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

First published online 10 July 2007
doi: 10.1242/jcs.002493

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 Google Scholar Google Scholar Articles by Dorsey, F. C. Articles by Cox, J. V. Search for Related Content PubMed PubMed Citation Articles by Dorsey, F. C. Articles by Cox, J. V.

A novel role for a YXX motif in directing the caveolin-dependent sorting of membrane-spanning proteins

Frank C. Dorsey^*, Thangavel Muthusamy, Michael A. Whitt and John V. Cox

Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163, USA

View larger version (151K): [in this window] [in a new window]   Fig. 1. MDCK cells expressing Fc38-63 (A-C), Fc38-63L50A (D-F) or Fc38-63Y47A (G-I) were incubated with rat anti-Fc receptor antibodies for 30 minutes at 4°C and then shifted to 37°C for 45 minutes. Cells were then fixed, permeabilized and incubated with anti-rat IgG conjugated to Alexa Fluor-594 (A,D,G) and Alexa Fluor-488-phalloidin (B,E,H). The confocal images correspond to an xy-slice through the middle of the cells. Bars, 10 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 2. MDCK cells expressing Fc38-63 or the human transferrin receptor were transfected with EGFP-tagged Eps1595-295 (B,E). The cells were incubated with Alexa Fluor-594-transferrin (A) or anti-Fc receptor antibodies (D) for 30 minutes at 4°C and then shifted to 37°C for 30 minutes. Cells were then fixed and immunofluorescent molecules were directly visualized (A-C) or the cells were fixed, permeabilized and incubated with anti-rat IgG conjugated to Alexa Fluor-594 prior to microscopic analysis (D-F). Bars, 10 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 3. MDCK cells expressing Fc38-63 were incubated in serum-free DMEM that lacked (A-C) or contained (D-F) 10 mM methyl--cyclodextrin for 1 hour. Cells were then fixed, permeabilized and incubated with rat anti-Fc-receptor antibody and with a rabbit antibody directed against furin. After washing, cells were incubated with anti-rat IgG conjugated to Alexa Fluor-594 and anti-rabbit IgG conjugated to Alexa Fluor-488. Alternatively, after a 1-hour incubation in 10 mM methyl--cyclodextrin, the cells were washed with DMEM containing 5% fetal bovine serum and incubated for 20 minutes (G-I) or 60 minutes (J-L) with 10 mM methyl--cyclodextrin loaded with cholesterol and processed as described above. The upper panel in each confocal image corresponds to an xy-slice near the middle of the cells, whereas the lower panel corresponds to an xz-slice. Black bars mark the position of the basal membrane. Bars, 10 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 4. MDCK cells expressing Fc38-63 were processed as described in the legend to Fig. 3 except the cells were incubated with caveolin 1 antibodies rather than antibodies to furin. The upper panel in each confocal image corresponds to an xy-slice near the middle of the cells, whereas the lower panel corresponds to an xz-slice. Black bars mark the position of the basal membrane. Bars, 10 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 5. MDCK cells expressing Fc38-63 were transfected with wild-type dynamin 2 (A-C) or the dynamin 2 (K44A) mutant (D-F) in a vector that also expresses EGFP. In each instance, the cells were incubated with anti-Fc receptor antibodies for 30 minutes at 4°C and then shifted to 37°C for 30 minutes. Cells were then fixed, permeabilized and incubated with anti-rat IgG conjugated to Alexa Fluor-594. Bars, 10 µm.

View larger version (120K): [in this window] [in a new window]   Fig. 6. Polarized MDCK cells expressing Fc38-63 were incubated in the presence of 10 mM methyl--cyclodextrin for 1 hour at 37°C. The cells were then fixed, permeabilized and incubated with antibodies specific for the Fc receptor (A) and caveolin 1 (B). Alternatively, untreated cells were incubated with anti-Fc receptor antibodies (D-F) for 30 minutes at 4°C. The cells were then washed, fixed, permeabilized and incubated with antibodies specific for caveolin 1 (E). In both instances, the cells were washed and incubated with anti-rat IgG conjugated to Alexa Fluor-594 (A,D) and anti-rabbit IgG conjugated to Alexa Fluor-488 (B,E). Bars, 10 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 7. Polarized MDCK cells expressing Fc38-63 were grown on permeable supports. The basolateral surface of the cells was incubated with anti-Fc receptor antibodies (A) for 30 minutes at 4°C. The cells were then washed and shifted to 37°C for 2 minutes. After 2 minutes of incubation, the cells were shifted to citrate buffer, pH 1.5, for 5 minutes at 4°C to elute surface-bound antibodies. The cells were then rinsed in PBS and fixed. Following permeabilization, the cells were incubated with antibodies specific for caveolin 1 (B). The cells were again washed and incubated with anti-rat IgG conjugated to Alexa Fluor-594 (A) and anti-rabbit IgG conjugated to Alexa Fluor-488 (B). The merged image (C) illustrates the overlap between the chimera and caveolin. The xy-slice in each confocal image is approximately 1 µm above the basal membrane of the cell. Similar analyses have quantified the colocalization of Fc38-63 with clathrin, transferrin, Ctx-B or EEA1. The bar graph in D shows the percentage of the total number of pixels resulting from Fc38-63 staining that colocalized with these various endocytic markers on the cell surface (0 Min.), and at 2 and 5 minutes post-internalization. This quantification reflects the average result from at least 25 cells from two independent experiments. Bar, 1 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 8. Subconfluent MDCK cells expressing Fc38-63 were transfected with a vector expressing the caveolin 1 siRNA. This vector also expressed EGFP under the control of a separate promoter. Forty-eight hours post-transfection, the cells were fixed (A-C) or incubated with anti-Fc receptor antibodies for 30 minutes at 4°C, washed and then fixed (D-F). Following permeabilization, the cells in A-C were incubated with caveolin 1 antibodies (A) followed by anti-rabbit IgG conjugated to Alexa Fluor-594. The cells in D-F were directly incubated with anti-rat IgG conjugated to Alexa Fluor-594. Following washing, cells were analyzed by confocal microscopy. Arrows in A and B mark the border of the cell expressing the caveolin 1 siRNA. Note the absence of caveolin staining in the plasma membrane. The bar graph in G has quantified the average effect of the caveolin 1 siRNA on the caveolin 1 protein level and Fc38-63 surface expression in 20 randomly chosen cells from two independent experiments. Bars, 10 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 9. Subconfluent MDCK cells expressing Fc38-63 were transfected with the caveolin 1 siRNA-expressing vector. Forty-eight hours post-transfection, the cells were incubated with anti-Fc receptor antibodies, Ctx-B conjugated to Alexa Fluor-594 or transferrin conjugated to Alexa Fluor-594 for 30 minutes at 4°C. The cells were then washed and shifted to 37°C for 2 minutes. At this time, the cells were shifted to citrate buffer, pH 1.5, for 5 minutes at 4°C to elute surface-bound molecules. The cells were then rinsed in PBS and fixed. Following permeabilization, cells that had been incubated with anti-Fc receptor antibodies were incubated with anti-rat IgG conjugated to Alexa Fluor-594. Immunofluorescent molecules were visualized on a Zeiss LSM 510 confocal microscope. The bar graph in B has quantified the effect of the caveolin 1 siRNA on the endocytosis of Fc38-63 and transferrin in 20 randomly chosen cells. The effect of a control siRNA directed against EGFP on Fc38-63 endocytosis is also illustrated (B). The standard error in B is indicated. Bars, 10 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 10. MDCK cells expressing V5-tagged versions of Fc38-63, Fc38-63Y47A, Fc38-63L50A were lysed and immunoprecipitates were prepared with V5-specific antibodies. The immunoprecipitates were incubated in the absence or presence of a mixture of glycosidases that removes all O-linked and N-linked sugars prior to immunoblotting analysis with V5 antibodies (A). V5 immunoprecipitates prepared from MDCK cells expressing V5-tagged versions of Fc38-63, Fc38-63Y47A, Fc38-63L50A were also subjected to immunoblotting analysis with V5- and caveolin 1-specific antibodies (B). Alternatively, cells expressing V5-tagged (+Tag) and untagged (–Tag) versions of Fc38-63 were incubated with anti-V5 antibodies for 30 minutes at 4°C. The cells were then washed and lysed. Immune complexes were captured with protein A agarose and subjected to immunoblotting analysis with V5- or caveolin 1-specific antibodies (B). V5 immunoprecipitates prepared from MDCK cells expressing V5-tagged AE1-4 or AE1-4L50A were also processed for immunoblotting analysis with AE1- and caveolin 1-specific antibodies (C). Finally, MDCK cells expressing the wild-type (WT) and mutant V5-tagged Fc38-63 or V5-tagged versions of AE1-4 or AE1-4L50A were lysed in 2 ml of isotonic buffer containing 1% LubrolWX. The lysates were fractionated on discontinuous sucrose gradients, and 1 ml fractions were collected from the top of the gradient (fraction 10 includes the pellet). These samples were either directly analyzed by immunoblotting analysis using caveolin 1, furin, or -COP antibodies (D), or immunoprecipitates were prepared from each fraction using V5-specific antibodies. The immunoprecipitates were then analyzed by immunoblotting analysis using V5-specific antibodies (D). The top and bottom of the gradient in D are indicated.

View larger version (52K): [in this window] [in a new window]   Fig. 11. Polarized MDCK cells expressing V5-tagged AE1-4 or AE1-4L50A were fixed and stained with AE1-specific antibodies (A). Alternatively, polarized cells expressing V5-tagged AE1-4 were incubated at 4°C for 30 minutes in the absence (B-D) or presence (E-J) of 10 mM methyl--cyclodextrin. During the 4°C incubation, V5-specific monoclonal antibodies were added to the basolateral surface of these polarized cells. The cells were then washed and shifted to 37°C for 20 minutes in the absence (B-D) or presence (E-J) of 10 mM methyl--cyclodextrin/cholesterol. At this time, the cells were directly fixed (B-D and H-J) or they were shifted to citrate buffer, pH 1.5, for 5 minutes at 4°C to elute surface-bound antibodies prior to fixation (E-G). The cells were then permeabilized and incubated with a rabbit caveolin-1 antibody, followed by anti-mouse IgG conjugated to Alexa Fluor-594 and anti-rabbit IgG conjugated to Alexa Fluor-488. The xy-slice in each confocal image is approximately in the middle of the cell with the corresponding xz-slice below. The merged images in G and J indicate that AE1-4V5 polypeptides internalized as a result of cholesterol depletion/repletion accumulate in caveolin 1-positive endosomes. Black bars mark the position of the basal membrane. Bars, 10 µm.