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A proteolytic cleavage assay to monitor autophagy in Dictyostelium discoideum

Calvo-Garrido, Javier et al.

Autophagy -- Taylor & Francis -- Volume: 7 Issue: 9; (pages 1063-1068) -- 2011

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  • Title:
    A proteolytic cleavage assay to monitor autophagy in Dictyostelium discoideum
  • Author: Calvo-Garrido, Javier;
    Carilla-Latorre, Sergio;
    Mesquita, Ana;
    Escalante, Ricardo
  • Found In: Autophagy. Volume 7:Number 9(2011); 20110901; 1063-1068
  • Journal Title: Autophagy
  • Subjects: Autophagic vacuoles; Apoptosis; Cell death; Lysosomes; Degeneration (Pathology); Autophagy; Cell Death; Lysosomes; LCSH: Autophagic vacuoles; LCSH: Apoptosis; LCSH: Cell death; LCSH: Lysosomes; LCSH: Degeneration (Pathology); Dewey: 571.936
  • Rights: Licensed
  • Publication Details: Taylor & Francis
  • Abstract:

    Dictyostelium discoideumis 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 NH4Cl 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 NH4Cl also facilitates the assessment of autophagic flux by confocal microscopy using the marker RFP-GFP-Atg8.

  • Identifier: Journal ISSN: 1554-8627
  • Publication Date: 2011
  • Physical Description: Electronic
  • Shelfmark(s): 1835.065800
    ELD Digital store
  • UIN: ETOCvdc_100053902525.0x000001

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