Quantitative Super-Resolution Imaging of ER-Phagy Initiation in Cells.
DNA-PAINT
ER-phagy
Exchange-PAINT
Quantitative microscopy
Super-resolution microscopy
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2024
2024
Historique:
medline:
8
8
2024
pubmed:
8
8
2024
entrez:
8
8
2024
Statut:
ppublish
Résumé
Selective autophagy of the endoplasmic reticulum (ER-phagy) is a mechanism that is necessary for degrading damaged ER components and preventing cells from experiencing ER stress. Various ER-phagy receptors orchestrate this process by building protein assemblies with dedicated functions. In order to understand the molecular building principles of ER-phagy, it is important to reveal the assembly of ER-phagy receptors in a temporal and functional context. However, direct visualization is hampered by the diffraction limit in light microscopy. Super-resolution microscopy (SRM) can bypass this limitation and resolve single proteins and nanoscale protein clusters in cells. In particular, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful technology that can resolve individual protein clusters in cells and provide information on their molecular composition. Here, we report a step-by-step protocol on how to utilize DNA-PAINT to perform super-resolution imaging of ER-phagy receptors in fixed cells. In addition, we provide a detailed explanation of image generation, cluster analysis, and molecular quantification.
Identifiants
pubmed: 39115662
doi: 10.1007/978-1-0716-4067-8_10
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
127-140Informations de copyright
© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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