconvertible fluorescent protein. Following the photoconversion of PM-localized Dendra2, both the recovery of the green signal and the changes in the photoconverted red signal are measured, and the rate of exocytosis is calculated from the changing rates of these two signals.
Additional Information
© 2016 John Wiley & Sons. Issue Online: 19 April 2016; Version of Record online: 31 March 2016; Accepted manuscript online: 29 January 2016; Manuscript accepted: 25 January 2016; Manuscript revised: 25 January 2016; Manuscript received: 30 October 2015. We thank members of the Yang Laboratory for stimulating discussion of this work. The work is supported by the U.S. National Institute of General Medical Sciences to Z. Y. (GM100130).Attached Files
Accepted Version - nihms-756400.pdf
Supplemental Material - tra12380-sup-0001-appendixs1.docx
Supplemental Material - tra12380-sup-0002-movies1.mov
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Additional details
- PMCID
- PMC7513930
- Eprint ID
- 66433
- DOI
- 10.1111/tra.12380
- Resolver ID
- CaltechAUTHORS:20160425-075557290
- GM100130
- NIH
- Created
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2016-04-27Created from EPrint's datestamp field
- Updated
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2022-05-05Created from EPrint's last_modified field