title: Rab-NANOPS: FRET biosensors for Rab membrane nanoclustering and prenylation detection in mammalian cells.
abstract: Rab proteins constitute the largest subfamily of Ras-like small GTPases. They are central to vesicular transport and organelle definition in eukaryotic cells. Unlike their Ras counterparts, they are not a hallmark of cancer. However, a number of diseases, including cancer, show a misregulation of Rab protein activity. As for all membrane-anchored signaling proteins, correct membrane organization is critical for Rabs to operate. In this chapter, we provide a detailed protocol for the use of a flow cytometry-based Fluorescence Resonance Energy Transfer (FRET)-biosensors assay, which allows to detect changes in membrane anchorage, subcellular distribution, and of the nanoscale organization of Rab-GTPases in mammalian cell lines. This assay is high-throughput amenable and can therefore be utilized in chemical-genomic and drug discovery efforts.
authors: Turku Centre for Biotechnology, Åbo Akademi University, Tykistökatu 6B, 20520, Turku, Finland.
authors: Arafath Kaja
authors: AK
authors: Najumudeen
authors: Camilo
authors: C
authors: Guzmán
authors: Itziar M D
authors: IM
authors: Posada
authors: Daniel
authors: D
authors: Abankwa
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D036103:Nanotechnology / Q000379:methods*; D017368:Protein Prenylation; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D019883:Alkyl and Aryl Transferases; C075607:Rab geranylgeranyltransferase; D020691:rab GTP-Binding Proteins