Imaging the Intracellular Trafficking of APP with Photoactivatable GFP
| Autor: | Joshua H K Tam, Stephen H. Pasternak |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Endosome
General Chemical Engineering Green Fluorescent Proteins Golgi Apparatus Endosomes General Biochemistry Genetics and Molecular Biology Green fluorescent protein Amyloid beta-Protein Precursor Mice symbols.namesake Lysosome mental disorders medicine Amyloid precursor protein Animals Late endosome General Immunology and Microbiology biology General Neuroscience Cell Membrane Golgi apparatus Photochemical Processes Subcellular localization Cell biology Protein Transport Cellular Biology medicine.anatomical_structure Microscopy Fluorescence Membrane protein biology.protein symbols Amyloid Precursor Protein Secretases Lysosomes |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| Popis: | Beta-amyloid (Aβ) is the major constituent of senile plaques found in the brains of Alzheimer's disease patients. Aβ is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by β and γ-secretases. Despite the importance of Aβ to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied. While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the γ-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells. We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments. |
| Databáze: | OpenAIRE |
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