Visualization of Rab5 activity in living cells using FRET microscopy.
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.
Galperin
Emilia
E
Sorkin
Alexander
A
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D017931:DNA Primers; D011992:Endosomes / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D008856:Microscopy, Fluorescence / Q000379:methods*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D020696:rab5 GTP-Binding Proteins