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First published online 25 November 2008
doi: 10.1242/jcs.033522

Journal of Cell Science 121, 4008-4017 (2008)
Published by The Company of Biologists 2008
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The adaptor complex AP-2 regulates post-endocytic trafficking through the non-clathrin Arf6-dependent endocytic pathway

Alan W. Lau1 and Margaret M. Chou2,*

1 Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
2 Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA


Figure 1
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  		Fig. 1. siRNA-mediated depletion of clathrin heavy chain blocks endocytosis of TfnR. (A) HeLa cells were transfected with siRNA oligonucleotides targeting clathrin heavy chain (CHC). Whole-cell extracts were prepared and subjected to immunoblotting using anti-CHC or actin antibodies. (B,C) Control siRNA (B) or CHC siRNA (C) cells were incubated with Alexa-Fluor-546-labeled Tfn before being fixed and subjected to indirect immunofluorescence using anti-CHC antibody. (D) HA-tagged dynamin(PH*)-transfected cells were incubated with Alexa-Fluor-546-labelled Tfn, then fixed and subjected to indirect immunofluorescence using anti-HA antibody. (E) CHC siRNA cells were incubated with Alexa-Fluor-488-labeled Tfn, then fixed and subjected to indirect immunofluorescence using anti-Tfn receptor antibody. All samples were analyzed by confocal microscopy. Arrows indicate depleted cells.

 

Figure 2
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  		Fig. 2. Clathrin depletion does not appear to block internalization of Arf6 pathway cargoes, but alters their intracellular distribution. (A) Live clathrin-depleted cells were incubated with an antibody against the extracellular domain of MHCI for 4 hours, then fixed and subjected to immunofluorescence using anti-CHC antibody. (B) Clathrin-depleted cells were incubated with antibody against the extracellular domain of β1 integrin for 3.5 hours. Alexa-Fluor-546–Tfn was then added and cells were incubated for an additional 30 minutes. Samples were fixed and analyzed by confocal microscopy. Arrows indicate depleted cells.

 

Figure 3
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  		Fig. 3. Depletion of {alpha}-adaptin and µ2 subunits of AP-2 blocks endocytosis of TfnR. (A) HeLa cells were transfected with the indicated siRNA oligonucleotides. Whole-cell lysates were prepared and immunoblotted with antibodies against {alpha}-adaptin, µ2-subunit or TfnR. (B) Live control cells were incubated with Alexa-Fluor-546-conjugated Tfn, then subjected to immunofluorescence using anti-{alpha}-adaptin antibody. (C) Live {alpha}-adaptin-depleted cells were incubated with Alexa-Fluor-488–Tfn, then stained with antibodies against {alpha}-adaptin and TfnR. (D) µ2-depleted cells were incubated with Alexa-Fluor-546-labeled Tfn, fixed, and then subjected to indirect immunofluorescence using anti-{alpha}-adaptin antibody. In C and D, the depleted cells are in the upper left parts of the panels.

 

Figure 4
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  		Fig. 4. Depletion of AP-2 subunits perturbs endocytic trafficking of Arf6-pathway cargoes. Live cells depleted of {alpha}-adaptin (left) or µ2-subunit (middle) were incubated with antibody against β1 integrin. Samples were fixed and stained with {alpha}-adaptin antibody (top) to identify depleted cells. Right, live {alpha}-adaptin-depleted cells were incubated with antibody against MHCI. Cells were fixed and stained with {alpha}-adaptin antibody. Arrows indicate depleted cells.

 

Figure 5
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  		Fig. 5. Depletion of {alpha}-adaptin does not significantly affect the steady-state distribution or initial endocytosis rate of MHCI. (A) Control, clathrin or {alpha}-adaptin-depleted cells were prepared, and relative steady-state levels of TfnR and MHCI at the cell surface were determined by flow cytometry (see Materials and Methods for details). (B,C) MHCI at the cell surface was labeled by incubation of cells with anti-MHCI antibody on ice. Cells were returned to 37°C for 5-10 minutes (B) or 20-120 minutes (C) to allow resumption of trafficking. MHCI that had remained at the cell surface was detected using FITC-conjugated secondary antibody, and levels were then quantified by flow cytometry. Data represent results from at least four independent experiments, each performed in duplicate; **P values (ranging from P=0.001 to P=0.01) indicating statistically significant differences.

 

Figure 6
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  		Fig. 6. Lysosomal targeting of Arf6-pathway cargo is enhanced in {alpha}-adaptin-depleted cells. (A) Internal pools of MHCI were measured by uptake of Alexa-Fluor-488-conjugated anti-MHCI antibody for 2 hours at 37°C. Antibody remaining at the cell surface was quenched using anti-Alexa-Fluor-488 antibody, and the internal pool was quantified by flow cytometry. Levels in clathrin and {alpha}-adaptin-depleted cells are reported relative to those in control cells. Data represent results from four independent experiments, each performed in duplicate; **P=0.0003, statistically significant difference. (B) Cell extracts were prepared from control or {alpha}-adaptin-depleted cells, treated with bafilomycin A1 (BafA) at 250 nM or not treated, as indicated. Immunoblotting was performed using antibodies against MHCI, {alpha}-adaptin and p70S6k. (C-E) Cell-surface proteins were biotinylated at 4°C, then returned to 37°C for 4 hours. Biotinylated proteins were immobilized using NeutraAvidin beads (Pdn), and subjected to immunoblotting with the indicated antibodies. In C, t=0 samples were immunoblotted with anti-TfnR antibody to confirm significant inactivation of clathrin and AP-2. In D and E, anti-MHCI immunoblotting was performed on cells depleted of {alpha}-adaptin (D) or clathrin (E). WCL, whole-cell lysate. (F) Quantification of MHCI levels from biotinylation experiments. Levels remaining after the 4-hour chase period were normalized against the starting levels (t=0) for each respective siRNA. Data represent results from at least three independent experiments, each performed in duplicate.**P values (ranging from P=0.001 to P=0.01) indicating statistically significant differences.

 

Figure 7
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  		Fig. 7. AP-2 depletion specifically enhances co-staining of Arf6 cargo with the late endosome and lysosome marker CD63. (A-C) Cells depleted of {alpha}-adaptin (A), µ2 (B) or clathrin (C) were incubated with antibody against β1 integrin for 4 hours. In C, Alexa-Fluor-546-labelled Tfn was added during the final 30 minutes to identify clathrin-depleted cells. Cells were fixed and subjected to indirect immunofluorescence using anti-CD63 and, for A and B, anti-{alpha}-adaptin. (D) The degree of β1 integrin colocalization with CD63 was quantified using MetaMorph Analysis Software, as detailed in Materials and Methods. The percentage of β1 integrin vesicles colocalizating with CD63 in control cells was determined, then arbitrarily set at 100; the levels in AP-2- and clathrin-depleted cells were expressed relative to this value. All differences were statistically significant (P values ranging from P=0.003 to P=0.009). (E) Enlarged view of the cell labeled with asterisk in the Merge panel of B. CD63, red;, β1 integrin; green. Magnifications of insets are x1.5 (B) and x2 (A,C). Arrows indicate depleted cells.

 

Figure 8
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  		Fig. 8. AP-2 depletion targets Arf6 cargo to CD63- and Lamp1-positive endosomes. (A) Cells depleted of {alpha}-adaptin were subjected to indirect immunofluorescence using antibodies against (A) CD63 and Lamp1, (B) EEA1, (C) TfnR or (D) TfnR and CD63. In B and C, live cells were incubated with antibody against β1 integrin for 4 hours prior to fixation. Arrows in A and D indicate depleted cells; cells shown in B and C are depleted of {alpha}-adaptin.

 

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