Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast
| Autor: | Beverly Wendland, Derek C. Prosser, Allyson F. O'Donnell, Kristie Wrasman, Thaddeus K Woodard |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Endosome General Chemical Engineering Endocytic cycle Population Vacuole Biology Endocytosis General Biochemistry Genetics and Molecular Biology Green fluorescent protein 03 medical and health sciences Lysosome medicine Fluorescence microscope education Molecular Biology education.field_of_study General Immunology and Microbiology General Neuroscience Cell biology Protein Transport 030104 developmental biology medicine.anatomical_structure Microscopy Fluorescence Saccharomycetales Vacuoles |
| Zdroj: | Journal of visualized experiments : JoVE. (116) |
| ISSN: | 1940-087X |
| Popis: | Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry. |
| Databáze: | OpenAIRE |
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