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First published online 30 September 2008
doi: 10.1242/jcs.031484

Journal of Cell Science 121, 3445-3458 (2008)
Published by The Company of Biologists 2008
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Sorting of EGF and transferrin at the plasma membrane and by cargo-specific signaling to EEA1-enriched endosomes

Deborah Leonard1, Akira Hayakawa1, Deirdre Lawe1, David Lambright1, Karl D. Bellve2, Clive Standley2, Lawrence M. Lifshitz2, Kevin E. Fogarty2 and Silvia Corvera1,*

1 Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
2 Biomedical Imaging Group, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA


Figure 1
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  		Fig. 1. Binding and internalization of EGF and Tf in COS-7 cells. COS-7 cells grown on coverslips were placed in KRH buffer at 35°C, and exposed to 50 ng/ml Alexa-Fluor-568-EGF and 20 µg/ml Alexa-Fluor-488-Tf. Image capture was started immediately after ligand addition. Background is pseudo colored in yellow to enhance low level signal. Colocalized signal (arrows) is pseudo colored in white. The complete time series can be seen in Movie 1 in supplementary material.

 

Figure 2
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  		Fig. 2. Imaging of ligand pairs conjugated to different fluorophores. (A) COS-7 cells grown on coverslips were placed in KRH buffer at 35°C, and exposed to 10 µg/ml Alexa-Fluor-488-Tf + 10 µg/ml Alexa-Fluor-568-Tf (top panels), 50 ng/ml Alexa-Fluor-568-EGF + 50 ng/ml Alexa-Fluor-488-EGF (middle panels), or 50 ng/ml Alexa-Fluor-568-EGF + 10 µg/ml Alexa-Fluor-488-Tf (bottom panels) for 5 minutes. (Right panels) Merged images showing the overlap. (B) Images from the time periods after addition of ligands indicated above the panels. Arrows indicate the clear segregation of signals seen in cells incubated with different ligands, that is not seen with the same ligand conjugated to two different fluorophores. (C) Quantification of signal overlap between fluorophores after 5 minutes of exposure. Values are mean ± s.e.m. of five different experiments. Statistical significance of the differences between groups was estimated using two-tailed Student's t-tests.

 

Figure 3
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  		Fig. 3. Quantification of the dynamics of EGF and Tf binding, internalization and colocalization. COS-7 cells grown on coverslips were placed in KRH buffer at 35°C, and exposed to 50 ng/ml Alexa-Fluor-568-EGF and 20 µg/ml Alexa-Fluor-488-Tf. Image capture was started immediately after ligand addition, with the incident angle of the laser set to visualize ~100 nm from the coverslip. After 2 minutes, cells were washed twice with KRH and the incident angle modified to visualize ~300 nm into the cell. Masked, overlapped images from several time points are shown in A,D and E. The number of total pixels of each fluorophore (B,F), as well as the number of colocalized pixels (C,G) seen at early time points after ligand addition (B,C), and after the wash step (F,G) are plotted over time after ligand addition. Results are from a single image set which is representative of a minimum of five independent experiments. (H) Cells were exposed to ligands for 90 seconds, imaged, washed in cold PBS, exposed to an acid wash solution for 3 minutes, and re-imaged.

 

Figure 4
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  		Fig. 4. (A) COS-7 cells were exposed to unlabeled EGF and Tf for the times indicated, fixed and stained with antibodies to the EGFR and the TFR. Optical sections were obtained at 250 nm intervals through the entire volume of the cell, and projected into a single 2D image. A clear demarcation of the cell edge was seen with antibodies to EGFR (arrows), but not to the TfR. (B) 130 nm optical slices through the thickest part of the nuclei of cells treated and stained as in A (arrows indicate localization of EGFR). (C) Cells were exposed to Alexa-Fluor-568-EGF for 20 minutes, fixed, and stained with antibodies to the EGFR. Optical sections were obtained at 250 nm intervals through the entire volume of the cell, and projected into a single 2D image.

 

Figure 5
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  		Fig. 5. Imaging of Tf and EEA1. (A) COS-7 cells expressing GFP-EEA1 (green) were exposed to 10 µg/ml Alexa-Fluor-568-Tf (red) for the times indicated in each panel. After 60 seconds cells were washed, incubated with 200 µg/ml unlabeled Tf, and imaged by TIRF-M with an incident angle set to visualize 300 nm from the coverslip. Colocalized voxels are depicted in white. (B) Higher resolution series depicting the transient nature of the colocalization of Tf with EEA1. (C) TIRF images were obtained at 0.5 Hz, and at 60-second intervals illumination was switched to epifluorescence for the acquisition of optical sections at 250 nm intervals through the entire cell volume. Shown is the TIRF image preceding the acquisition of the image stack. Optical sections were projected into a single 2D image. Arrows in A and B indicate the movement of Tf-containing vesicles as they near EEA1-enriched endosomes; however, colocalization is brief because the Tf structures move away from the EEA1 signal.

 

Figure 6
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  		Fig. 6. Imaging of EGF and EEA1. (A) COS-7 cells expressing GFP-EEA1 (green) were exposed to 50 ng/ml Alexa-Fluor-568-EGF (red) and imaged for the times after ligand addition, as indicated in each panel. After 3 minutes cells were washed and imaged by TIRF-M with an incident angle set to visualize 300 nm from the coverslip. Colocalized voxels are depicted in white, and indicated with arrowheads. Arrows point to the appearance of a ring-like structure containing EEA1, to which EGF-containing vesicles appear to attach. (B) Higher resolution series depicting the association of EGF-containing vesicles (arrow) with EEA1-enriched endosomes. (C) Optical sections at 250 nm intervals through the entire cell volume were projected into a single 2D image.

 

Figure 7
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  		Fig. 7. Quantification of EGF and Tf colocalization with EEA1. COS-7 cells expressing GFP-EEA1 were exposed to 50 ng/ml Alexa-Fluor-568-EGF (A,C) or 10 µg/ml Alexa-Fluor-568-Tf (B,C) for 5 minutes, after which fluorophores were washed and unlabeled Tf added at a concentration of 200 µg/ml (B,C). Plotted are the percentage of total EEA1 pixels colocalized with EGF (A) or Tf (B) over time. (C) Number of total pixels of each fluorophore over time, normalized to the maximum seen immediately after the wash step. (D) Percentage of Tf or EGF pixels colocalized with EEA1 in regions containing both fluorophores at indicated times after exposure to ligands. Results are from a single image set which is representative of a minimum of five independent experiments.

 

Figure 8
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  		Fig. 8. Effect of EEA1 knockdown on Tf and EGF trafficking. (A) Real time quantitative PCR in HeLa cells stably expressing a scrambled control (C) or an EEA1-directed (KD) shRNA. The mRNAs examined are indicated along the x-axis. (B) Western blotting of EEA1 and TfR in two independent stable clones, and immunofluorescence analysis of EEA1. (C) Cells were serum starved and incubated in the presence of EGF for the times indicated. Extracts were analyzed by western blotting with anti-EGFR antibodies. Values are mean ± s.e.m. of four independent experiments analyzed by densitometric scanning. (D) Kinetics of Tf uptake and recycling in C and KD cells. Values are mean ± s.e.m. of three experiments performed in duplicate.

 

Figure 9
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  		Fig. 9. Imaging of GFP-Rab5c, Tf and EGF. (A-C) COS-7 cells expressing GFP-Rab5c were exposed to Alexa-Fluor-568-Tf for 3 minutes followed by a wash and addition of unlabeled Tf at a concentration of 200 µg/ml. Cells were imaged continuously by TIRF-M alternating with epifluorescence as described above. After 30 minutes, when the vast majority of the Tf signal had disappeared from the cell, Alexa-Fluor-568-EGF was added, and imaging resumed. (A) Optical sections through the cell in the GFP channel, illustrating the distribution of Rab5 in the three-dimensional volume of the cell. Arrows point to the redistribution of Rab5 to the cell periphery in response to EGF. (B) The mean intensity of Rab5 in the peripheral and juxtanuclear regions over time after exposure to EGF was quantified in five independent cells. (C) TIRF images of the fluorescent ligands superimposed on the Rab5 image. Arrows point to regions of colocalization. (D) Colocalization between Rab5-GFP and Alexa-Fluor-568-EGF or Alexa-Fluor-568-Tf over time. Plotted are the percentage of Rab5 colocalized with each ligand in pixel-dense regions (two to three regions per cell) over the indicated time intervals. Values are mean ± s.e.m. of three independent experiments. Statistical significance was calculated from two-tailed paired Student's t-tests. *P<0.001. (E) Sequence of images of a cell expressing GFP-Rab5 and exposed to Alexa-Fluor-568-EGF for the times shown in each panel. Arrows point to regions containing Alexa-Fluor-568-EGF, which acquire GFP-Rab5. Similar results were observed in a minimum of five independent experiments.

 

Figure 10
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  		Fig. 10. Model for Tf and EGF internalization and sorting relative to EEA1. The majority of liganded EGFR and TfR internalize through distinct plasma membrane regions, entering into vesicles that contain Rab5. The active EGFR recruits more Rab5 resulting in a higher density of this GTPase in EGF-containing vesicles compared to Tf-containing vesicles. Both types of vesicles interact with EEA1-enriched endosomes, but only EGF-containing vesicles can interact strongly, and fuse. Tf-TfR-containing vesicles move to the perinuclear recycling endosome, while the EGF-EGFR complex is now sequestered by EEA1-enriched endosomes. In this model, EEA1 marks the entry point into the degradative pathway.

 

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© The Company of Biologists Ltd 2008