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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Acknowledgments
This work was supported by grants SAF2009-11642 (financed in part by Plan E), and SAF2012-36519 (MINECO), RETIC RD07/0007/64 and PIE201020E031 to DPS, and by SAF2009-08086 to PB. CLO is the recipient of a fellowship from the FPI Program (MINECO).
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418_2012_1057_MOESM1_ESM.pptx
Fig. S1 Distribution of LTR, Lamp1-GFP and GFP-8. BAEC transiently transfected with Lamp1-GFP (a) or GFP-8 (b) were treated with the indicated agents in the absence of serum for 20 h. After treatment, cells were stained with LTR. Results shown are representative of three assays with similar results. Scale bar, 20 µm (PPTX 2049 kb)
418_2012_1057_MOESM2_ESM.pptx
Fig. S2 Kinetics and distribution of RFP-LC3 and Cyto-ID upon autophagy induction. a BAEC transiently transfected with RFP-LC3 were amino acid-starved (EBSS) for the indicated times. Individual sections are shown (top panels). Broad field images show heterogeneity in RFP-LC3 localization within the cell population (bottom panels), which switches from a mainly diffuse to a particulate pattern with longer EBSS incubation periods. b BAEC were incubated in EBSS for the indicated times and stained with Cyto-ID as described in Materials and methods. Scale bar, 20 µm (PPTX 977 kb)
418_2012_1057_MOESM3_ESM.pptx
Fig. S3 Distribution of MDC and Cyto-ID in BAEC. Cells were cultured in serum-free medium (control) or in EBSS for 2 h, after which, labelling with Cyto-ID and MDC was carried out as detailed in Materials and methods. MDC was added for the last 10 min of incubation with Cyto-ID. The patterns obtained with both dyes were undistinguishable in serum-free (figure) and in serum-containing medium (not shown). Images shown are representative from two independent experiments performed in duplicate and correspond to single confocal sections. Scale bar, 20 µm (PPTX 485 kb)
418_2012_1057_MOESM4_ESM.pptx
Fig. S4 Effect of U18666A on the distribution of Cyto-ID and lysosomal probes. Cells were incubated in the absence or presence of 10 μM U18666A for 20 h, after which cells were stained with Cyto-ID and LTR (a) or Lyso-ID (b). In (c) cells were stained only with Cyto-ID (left panels) or with Cyto-ID plus Lyso-ID (right panels) and images were obtained using conditions so that the Cyto-ID fluorescence in Cyto-ID/Lyso-ID-stained cells was close to background in order to maximize differences with the signal given by Cyto-ID alone (PPTX 1078 kb)
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Oeste, C.L., Seco, E., Patton, W.F. et al. Interactions between autophagic and endo-lysosomal markers in endothelial cells. Histochem Cell Biol 139, 659–670 (2013). https://doi.org/10.1007/s00418-012-1057-6
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DOI: https://doi.org/10.1007/s00418-012-1057-6