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- Fig. 1.
Effect of bound antibodies, GA and CPZ on ErbB2 localization in SK-BR-3 cells. (A) ErbB2 in fixed, permeabilized cells detected by immunofluorescence. (B) Cells were warmed for 2 hours after binding Fl-anti-ErbB2 before fixation. (C) Cells were treated with GA for 2 hours before detecting ErbB2 by immunofluorescence. (D,E) Cells were pre-treated for 45 minutes at 37°C with GA, with (D) or without (E) 12 μg/ml CPZ, before binding anti-ErBb2 antibodies (left, green) and Alexa-Fluor-594-Tf (middle, red) for 1 hour on ice and warming for 2 minutes with the same drugs. Cells were acid-washed and processed for immunofluorescence, detecting ErbB2 with the Alexa-Fluor-488 Zenon mouse IgG labeling kit. Merged images are shown on the right. Scale bar: 10 μm. (F,G) Internalization of biotinylated Tf (F) or biotinylated anti-ErbB2 antibodies (G) was measured by CELISA after treatment with GA (circles) or both GA and CPZ (squares). Values shown are the mean ± s.e.m. of three experiments.
- Fig. 2.
Localization of internalized ErbB2, Rh-Tf, EGFR and clathrin. SK-BR-3 cells were pretreated with GA for 1 hour before binding Fl-anti-ErbB2 (A,C,E,F) or Fl-anti-EGFR (B,D,G) and warming for 5 minutes (with Rh-Tf in A,B and E) before acid washing and immunofluorescence analysis. (C,D) Clathrin heavy chain (CHC, red) was detected by immunofluorescence. (E-G) High-magnification views of the boxed regions in A,C and D, respectively. Right-hand panels in A-D and bottom panels in E-G show merged images. (A) Epifluorescence images. All other panels show maximum-intensity projections of deconvolved z-stacks. Scale bars: 10 μm (bar in B applies to B-D). (H) Quantification of colocalization of Rh-Tf with ErbB2 or EGFR in cells treated as in A,B, except that internalization was for 2 minutes.
- Fig. 3.
Dominant-negative forms of Eps15 and dynamin inhibit internalization of Rh-Tf but not ErbB2. SK-BR-3 cells transfected with EGFP–DN-Eps15 (A,C) or HA–DN-dynamin-1 (B,D) were pretreated with GA for 2 hours before binding unlabeled anti-ErbB2 antibodies (A,C) or Fl-anti-ErbB2 antibodies (B,D). Cells were then warmed for 30 minutes with Rh-Tf, fixed and permeabilized. (A,B) EGFP–DN-Eps15 (A, green) or HA–DN-dynamin (B, blue) are shown with Rh-TF and ErbB2 in deconvolved images, each from a z-stack of a field in which one cell expressed DN-Eps15 (A) or DN-dynamin (B). ErbB2 was detected with Alexa-Fluor-350 goat anti-mouse antibodies (A, blue; C) or by fluorescein fluorescence (B, green; D). Scale bar: 10 μm. (C,D) Internalization of ErbB2 and Rh-Tf in cells expressing EGFP–DN-Eps15 (C) or HA–DN-dynamin (D) (+), and untransfected cells on the same coverslips (–). Cells showing at least three intracellular punctae were scored positive. Numbers shown are averages of two experiments (counting at least 100 transfected and 100 untransfected cells in each experiment), the data from which varied by <10%.
- Fig. 4.
Genistein inhibits EGF-stimulated tyrosine-kinase activity but not ErbB2 internalization. SK-BR-3 cells were serum-starved overnight, treated as described below for the individual panels, fixed and permeabilized. Cells were treated with: (A) GA for 2 hours; (B) 100 ng/ml EGF for 10 minutes; (C) GA for 2 hours, with 100 ng/ml EGF added for the last 10 minutes; and (D) 100 μg/ml genistein for 1 hour, then GA added for another 2 hours and 100 ng/ml EGF added for the last 10 minutes. Deconvolved images from z-stacks are shown. ErbB2 (green, top) and phosphotyrosine (P-Tyr; red, middle) were detected by immunofluorescence. Bottom: merged images. Scale bar: 10 μm. (E) SK-BR-3 cells were pretreated with GA (circles) or GA + 100 μg/ml genistein (squares) for 45 minutes before binding biotinylated anti-ErbB2 antibodies and warming for 0-5 minutes. Internalized antibodies were quantified by CELISA. Values shown are the mean ± s.e.m. of three experiments.
- Fig. 5.
Internalized ErbB2 colocalizes with Alexa-Fluor-594-CtxB, GPI-anchored proteins and dextran in GA-treated SK-BR-3 cells. Cells were pretreated with GA for 1 hour, subjected to antibody and/or toxin binding, warmed for 5 minutes, acid-stripped and fixed. (A) Fl-anti-ErbB2 (green) and Alexa-Fluor-594-CtxB (0.5 μg/ml; red) were bound to cells. A merged maximum-intensity projection image of a deconvolved z-stack is shown. Asterisks delimit the region shown enlarged in B (ErbB2, left; Alexa-Fluor-594-CtxB, middle; merged image, right). (C) Fl-anti-PLAP Fab fragments and Rh-anti-ErbB2 antibodies were bound to cells. A deconvolved image from a z-stack, showing part of the edge of one cell, is shown. ErbB2, left; PLAP, middle; merged image, right. (D) Fl-anti-ErbB2 was bound to cells, which were warmed with 1 mg/ml FluoroRuby dextran. An epifluorescence image, showing part of the edge of one cell, is shown. ErbB2, left; dextran, middle; merged image, right. Scale bars: 10 μm (A); 5 μm (D; applies to B-D). (E) Colocalization of ErbB2 and CtxB, or ErbB2 and PLAP, in cells treated as in A-C (except that internalization was for 2 minutes) was quantified. To measure the colocalization of ErbB2 and Thy1.1, SK-BR-3 cells transfected with Thy1.1 were treated with GA for 1 hour. Fl-anti-ErbB2 and Alexa-Fluor-594-anti-Thy1 Fab fragments were bound on ice, and cells warmed for 2 minutes. Residual surface-bound antibodies were acid-stripped before fixation, visualization and quantification.
- Fig. 6.
Internalized ErbB2 colocalizes with CtxB, GPI-anchored proteins and dextran in SK-BR-3 cells without GA. (A-D) Cells were subjected to antibody and/or toxin binding on ice for 1 hour, warmed for 2 minutes, acid-stripped and fixed. (A) Fl-anti-ErbB2 and Alexa-Fluor-594-CtxB (0.5 μg/ml) were bound to cells. (B) Fl-anti-PLAP Fab fragments and Rh-anti-ErbB2 antibodies were bound to cells. (C) Fl-anti-ErbB2 was bound to cells, which were warmed with 1 mg/ml FluoroRuby dextran. (D) Fl-anti-ErbB2 and Rh-Tf were bound to cells. (A-D) Deconvolved images from z-stacks are shown. ErbB2, left; CtxB, PLAP, dextran or Tf, middle; merged images, right. Scale bar: 10 μm. (E) Internalization of biotinylated anti-ErbB2 antibodies was measured by CELISA in cells treated with (squares) or without (circles) GA. Values shown are the mean ± s.e.m. of three experiments.
- Fig. 7.
C. difficile toxin B does not inhibit internalization of ErbB2 or fluid in SK-BR-3 cells. (A) SK-BR-3 cells grown for 48 hours on poly-Lys-coated coverslips were left untreated (Con) or were treated for 2 hours with 0.5 μg/ml C. difficile toxin B (Tox), fixed, permeabilized and incubated with rhodamine phalloidin (4 U/ml). Stress fibers were seen in about half of the control cells and <1% of treated cells. (B,C) Serum-starved SK-BR-3 cells on poly-Lys-coated coverslips were left untreated (B) or were treated for 2 hours with 0.5 μg/ml C. difficile toxin B (C) before addition of FluoroRuby dextran (1 mg/ml) for 10 minutes. After fixation, surface morphology was visualized with anti-ErbB2 antibodies and green secondary antibodies. PM, plasma membrane. Scale bar: 10 μm. (D,E) Internalization of biotinylated anti-ErbB2 antibodies (D) or biotinylated BSA (E) was measured by CELISA in serum-starved SK-BR-3 cells treated with GA (D, circles), GA + C. difficile toxin B (D, squares), C. difficile toxin alone (E, squares) or left untreated (E, circles) as described in the Materials and Methods. Where appropriate, cells were pre-incubated with C. difficile toxin B (0.5 μg/ml) for 2 hours at 37°C with the addition of GA for the last 45 minutes. Values shown are the mean ± s.e.m. of three experiments.
- Fig. 8.
ErbB2 is delivered to early endosomes after GA treatment. (A-D) SK-BR-3 cells were left untransfected (A,B), or were transiently transfected with GFP-Rab5 (C) or GFP–Rab5-Q79L (D), and then treated with GA for 2 hours (with Rh-Tf added for the last 30 minutes in A), fixed and permeabilized. Left panels: ErbB2, detected with polyclonal antibodies. Center panels: (A) Rh-Tf fluorescence; (B) endogenous EEA1; (C,D) GFP fluorescence. Right panels: merged images. (A) Deconvolved image from a z-stack; (B-D) epifluorescence images. Scale bars: 10 μm (A); 5 μm (D, applies to B-D).
- Fig. 9.
ErbB2 is delivered to late endosomes and lysosomes after GA treatment. After transient expression of GFP-Rab7 (A only), SK-BR-3 cells were treated with GA for 5 hours (together with 0.1 mg/ml leupeptin, D only), fixed and permeabilized. Left panels: ErbB2 was detected with polyclonal (A,B) or monoclonal (C,D) antibodies and appropriate secondary antibodies. Middle panels: (A) Rab7 (GFP fluorescence); (B) endogenous CD63; (C,D) endogenous LAMP1. Right panels: merged images. Epifluorescence images are shown. Scale bar: 5 μm.
- Fig. 10.
GA-induced ErbB2 degradation is sensitive to chloroquine. SK-BR-3 cells were incubated with GA with or without chloroquine (CQ) for the times indicated and lysed. Proteins were separated by SDS-PAGE and transferred to membranes for western blotting and detection of ErbB2. (A) Western blots. Top, GA alone; bottom, GA and CQ. Arrow: ca. 135-kDa ErbB2 fragment. (B) Bands were quantified by scanning densitometry and plotted as the percentage of the signal at time=0 remaining at each time point.
- Fig. 11.
Accumulation of ErbB2 in Arf-Q67L-positive endosomes occurs only without GA. (A-F) SK-BR-3 cells were transfected with Arf6-Q67L alone (A,D) or in combination with either GFP-Rab5 (B,E) or GFP-Rab7 (C,F). Fl-anti-ErbB2 (A,D) or unlabeled anti-ErbB2 (B,C,E,F) antibodies were bound for 1 hour before cells were warmed for 2 hours with (A-C) or without (D-F) GA. Internalized anti-ErbB2 was detected in fixed and permeabilized cells by Fl-anti-ErbB2 fluorescence (A,D) or with Texas-Red goat-anti-mouse antibodies (B,C,E,F). Although Arf6-Q67L was not visualized in B,C,E or F, vacuoles characteristic of Arf6-Q67L expression were seen. Scale bar: 10 μm. (G,H) COS-7 cells were transfected with ErbB2 alone or together with Arf6-Q67L as indicated. Cells were incubated with GA for the indicated times and solubilized in gel loading buffer. Proteins were separated by SDS-PAGE and transferred to nitrocellulose. ErbB2 and Arf6-Q67L were detected by immunoblotting as described in the Materials and Methods. (H) A representative Arf6-Q67L blot is shown, demonstrating expression in co-transfected cells (+Arf6-Q67L), but not in cells expressing ErbB2 alone (–Arf6-Q67L).
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