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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

Abstract

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.

Keywords

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.

Notes

Acknowledgments

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

None.

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)
436_2012_3253_MOESM2_ESM.mov (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)
436_2012_3253_MOESM3_ESM.mov (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)

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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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