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

This Article Summary Full Text Full Text (PDF) 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 Savina, A. Articles by Colombo, M. I. Search for Related Content PubMed PubMed Citation Articles by Savina, A. Articles by Colombo, M. I.

The exosome pathway in K562 cells is regulated by Rab11

¹ Laboratorio de Biología Celular y Molecular-Instituto de Histología y Embriología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo-CONICET, Mendozam 5500, Argentina
² UMR 5539, Université Montpellier II, Montpellier 34095, France

View larger version (47K): [in a new window]   Fig. 1. Subcellular distribution of GFP-Rab11 in K562 cells. K562 cells were transfected with the plasmid pEGFP (control) or with the constructs pEGFP-Rab11wt, pEGFP-Rab11Q70L and pEGFP-Rab11S25N. The stably transfected cells were mounted on coverslips and immediately analyzed by fluorescence microscopy without fixation using an inverted microscope (Nikon Eclipse TE 300, Germany) equipped with a filter system (excitation filter 450-490, barrier filter 515). Images were obtained with a CCD camera (Orca I, Hamamatsu) and processed using MetaMorph 4.5 (Universal Images Corporation).

View larger version (25K): [in a new window]   Fig. 2. Expression levels of GFP-Rab11 wt and mutants. Stably transfected K562 cells (1x105 cells) were lysed with PBS containing 1% Triton X100. Samples were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane as described in the Materials and Methods. The membrane was incubated with a rabbit anti-Rab11 serum and a corresponding HRP-labeled secondary antibody and developed with a enhanced chemiluminescence detection kit (Pierce). The intensity of the bands was quantified by densitometry and was expressed as arbitrary units.

View larger version (67K): [in a new window]   Fig. 3. Colocalization of GFP-Rab11 wt or mutants with rhodamine-transferrin. Cells were incubated in RPMI (serum free) containing 20 µg/ml human Tf (tetramethyl rhodamine-conjugated) for 45 minutes at 37°C and washed twice with ice-cold PBS. Cells were mounted on coverslips and immediately analyzed by fluorescence microscopy. Images were obtained with a CCD camera (Orca I, Hamamatsu) and processed using MetaMorph 4.5 (Universal Images Corporation). Left panels: GFP-Rab11wt and mutants. Middle panels: Rhodamine-transferrin (Rh-Tf). Right panels: overlay. In the first three panels, the focus was set on the small Rab11 structures that colocalize with the internalized Rh-Tf. In contrast, in the bottom panels the focus was set on the large ring-shaped structures formed in cells transfected with the mutant Q70L. Rh-Tf was not detected in these structures.

View larger version (51K): [in a new window]   Fig. 4. The large ring-shaped vesicles observed in the GFP-Rab11Q70L-transfected cells are labeled with Bodipy-ceramide. Stably transfected cells overexpressing the mutant GFP-Rab11Q70L were labeled with Lysotracker (Lys) or monodansylcadaverine (MDC) as described in the Materials and Methods. Late endosomes were labeled using an antibody against the mannose-6-phosphate receptor (M6PR) and a Texas-Red-conjugated goat-anti rabbit secondary antibody. Labeling with Bodipy-TR ceramide (B-Cer) was performed as described in the Materials and Methods. Cells were mounted on coverslips and analyzed by fluorescence microscopy. Images were obtained as described in Fig. 3.

View larger version (54K): [in a new window]   Fig. 5. Monensin causes the formation of large MVBs labeled with the trans-Golgi marker TGN46. (A) Stably transfected K562 cells overexpressing Rab11Q70L were incubated with monensin (1 µM) for 4 hours and with the fluorescent lipid analog N-(lissamine rhodamine B sulfonyl) phosphatidyl ethanolamine (N-Rh-PE) to label exosomes. The lipid was internalized via endocytosis. Cells were mounted on coverslips and immediately analyzed by fluorescence microscopy. Images were obtained as described in Fig. 3. Two sets of images show MVBs that are labeled with the lipid N-Rh-PE (depicted in red) and decorated with GFP-Rab11Q70L (green). (B) Cells overexpressing Rab11Q70L were incubated with monensin (1 µM) for 4 hours. Cells were mounted on coverslips, fixed and subjected to immunofluorescence with a rabbit antibody against TGN46 and a Texas-Red-conjugated goat-anti rabbit secondary antibody. Cells were analyzed by fluorescence microscopy. Two sets of images clearly show that TGN46 (red) colocalizes with the ring-shaped structures decorated by GFP-Rab11Q70L.

View larger version (14K): [in a new window]   Fig. 6. Overexpression of GFP-Rab11wt and mutants inhibits transferrin recycling. Cells were first pre-incubated for 1 hour at 37°C in serum-free RPMI medium supplemented with 1% BSA to deplete endogenous transferrin (Tf) and then were allowed to internalize 125I-labeled human Tf continuously for 1 hour at 37°C. Cells were washed at 4°C, and surface-bound transferrin was dissociated by acid treatment as described in the Materials and Methods. Cells were then further incubated at 37°C in serum-free RPMI medium containing 100 µM deferoxamine mesylate and 20 µg/ml unlabeled human Tf. At different times, the incubations were stopped by adding ice-cold PBS and placing the samples on ice. The samples were centrifuged to sediment the cells, and the medium was collected. The cell pellet was washed with the acetate buffer to remove surface-bound Tf. The radioactivity in both the medium and the cell pellet was determined by -counting. Data represent the mean±s.e.m. of three independent experiments.

View larger version (18K): [in a new window]   Fig. 7. Surface transferrin receptor measurements. Ligand binding studies were performed as described in the Materials and Methods. Briefly, control cells transfected with the vector alone (white bar) or with Rab11 wt (black bar) or mutants (hatched bars) were incubated with increasing concentrations of 125I-transferrin for 90 minutes at 4°C. Cells were washed, and binding was determined by spinning the cells through dibutyl phthalate oil. Nonspecific binding was determined and subtracted for each point. Specific binding data were analyzed by the Scatchard method to determine the number of Tf-binding sites. The data represent the mean±s.e.m. of four independent experiments. Statistical analysis: t-test for single group mean. *Mean differs from control P<0.05; **mean differs from control P<0.01.

View larger version (37K): [in a new window]   Fig. 8. Quantitation of exosome release by western blot and by measuring exosome-associated acetyl-cholinesterase and N-Rh-PE. (A) Samples of the exosomal fraction (see Materials and Methods) were solubilized in reducing SDS loading buffer, boiled and subjected to SDS-PAGE. The proteins were transferred to nitrocellulose membranes and detected with antibodies against the transferrin receptor (TfR), the protein Lyn and the heat shock protein Hsc70, respectively. The corresponding bands were detected using an enhanced chemiluminescence detection kit. A shows representative western blots of at least three independent experiments. Densitometric quantitation of the bands is depicted in B. Data are the mean±s.e.m. of three experiments expressed as a percentage of the control cells transfected with the vector alone. *P<0.05. (C) An aliquot of the exosome fraction (see Materials and Methods) was suspended in 100 µl phosphate buffer and incubated with 1.25 mM acetylthiocholine and 0.1 mM 5,5'-dithio-bis(2-nitrobenzoic acid) in a final volume of 1 ml. The incubation was carried out in cuvettes at 37°C, and the change in absorbance at 412 nm was followed continuously for 30 minutes. Data were normalized as the percentage of the maximal activity measured at 30 minutes in the vector-transfected cells. Data represent the mean±s.e.m. of four independent experiments. (D) Cells were labeled for 60 minutes at 4°C with the fluorescent lipid analog N-(lissamine rhodamine B sulfonyl) phosphatidyl ethanolamine (N-Rh-PE) as described in the Materials and Methods. Cells were extensively washed with cold PBS to remove excess unbound lipid and were then cultured in complete RPMI medium to collect exosomes. A sample of the exosomal fraction was suspended in 1.5 ml PBS-TX100 (0.1%) and N-Rh-PE was measured by fluorometry. Data represent the mean±s.e.m. of three independent experiments expressed as a percentage of the control (vector transfected cells). Statistical analysis: t-test for single group mean. *Mean differs from control P<0.05; **mean differs from control P<0.01.

View larger version (61K): [in a new window]   Fig. 9. Transferrin-coated colloidal gold is targeted to MVBs in K562 cells. Stably transfected K562 cells overexpressing GFP-Rab11 wt (A,B) or Rab11 S25N (C) were incubated with 10 nm Tf-coated colloidal gold particles (Tf-gold) for 45 minutes at 37°C and processed for transmission electron microscopy following standard techniques (see the Materials and Methods). (A) A typical multivesicular body (MVB) that contains vesicles labeled with Tf-gold is shown. A well defined membrane can often be seen surrounding these vesicles but since exosomes are small the large degree of curvature of this membrane might not be well cross-sectioned and consequently would not always be discernable. Magnification: 100,000x. (B) Two MVBs are shown, one of them has already fused with the plasma membrane, and Tf-receptor-containing exosomes are shed from the cell. Magnification: 100,000x. (C,D) Sections of a Rab11-S25N-transfected cell showing MVBs containing vesicles labeled with Tf-gold are depicted. Magnification: 110,000x.