Visualization of Rab5 activity in living cells using FRET microscopy

Emilia Galperin, Alexander Sorkin

Research output: Contribution to journalReview articlepeer-review

9 Scopus citations

Abstract

Rab5 is a member of the large family of small GTPases involved in membrane trafficking. Two genetically encoded sensors were developed to visualize Rab5 in its GTP-bound conformation in living cells. Rab5-binding fragments of Rabaptin5 or early endosomal antigen 1 (EEA.1) were fused to yellow fluorescent protein (YFP) and used in the fluorescent resonance energy transfer (FRET) assay together with Rab5-tagged cyan fluorescent protein (CFP). The presence of energy transfer between CFP-Rab5 and YFP-Rab5 binding fragments detected by sensitized FRET microscopy has validated the utility of these generated sensors to visualize the localization of GTP-bound Rab5. GTP-bound Rab5 was found in endosomes, often concentrated in distinct microdomains. Molecular architecture of the Rab5 microdomains was analyzed by three-chromophore FRET (3-FRET) microscopy, utilizing YFP, CFP, and monomeric red fluorescent proteins (mRFP.l). The results of the 3-FRET analysis suggest that GTP-bound Rab5 is capable of oligomerization and present in multiprotein complexes.

Original languageEnglish
Article number11
Pages (from-to)119-134
Number of pages16
JournalMethods in Enzymology
Volume403
DOIs
StatePublished - 2005

Bibliographical note

Funding Information:
This work was supported by grants from National Cancer Institute, National Institute of Drug Abuse (A.S.), and American Cancer Society (A.S. and E.G.), and a postdoctoral fellowship from the American Heart Association (to E.G.).

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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