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Interactions between autophagic and endo-lysosomal markers in endothelial cells. | LitMetric

Interactions between autophagic and endo-lysosomal markers in endothelial cells.

Histochem Cell Biol

Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.

Published: May 2013

AI Article Synopsis

  • Autophagic and endo-lysosomal pathways are important for maintaining cell balance, and effective tools are needed to study their roles in health and disease.
  • This study assesses the specificity of two probes, Lyso-ID for lysosomes and Cyto-ID for autophagosomes, by observing their behavior in live cells during changes in autophagy and endo-lysosomal function.
  • The findings indicate that both probes can successfully identify and co-localize with specific cellular markers, highlighting their potential for studying the interactions between autophagy and lysosomal pathways in real-time experiments.

Article Abstract

Autophagic and endo-lysosomal degradative pathways are essential for cell homeostasis. Availability of reliable tools to interrogate these pathways is critical to unveil their involvement in physiology and pathophysiology. Although several probes have been recently developed to monitor autophagic or lysosomal compartments, their specificity has not been validated through co-localization studies with well-known markers. Here, we evaluate the selectivity and interactions between one lysosomal (Lyso-ID) and one autophagosomal (Cyto-ID) probe under conditions modulating autophagy and/or endo-lysosomal function in live cells. The probe for acidic compartments Lyso-ID was fully localized inside vesicles positive for markers of late endosome-lysosomes, including Lamp1-GFP and GFP-CINCCKVL. Induction of autophagy by amino acid deprivation in bovine aortic endothelial cells caused an early and potent increase in the fluorescence of the proposed autophagy dye Cyto-ID. Cyto-ID-positive compartments extensively co-localized with the autophagosomal fluorescent reporter RFP-LC3, although the time and/or threshold for organelle detection was different for each probe. Interestingly, use of Cyto-ID in combination with Lysotracker Red or Lyso-ID allowed the observation of structures labeled with either one or both probes, the extent of co-localization increasing upon treatment with protease inhibitors. Inhibition of the endo-lysosomal pathway with chloroquine or U18666A resulted in the formation of large Cyto-ID and Lyso-ID-positive compartments. These results constitute the first assessment of the selectivity of Cyto-ID and Lyso-ID as probes for the autophagic and lysosomal pathways, respectively. Our observations show that these probes can be used in combination with protein-based markers for monitoring the interactions of both pathways in live cells.

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Source
http://dx.doi.org/10.1007/s00418-012-1057-6DOI Listing

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