QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 20 March 2007
doi: 10.1242/jcs.03427

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rodríguez, M. Articles by Beaumelle, B. Search for Related Content PubMed PubMed Citation Articles by Rodríguez, M. Articles by Beaumelle, B.

Intracellular pathway of Onconase that enables its delivery to the cytosol

¹ Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi s/n E-17071 Girona, Spain
² UMR 5539 CNRS, Département Biologie-Santé, Université Montpellier II, 34095 Montpellier Cedex 05, France

View larger version (48K): [in this window] [in a new window]   Fig. 1. Onco enters cells using the clathrin-mediated pathway. (A) Inhibition of AP-2-dependent endocytosis prevents Onco internalization. Jurkat cells were transiently transfected with a vector coding for an EGFP-tagged protein (Eps15D32, Eps1595/295 or intersectin SH3A), or with both an EGFP vector and another coding for dynamin II-WT or dynamin II-K44A. After 24 hours, 250 nM Onco-Red and 100 nM Tf-Cy5 were added for 45 minutes. Cells were then washed, fixed and examined under a confocal microscope. Representative sections through the middle of the cell. Bar, 10 µm. (B) Onco is found within coated pits at the plasma membrane. Jurkat cells were labeled for 15 minutes at 37°C with Onco conjugated to 10-nm diameter colloidal gold. They were then cooled to 4°C, washed, fixed and processed for conventional electronmicroscopic examination. Bar, 50 nm. (C) Inhibition of clathrin-mediated endocytosis protects cells against Onco-induced apoptosis. Jurkat cells were transfected with a vector coding for EGFP alone or EGFP-tagged proteins, as indicated. After 24 hours, 6 µM Onco was added. Apoptosis was quantified 24 hours later by monitoring annexin-V-Cy5 binding to transfected cells by FACS. The results are expressed as the mean ± s.e.m. of three separate experiments.

View larger version (52K): [in this window] [in a new window]   Fig. 2. Onco intracellular pathway. HeLa cells were incubated for 45 minutes with Onco-Red and Tf-Cy5 (to label early endosomes). When indicated, cells were also labeled with dextran-FITC, a tracer that is addressed to lysosomes. Cells were then processed for immunodetection of markers for late endosome/lysosomes [Lamp-1, Lamp-2 or lysobisphosphatidic acid (LBPA)], the ER (calnexin) or the trans-Golgi network (TGN46), before mounting and observation under a confocal microscope. Median optical sections; bar, 10 µm. Boxed areas in LBPA and Lamp-2 images indicate regions enlarged on the right.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Onco endocytosis and toxicity are regulated by Rab5. (A) Onco internalization depends on Rab5. Jurkat cells were transfected with the indicated Rab5 or Rab7 expression vectors, together with EGFP. After 24 hours, Onco-Red and Tf-Cy5 were added for 45 minutes before fixation and confocal microscopic examination. Bar, 10 µm. (B) Cellubrevin and Rab5 regulate Onco delivery to the cytosol. Jurkat cells were transfected with EGFP and either the wild type or a mutated version of Rab5, Rab7 or TeNT-LC, as indicated. After 24 hours, 6 µM Onco was added. Cytotoxicity was measured after 24 hours, on transfected cells, using annexin-V-Cy5 labeling and FACS analysis.

View larger version (36K): [in this window] [in a new window]   Fig. 4. Onco toxicity and translocation are enhanced by endosome neutralization. (A) Cytotoxicity to HeLa and A431 cells. Cells were pre-incubated for 30 minutes with 0.25 µM monensin, 20 mM NH4Cl (Amm Cl) or 100 nM bafilomycin (Baf), as indicated, before adding various concentrations of Onco. Cell viability was measured after 24 hours (HeLa) or 36 hours (A431). The results are expressed as the Onco concentration generating a growth inhibition of 50% (IC50). Mean ± s.e.m. of at least three separate experiments. (B) Endosomal pH measurements. A431 cells were labeled for 40 minutes with Fl-Tf-Rh before acquiring Fl and Rh fluorescence images using a confocal microscope. The Fl/Rh intracellular intensity ratio was then used together with a calibration curve to determine the endosomal pH (Dunn et al., 1994; Presley et al., 1997) in living cells treated with the indicated drug. (C) Endosome neutralization enhances Onco translocation. 125I-Onco- or 125I-Tf-loaded lymphocyte endosomes were purified and resuspended in translocation buffer supplemented with 10 mM ATP. Translocation assays (Morlon-Guyot et al., 2003; Vendeville et al., 2004) were performed for 1 hour in the presence or absence of 400 nM Baf or 20 µM monensin, as indicated. Translocation activity corresponds to the time course increase in the supernatant/(endosome plus supernatant) radioactivity ratio. (D) Onco translocation requires cytosolic ATP hydrolysis. Translocation was assayed in the presence of Baf and 10 mM ATP or ATPS, as indicated.