Parasitology Research

, Volume 112, Issue 4, pp 1813–1818 | Cite as

Receptor-mediated endocytosis and trafficking between endosomal–lysosomal vacuoles in Giardia lamblia

  • Maria R. Rivero
  • Ignacio Jausoro
  • Mariano Bisbal
  • Constanza Feliziani
  • Adriana Lanfredi-Rangel
  • Maria C. TouzEmail author
Short Communication


The early branching Giardia lamblia has highly polarized vacuoles, located underneath the plasma membrane, which have at least some of the characteristics of endosomes and of lysosomes. These peripheral vacuoles (PVs) are necessary for nutrient uptake and the maintenance of plasma membrane composition, but whether they carry out sorting and segregation of receptors and ligands is a matter of debate. Here, we showed that the internalization of low-density lipoprotein (LDL) to the PVs is highly dynamic in trophozoites with a rate similar to the internalization of the low-density lipoprotein receptor-related protein 1. Moreover, by analyzing receptor-mediated and fluid-phase endocytosis in living cells, we showed that after endocytosis LDL but not dextran moved laterally between the PVs. We speculate on PV functional heterogeneity and maturation in this parasite.


Giardia Lamblia Total Internal Reflection Fluorescence Microscopy Chroma Technology Numerical Aperture Objective Giardia Trophozoite 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The project was supported by grant number R01TW00724 from the Fogarty International Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Fogarty International Center or the National Institutes of Health. This research was also supported in part by the Argentine Agencia Nacional para la Promoción de la Ciencia y Tecnología (FONCyT-PICT698).

Conflict of interest


Supplementary material

436_2012_3253_Fig6_ESM.gif (0 kb)
Fig. S1

Total Internal Reflection Microscopy: Basic concept. Schematic illustration showing the internalization of fluorescent molecules in a living Giardia trophozoite. Trophozoites were grown in complete medium, suspended in 1 ml of labeling buffer (50 mM glucose, 10 mM cysteine, 2 mM ascorbic acid in PBS, pH 7.2), and attached to polylysine-treated cover slips for 30 min at 37 °C. 7.5 μg of BODIPY-LDL (Molecular Probes, Invitrogen) was loaded before chamber assembly. Live trophozoites were observed using a ×60 1.45 numerical aperture objective equipped for through-the-objective TIRF illumination (for TIRFM assays) using a 488-nm argon laser on a Nikon TE2000-U microscope with filter cubes optimized for fluorescein/GFP (Chroma Technology, Rockingham, VT). When the incidence angle of laser excitation is the correct one to entirely reflect back the illuminating beam, specific fluorescent excitation (evanescent wave) is induced in a very thin optical section from the glass surface (≤100 nm in depth). This evanescent wave is an electromagnetic field which decays exponentially, thus only the fluorophores nearest the glass surface are selectively excited (

), and fluorophores located outside the field ( ) do not compose TIRF images (Adapted from Axelrod 2003). Images were captured with a cooled CCD ORCA II-ER (Hamamatsu) camera and MetaMorph software (Molecular Devices). The uptake events were captured 5 min post-BODYPI-LDL addition, with images being taken at the rate of 1 frame per 5 s. The same imaging parameters were used to photograph trophozoites without BODIPY-LDL as a control for autofluorescence (not shown). Other controls include the use of 20,000 MW FITC-dextran (Sigma-Aldrich) (does not adsorb to the surface, not shown) and BODIPY-LDL addition to fixed trophozoites (does adsorb to the surface but is not internalized, M2). The same procedure was used for time-lapse epifluorescence recording, just by configuring the TE2000 platform for epifluorescence illumination. These experiments were performed in triplicate (JPEG 15 kb)

436_2012_3253_MOESM1_ESM.tif (244 kb)
High resolution image (TIFF 243 kb) (3.4 mb)
Movie 1 Detailed picture of BODIPY-LDL uptake in living Giardia trophozoites. The TIRFM images depicted in pseudocolor detail the LDL internalization and indicate the estimation of the surface intensity: bright red areas represent high LDL cluster formation at the surface of the plasma membrane, and as the LDL molecules are endocytosed, this color becomes yellow and finally dark blue when PV delivery occurs (M1: ∼3.5-Mb QuickTime movie) (MOV 3444 kb) (849 kb)
Movie 2 BODIPY-LDL imaging near the surface of fixed trophozoite. Binding but not internalization of BODIPY-LDL is observed. The pattern of intensity is low (blue) and uniform in all cells. One trophozoite is shown as an example (M2: ∼3.5-Mb QuickTime movie) (MOV 848 kb)
Movie 3

Uptake and PV-to-PV movement is observed in live Giardia trophozoites by epifluorescence microscopy. (M3: ∼4-Mb QuickTime movie) (MOV 4689 kb)


  1. Axelrod D (2003) Total internal reflection fluorescence microscopy in cell biology. Methods Enzymol 361:1–33PubMedCrossRefGoogle Scholar
  2. Bisbal M, Conde C, Donoso M, Bollati F, Sesma J, Quiroga S, Diaz Anel A, Malhotra V, Marzolo MP, Caceres A (2008) Protein kinase d regulates trafficking of dendritic membrane proteins in developing neurons. J Neurosci 28:9297–9308PubMedCrossRefGoogle Scholar
  3. Bu G, Maksymovitch EA, Geuze H, Schwartz AL (1994) Subcellular localization and endocytic function of low density lipoprotein receptor-related protein in human glioblastoma cells. J Biol Chem 269:29874–29882PubMedGoogle Scholar
  4. Dupraz S, Grassi D, Bernis ME, Sosa L, Bisbal M, Gastaldi L, Jausoro I, Caceres A, Pfenninger KH, Quiroga S (2009) The TC10-Exo70 complex is essential for membrane expansion and axonal specification in developing neurons. J Neurosci 29:13292–13301PubMedCrossRefGoogle Scholar
  5. Feely DE, Dyer JK (1987) Localization of acid phosphatase activity in Giardia lamblia and Giardia muris trophozoites. J Protozool 34:80–83PubMedGoogle Scholar
  6. Gaechter V, Schraner E, Wild P, Hehl AB (2008) The single dynamin family protein in the primitive protozoan Giardia lamblia is essential for stage conversion and endocytic transport. Traffic 9:57–71PubMedCrossRefGoogle Scholar
  7. Goldstein JL, Basu SK, Brown MS (1983) Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol 98:241–260PubMedCrossRefGoogle Scholar
  8. House SA, Richter DJ, Pham JK, Dawson SC (2011) Giardia flagellar motility is not directly required to maintain attachment to surfaces. PLoS Pathog 7:e1002167PubMedCrossRefGoogle Scholar
  9. Kattenbach WM, Pimenta PF, De Souza W, Pinto Da Silva P (1991) Giardia duodenalis: a freeze-fracture, fracture-flip and cytochemistry study. Parasitol Res 77:651–658PubMedCrossRefGoogle Scholar
  10. Keister DB (1983) Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans R Soc Trop Med Hyg 77:487–488PubMedCrossRefGoogle Scholar
  11. Lanfredi-Rangel A, Attias M, De Carvalho TM, Kattenbach WM, De Souza W (1998) The peripheral vesicles of trophozoites of the primitive protozoan Giardia lamblia may correspond to early and late endosomes and to lysosomes. J Struct Biol 123:225–235PubMedCrossRefGoogle Scholar
  12. Lindmark DG (1988) Giardia lamblia: localization of hydrolase activities in lysosome-like organelles of trophozoites. Exp Parasitol 65:141–147PubMedCrossRefGoogle Scholar
  13. Nash TE, Aggarwal A, Adam RD, Conrad JT, Merritt JW Jr (1988) Antigenic variation in Giardia lamblia. J Immunol 141:636–641PubMedGoogle Scholar
  14. Rivero MR, Miras SL, Quiroga R, Ropolo AS, Touz MC (2011) Giardia lamblia low-density lipoprotein receptor-related protein is involved in selective lipoprotein endocytosis and parasite replication. Mol Microbiol 79:1204–1219PubMedCrossRefGoogle Scholar
  15. Rivero MR, Vranych CV, Bisbal M, Maletto BA, Ropolo AS, Touz MC (2010) Adaptor protein 2 regulates receptor-mediated endocytosis and cyst formation in Giardia lamblia. Biochem J 428:33–45PubMedCrossRefGoogle Scholar
  16. Touz MC (2012) The unique endosomal/lysosomal system of Giardia lamblia in molecular regulation of endocytosis, Vol. 1 (ed. Ceresa, B.), InTech, Croatia.pp. Accessed 6 July 2012
  17. Touz MC, Lujan HD, Hayes SF, Nash TE (2003) Sorting of encystation-specific cysteine protease to lysosome-like peripheral vacuoles in Giardia lamblia requires a conserved tyrosine-based motif. J Biol Chem 278:6420–6426PubMedCrossRefGoogle Scholar
  18. Touz MC, Nores MJ, Slavin I, Carmona C, Conrad JT, Mowatt MR, Nash TE, Coronel CE, Lujan HD (2002) The activity of a developmentally regulated cysteine proteinase is required for cyst wall formation in the primitive eukaryote Giardia lamblia. J Biol Chem 277:8474–8481PubMedCrossRefGoogle Scholar
  19. Ward W, Alvarado L, Rawlings ND, Engel JC, Franklin C, Mckerrow JH (1997) A primitive enzyme for a primitive cell: the protease required for excystation of Giardia. Cell 89:437–444PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Maria R. Rivero
    • 1
    • 3
  • Ignacio Jausoro
    • 1
  • Mariano Bisbal
    • 1
  • Constanza Feliziani
    • 1
  • Adriana Lanfredi-Rangel
    • 2
  • Maria C. Touz
    • 1
    Email author
  1. 1.Instituto de Investigación Médica Mercedes y Martín Ferreyra, INIMEC–CONICETUniversidad Nacional de CórdobaCórdobaArgentina
  2. 2.Serviço de Microscopia Eletrônica, Centro de Pesquisas Gonçalo MonizFIOCRUZ-BASalvadorBrazil
  3. 3.Dto. de Cs. Naturales, Fac. de Cs. Exactas Fisicoquímicas y NaturalesUniversidad Nacional de Rio CuartoRio CuartoArgentina

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