Histochemistry and Cell Biology

, Volume 139, Issue 5, pp 659–670 | Cite as

Interactions between autophagic and endo-lysosomal markers in endothelial cells

  • Clara L. Oeste
  • Esther Seco
  • Wayne F. Patton
  • Patricia Boya
  • Dolores Pérez-Sala
Original Paper


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.


Endo-lysosomal pathway Autophagy Amino acid deprivation Live cell fluorescence Autophagosome marker Endothelial cell 



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).

Supplementary material

418_2012_1057_MOESM1_ESM.pptx (2 mb)
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 (978 kb)
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 (485 kb)
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 (1.1 mb)
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)


  1. Alvarez M, Villanueva A, Acedo P, Canete M, Stockert JC (2011) Cell death causes relocalization of photosensitizing fluorescent probes. Acta Histochem 113:363–368. doi: 10.1016/j.acthis.2010.01.008 PubMedCrossRefGoogle Scholar
  2. Bampton ET, Goemans CG, Niranjan D, Mizushima N, Tolkovsky AM (2005) The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes. Autophagy 1:23–36PubMedCrossRefGoogle Scholar
  3. Barth S, Glick D, Macleod KF (2010) Autophagy: assays and artifacts. J Pathol 221:117–124. doi: 10.1002/path.2694 PubMedCrossRefGoogle Scholar
  4. Berg TO, Fengsrud M, Stromhaug PE, Berg T, Seglen PO (1998) Isolation and characterization of rat liver amphisomes. Evidence for fusion of autophagosomes with both early and late endosomes. J Biol Chem 273:21883–21892PubMedCrossRefGoogle Scholar
  5. Biederbick A, Kern HF, Elsasser HP (1995) Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 66:3–14PubMedGoogle Scholar
  6. Bucci C, Parton RG, Mather IH, Stunnenberg H, Simons K, Hoflack B, Zerial M (1992) The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell 70:715–728PubMedCrossRefGoogle Scholar
  7. Bucci C, Thomsen P, Nicoziani P, McCarthy J, van Deurs B (2000) Rab7: a key to lysosome biogenesis. Mol Biol Cell 11:467–480PubMedGoogle Scholar
  8. Chen Y, Klionsky DJ (2011) The regulation of autophagy—unanswered questions. J Cell Sci 124:161–170. doi: 10.1242/jcs.064576 PubMedCrossRefGoogle Scholar
  9. Coleman J, Xiang Y, Pande P, Shen D, Gatica D, Patton WF (2010) A live-cell fluorescence microplate assay suitable for monitoring vacuolation arising from drug or toxic agent treatment. J Biomol Screen 15:398–405PubMedCrossRefGoogle Scholar
  10. Du J, Teng RJ, Guan T, Eis A, Kaul S, Konduri GG, Shi Y (2012) Role of autophagy in angiogenesis in aortic endothelial cells. Am J Physiol Cell Physiol 302:C383–C391. doi: 10.1152/ajpcell.00164.2011 PubMedCrossRefGoogle Scholar
  11. Dunn WA Jr (1994) Autophagy and related mechanisms of lysosome-mediated protein degradation. Trends Cell Biol 4:139–143PubMedCrossRefGoogle Scholar
  12. Hernández-Perera O, Pérez-Sala D, Navarro-Antolín J, Sánchez-Pascuala R, Hernández G, Díaz C, Lamas S (1998) Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells. J Clin Invest 101:2711–2719PubMedCrossRefGoogle Scholar
  13. Ishibashi S, Yamazaki T, Okamoto K (2009) Association of autophagy with cholesterol-accumulated compartments in Niemann–Pick disease type C cells. J Clin Neurosci 16:954–959PubMedCrossRefGoogle Scholar
  14. Iwai-Kanai E, Yuan H, Huang C, Sayen MR, Perry-Garza CN, Kim L, Gottlieb RA (2008) A method to measure cardiac autophagic flux in vivo. Autophagy 4:322–329PubMedGoogle Scholar
  15. Jaiswal JK, Fix M, Takano T, Nedergaard M, Simon SM (2007) Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes. Proc Natl Acad Sci USA 104:14151–14156. doi: 10.1073/pnas.0704935104 PubMedCrossRefGoogle Scholar
  16. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720–5728. doi: 10.1093/emboj/19.21.5720 PubMedCrossRefGoogle Scholar
  17. Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS, Baba M, Baehrecke EH, Bahr BA, Ballabio A, Bamber BA, Bassham DC, Bergamini E, Bi X, Biard-Piechaczyk M, Blum JS, Bredesen DE, Brodsky JL, Brumell JH, Brunk UT, Bursch W, Camougrand N, Cebollero E, Cecconi F, Chen Y, Chin LS, Choi A, Chu CT, Chung J, Clarke PG, Clark RS, Clarke SG, Clave C, Cleveland JL, Codogno P, Colombo MI, Coto-Montes A, Cregg JM, Cuervo AM, Debnath J, Demarchi F, Dennis PB, Dennis PA, Deretic V, Devenish RJ, Di Sano F, Dice JF, Difiglia M, Dinesh-Kumar S, Distelhorst CW, Djavaheri-Mergny M, Dorsey FC, Droge W, Dron M, Dunn WA, Jr., Duszenko M, Eissa NT, Elazar Z, Esclatine A, Eskelinen EL, Fesus L, Finley KD, Fuentes JM, Fueyo J, Fujisaki K, Galliot B, Gao FB, Gewirtz DA, Gibson SB, Gohla A, Goldberg AL, Gonzalez R, Gonzalez-Estevez C, Gorski S, Gottlieb RA, Haussinger D, He YW, Heidenreich K, Hill JA, Hoyer-Hansen M, Hu X, Huang WP, Iwasaki A, Jaattela M, Jackson WT, Jiang X, Jin S, Johansen T, Jung JU, Kadowaki M, Kang C, Kelekar A, Kessel DH, Kiel JA, Kim HP, Kimchi A, Kinsella TJ, Kiselyov K, Kitamoto K, Knecht E, Komatsu M, Kominami E, Kondo S, Kovacs AL, Kroemer G, Kuan CY, Kumar R, Kundu M, Landry J, Laporte M, Le W, Lei HY, Lenardo MJ, Levine B, Lieberman A, Lim KL, Lin FC, Liou W, Liu LF, Lopez-Berestein G, Lopez-Otin C, Lu B, Macleod KF, Malorni W, Martinet W, Matsuoka K, Mautner J, Meijer AJ, Melendez A, Michels P, Miotto G, Mistiaen WP, Mizushima N, Mograbi B, Monastyrska I, Moore MN, Moreira PI, Moriyasu Y, Motyl T, Munz C, Murphy LO, Naqvi NI, Neufeld TP, Nishino I, Nixon RA, Noda T, Nurnberg B, Ogawa M, Oleinick NL, Olsen LJ, Ozpolat B, Paglin S, Palmer GE, Papassideri I, Parkes M, Perlmutter DH, Perry G, Piacentini M, Pinkas-Kramarski R, Prescott M, Proikas-Cezanne T, Raben N, Rami A, Reggiori F, Rohrer B, Rubinsztein DC, Ryan KM, Sadoshima J, Sakagami H, Sakai Y, Sandri M, Sasakawa C, Sass M, Schneider C, Seglen PO, Seleverstov O, Settleman J, Shacka JJ, Shapiro IM, Sibirny A, Silva-Zacarin EC, Simon HU, Simone C, Simonsen A, Smith MA, Spanel-Borowski K, Srinivas V, Steeves M, Stenmark H, Stromhaug PE, Subauste CS, Sugimoto S, Sulzer D, Suzuki T, Swanson MS, Tabas I, Takeshita F, Talbot NJ, Talloczy Z, Tanaka K, Tanida I, Taylor GS, Taylor JP, Terman A, Tettamanti G, Thompson CB, Thumm M, Tolkovsky AM, Tooze SA, Truant R, Tumanovska LV, Uchiyama Y, Ueno T, Uzcategui NL, van der Klei I, Vaquero EC, Vellai T, Vogel MW, Wang HG, Webster P, Wiley JW, Xi Z, Xiao G, Yahalom J, Yang JM, Yap G, Yin XM, Yoshimori T, Yu L, Yue Z, Yuzaki M, Zabirnyk O, Zheng X, Zhu X, Deter RL (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4:151–175Google Scholar
  18. Klionsky DJ et al (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8:445–454PubMedCrossRefGoogle Scholar
  19. Kobayashi T, Beuchat MH, Lindsay M, Frias S, Palmiter RD, Sakuraba H, Parton RG, Gruenberg J (1999) Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nat Cell Biol 1:113–118PubMedCrossRefGoogle Scholar
  20. Lee J, Giordano S, Zhang J (2012) Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling. Biochem J 441:523–540. doi: 10.1042/BJ20111451 PubMedCrossRefGoogle Scholar
  21. Liou W, Geuze HJ, Geelen MJ, Slot JW (1997) The autophagic and endocytic pathways converge at the nascent autophagic vacuoles. J Cell Biol 136:61–70PubMedCrossRefGoogle Scholar
  22. Mizushima N, Yoshimori T (2007) How to interpret LC3 immunoblotting. Autophagy 3:542–545PubMedGoogle Scholar
  23. Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140:313–326. doi: 10.1016/j.cell.2010.01.028 PubMedCrossRefGoogle Scholar
  24. Munafo DB, Colombo MI (2001) A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci 114:3619–3629PubMedGoogle Scholar
  25. Niemann A, Takatsuki A, Elsasser HP (2000) The lysosomotropic agent monodansylcadaverine also acts as a solvent polarity probe. J Histochem Cytochem 48:251–258PubMedCrossRefGoogle Scholar
  26. Pacheco CD, Kunkel R, Lieberman AP (2007) Autophagy in Niemann–Pick C disease is dependent upon Beclin-1 and responsive to lipid trafficking defects. Hum Mol Genet 16:1495–1503PubMedCrossRefGoogle Scholar
  27. Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877PubMedCrossRefGoogle Scholar
  28. Pérez-Sala D, Boya P, Ramos I, Herrera M, Stamatakis K (2009) The C-terminal sequence of RhoB directs protein degradation through an endo-lysosomal pathway. PLoS ONE 4(12):e8117PubMedCrossRefGoogle Scholar
  29. Saftig P, Klumperman J (2009) Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nat Rev Mol Cell Biol 10:623–635. doi: 10.1038/nrm2745 PubMedCrossRefGoogle Scholar
  30. Sato K, Tsuchihara K, Fujii S, Sugiyama M, Goya T, Atomi Y, Ueno T, Ochiai A, Esumi H (2007) Autophagy is activated in colorectal cancer cells and contributes to the tolerance to nutrient deprivation. Cancer Res 67:9677–9684. doi: 10.1158/0008-5472.CAN-07-1462 PubMedCrossRefGoogle Scholar
  31. Sobo K, Le Blanc I, Luyet PP, Fivaz M, Ferguson C, Parton RG, Gruenberg J, van der Goot FG (2007) Late endosomal cholesterol accumulation leads to impaired intra-endosomal trafficking. PLoS One 2(9):e851PubMedCrossRefGoogle Scholar
  32. Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 10:513–525. doi: 10.1038/nrm2728 PubMedCrossRefGoogle Scholar
  33. Valero RA, Oeste CL, Stamatakis K, Ramos I, Herrera M, Boya P, Pérez-Sala D (2010) Structural determinants allowing endo-lysosomal sorting and degradation of endosomal GTPases. Traffic 11:1221–1233PubMedCrossRefGoogle Scholar
  34. Wang Q, Liang B, Shirwany NA, Zou MH (2011) 2-Deoxy-d-glucose treatment of endothelial cells induces autophagy by reactive oxygen species-mediated activation of the AMP-activated protein kinase. PLoS One 6(2):e17234PubMedCrossRefGoogle Scholar
  35. Warenius HM, Kilburn JD, Essex JW, Maurer RI, Blaydes JP, Agarwala U, Seabra LA (2011) Selective anticancer activity of a hexapeptide with sequence homology to a non-kinase domain of cyclin dependent kinase 4. Mol Cancer 10:72. doi: 10.1186/1476-4598-10-72 PubMedCrossRefGoogle Scholar
  36. Yang Z, Klionsky DJ (2010) Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol 22:124–131PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Clara L. Oeste
    • 1
  • Esther Seco
    • 1
  • Wayne F. Patton
    • 2
  • Patricia Boya
    • 1
  • Dolores Pérez-Sala
    • 1
  1. 1.Centro de Investigaciones BiológicasConsejo Superior de Investigaciones CientíficasMadridSpain
  2. 2.Enzo Life SciencesFarmingdaleUSA

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