Please use this identifier to cite or link to this item:
http://hdl.handle.net/10261/40098
Share/Export:
![]() ![]() |
|
Visualizar otros formatos: MARC | Dublin Core | RDF | ORE | MODS | METS | DIDL | DATACITE | |
Title: | A proteolytic cleavage assay to monitor autophagy in Dictyostelium discoideum |
Authors: | Calvo-Garrido, Javier CSIC; Carilla-Latorre, Sergio CSIC; Mesquita, Ana; Escalante, Ricardo CSIC ORCID | Keywords: | Protein Macroautophagy Genes |
Issue Date: | 1-Sep-2011 | Publisher: | Landes Bioscience | Citation: | Autophagy 7(9): 1063-1068 (2011) | Abstract: | Dictyostelium discoideum is a good model of autophagy. However, the lack of autophagic flux techniques hinders the assessment of new mutants or drugs. One of these techniques, which has been used successfully in yeast and mammalian cells, but has not yet been described in Dictyostelium, is based on the presence of proteolytic fragments derived from autophagic degradation of expressed fusion proteins. Lysosomotropic agents such as NH(4)Cl penetrate acidic compartments and raise their pH, thus allowing the accumulation and measurement of these cleaved fragments, which otherwise would be rapidly degraded. We have used this property to detect the presence of free GFP fragments derived from the fusion protein GFP-Tkt-1, a cytosolic marker. We demonstrate that this proteolytic event is dependent on autophagy and can be used to detect differences in the level of autophagic flux among different mutant strains. Moreover, treatment with NH(4)Cl also facilitates the assessment of autophagic flux by confocal microscopy using the marker RFP-GFP-Atg8. | Publisher version (URL): | http://dx.doi.org/10.4161/auto.7.9.16629 | URI: | http://hdl.handle.net/10261/40098 | DOI: | 10.4161/auto.7.9.16629 | ISSN: | 1554-8627 | E-ISSN: | 1554-8635 |
Appears in Collections: | (IIBM) Artículos |
Files in This Item:
File | Description | Size | Format | |
---|---|---|---|---|
Autophagy 2011 vol7 n9 pp1063.pdf | 1,12 MB | Adobe PDF | ![]() View/Open |
Review this work
SCOPUSTM
Citations
30
checked on May 22, 2022
WEB OF SCIENCETM
Citations
27
checked on May 26, 2022
Page view(s)
365
checked on May 27, 2022
Download(s)
197
checked on May 27, 2022
Google ScholarTM
Check
Altmetric
Dimensions
WARNING: Items in Digital.CSIC are protected by copyright, with all rights reserved, unless otherwise indicated.