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Illuminating Autophagy in Bovine Aortic Cells With Fluorescent Dyes

Degradation of cellular components is essential for homeostasis, and this complex highly regulated process involves both the endo-lysosomal and autophagic pathways. Researchers studying these pathways have a variety of reagents and reporter systems at their disposal, each with inherent advantages and limitations. In their recent publication in Histochemistry and Cell Biology, Oeste and co-workers investigated Enzo’s Cyto-ID® and Lyso-ID® dyes in bovine aortic endothelial cells (BAEC) to detect autophagic and endo-lysosomal vesicles, respectively. Their results must be taken in the context of the cell lines and conditions used, but their findings illuminate the utility and advantages of these selective dyes as alternatives to, or in combination with, LC3 reporter systems or dyes such as monodansyl cadaverine (MDC). Significant findings included:

  • Lyso-ID signal co-localized with both LampI-GFP and GFP8, a GFP construct containing the endolysosomal targeting signal of RhoB GTPase. Co-localization persisted when cells were treated with chloroquine or U18666A, a compound that disrupts endosome dynamics.
  • Using BAECs transduced with RFP-LC3, and inducing autophagy by amino acid starvation, the researchers showed extensive co-localization of Cyto-ID autophagy dye with RFP-LC3. The co-localization was more pronounced when inhibitors of lysosomal proteinases were added to prevent degradation of the RFP-LC3. Upon starvation, Cyto-ID signal preceded the formation of punctate signals by RFP-LC3, providing earlier detection of autophagic vesicle formation. Cyto-ID signal was shown to be responsive to the autophagy inducer Rapamycin, and to the autophagy inhibitor 3-methyladenine (3-ma) during starvation.
  • The authors noted reduced background staining of Cyto-ID compared to MDC, showing significant basal level MDC signal without induction of autophagy. Bampton and co-workers (2005) have previously proposed that MDC accumulates in acidic compartments due to ion trapping, yielding significant background signal. Following induction of autophagy, only partial overlap of MDC and Cyto-ID signal was seen, reflecting the more specific labeling of Cyto-ID dye.
  • Using Cyto-ID and Lyso-ID dyes in combination, Oeste and co-workers displayed a predictable co-localized signal resulting from autophagosome-lysosome fusion events, as well as unique subpopulations of Cyto-ID and Lyso-ID stained organelles.

Enzo Life Sciences offers an unrivaled portfolio of tools for autophagy research, including live cell analysis kits, immunoassays, compound libraries, and antibodies, some of which are briefly described below.

 

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