Discrepancy between fluorescence correlation spectroscopy and fluorescence recovery after photobleaching diffusion measurements of G-protein-coupled receptors

Rhodora Cristina Calizo, Suzanne Scarlata

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) are the two most direct methods to measure the diffusion of molecules in intact living cells. Ideally, these methods should produce similar results for an identical system. We have used these methods to monitor the diffusion of two G-protein-coupled receptors and their associated proteins in the plasma membranes of cells that do not or do contain invaginated protein domains called caveolae. FRAP studies show that caveolae domains increase the immobile fraction of receptors without significantly changing their mobility. On the other hand, FCS studies show an unexpected increase the mobility of caveolae-associated proteins. Our data suggest that the geometry of caveolae domains gives rise to a confined diffusion of its attached proteins, resulting in an apparent increase in mobility.

Original languageEnglish
Pages (from-to)40-48
Number of pages9
JournalAnalytical Biochemistry
Volume440
Issue number1
DOIs
StatePublished - 2013
Externally publishedYes

Keywords

  • Caveolae
  • Fluorescence correlation spectroscopy
  • Fluorescence recovery after photobleaching
  • G-protein-coupled receptors
  • Protein diffusion

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