title: High-throughput quantitation of intracellular trafficking and organelle disruption by flow cytometry.
abstract: Current methods for the quantitation of membrane protein trafficking rely heavily on microscopy, which has limited quantitative capacity for analyses of cell populations and is cumbersome to perform. Here we describe a simple flow cytometry-based method that circumvents these limitations. The method utilizes fluorescent pulse-width measurements as a highly sensitive indicator to monitor the changes in intracellular distributions of a fluorescently labelled molecule in a cell. Pulse-width analysis enabled us to discriminate cells with target proteins in different intracellular locations including Golgi, lyso-endosomal network and the plasma membrane, as well as detecting morphological changes in organelles such as Golgi perturbation. The movement of endogenous and exogenous retrograde cargo was tracked from the plasma membrane-to-endosomes-to-Golgi, by decreasing pulse-width values. A block in transport upon RNAi-mediated ablation of transport machinery was readily quantified, demonstrating the versatility of this technique to identify pathway inhibitors. We also showed that pulse-width can be exploited to sort and recover cells based on different intracellular staining patterns, e.g. early endosomes and Golgi, opening up novel downstream applications. Overall, the method provides new capabilities for viewing membrane transport in thousands of cells per minute, unbiased analysis of the trafficking of cargo, and the potential for rapid screening of inhibitors of trafficking pathways.
authors.affiliation: The Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, 3010, Australia.
authors.forename: Pei Zhi Cheryl
authors.initials: PZ
authors.lastname: Chia
authors.forename: Yasmin M
authors.initials: YM
authors.lastname: Ramdzan
authors.forename: Fiona J
authors.initials: FJ
authors.lastname: Houghton
authors.forename: Danny M
authors.initials: DM
authors.lastname: Hatters
authors.forename: Paul A
authors.initials: PA
authors.lastname: Gleeson
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D011992:Endosomes / Q000378:metabolism* / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism* / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology*
chemical_list: D008565:Membrane Proteins
keywords: Golgi; flow cytometry; fluorescence pulse-shape; intracellular location; membrane transport; retrograde transport