title: Real-time imaging of Rab5 activity using a prequenched biosensor.
abstract: A key regulator of receptor-mediated endocytosis, Rab5, plays a pivotal role in cargo receptor internalization, endosomal maturation, and transduction and degradation of internalized signaling molecules and recycling cargo receptor. Stressful conditions within cells lead to increased Rab5 activation, and increasing evidence correlates Rab5 activity abnormalities with certain diseases. Current antibody-based imaging methods cannot distinguish active Rab5 from total Rab5 population and provide dynamic information on magnitude and duration of Rab5 activation in cellular events and pathogenesis. We report here novel molecular imaging probes that specifically target GTP-bound Rab5 associated with the early endosome membrane in live cells and fixed mouse brain tissues. Our Rab5 activity fluorescent biosensor (RAFB) contains the Rab5 binding domain of the Rab5 effector Rabaptin 5, a fluorophore (a quantum dot or fluorescent dye) and a cell-penetrating peptide for live-cell delivery. The quantum dot conjugated RAFB was able to image the elevated Rab5 activity in both the cortex and hippocampi tissues of a Ts65Dn mouse. A prequenched RAFB based on fluorescence resonance energy transfer (FRET) can image cytosolic active Rab5 in single live cells. This novel method should enable imaging of the biological process in which Rab5 activity is regulated in various cellular systems.
authors.affiliation: Molecular Imaging Program at Stanford, Department of Radiology, Stanford University School of Medicine, 1201 Welch Road, California 94305-5484, United States.
authors.forename: Ke
authors.initials: K
authors.lastname: Zhan
authors.forename: Hexin
authors.initials: H
authors.lastname: Xie
authors.forename: Jessica
authors.initials: J
authors.lastname: Gall
authors.forename: Manlung
authors.initials: M
authors.lastname: Ma
authors.forename: Oliver
authors.initials: O
authors.lastname: Griesbeck
authors.forename: Ahmad
authors.initials: A
authors.lastname: Salehi
authors.forename: Jianghong
authors.initials: J
authors.lastname: Rao
mesh_terms: D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques; D001921:Brain / Q000378:metabolism; D057846:Cell-Penetrating Peptides / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D051379:Mice; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering; D045663:Quantum Dots; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*
chemical_list: D057846:Cell-Penetrating Peptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins; D016650:Fluorescein-5-isothiocyanate