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Brefeldin A rapidly disrupts plasma membrane polarity by blocking polar sorting in common endosomes of MDCK cells

¹ Department of Medicine, Division of Nephrology, Indiana University Medical Center, Indianapolis, IN 46202, USA
² Departments of Medicine, Surgery and Cellular Biology and Anatomy, Institute of Molecular Medicine and Genetics, Medical College of Georgia and the Augusta VA Medical Center, Augusta, GA 30912, USA
³ Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609

View larger version (109K): [in a new window]   Fig. 1. BFA induces apical redistribution of TfR. 3-D stereopair images of living, polarized PTR cells imaged in the presence of apical Alexa488-Tf and basolateral Alexa568-Tf after 30 minutes of uptake, following 15 minutes of pretreatment in medium lacking (A) or containing (B) 10 µM BFA, show that BFA significantly increases apical Tf uptake. (Note that these and all subsequent 3-D stereopair images are also presented as rotating animations at http://renal.nephrology.iupui.edu/wangetal2). Note that the background fluorescence in these and subsequent images of living cells results from the presence of fluorescent ligands in the medium. (C-E) The speed with which BFA induces apical redistribution of TfR is shown in extended focus projected volumes of living cells preincubated with apical Alexa488-Tf and basolateral Alexa568-Tf for 20 minutes, and then in probes plus BFA for the indicated times. (F-H) Uptake of Tf from the apical chamber does not result from leakage of dye across disrupted tight junctions. (F) Vertical cross section of an image volume collected of control cells incubated with basolateral 70 kDa fluorescein dextran for 20 minutes show that the dye is limited to the basolateral medium. (G) The tight junction barrier is likewise intact in cells pretreated with 10 µM BFA, then in dextran and BFA for another 30 minutes. (H) The junction is breached in cells pre-incubated in PBS lacking Ca2+ for 30 minutes, then incubated with dextran in PBS for 20 minutes, resulting in leakage of dextran into the apical space. Scale bar, 10 µm for all panels except C-E (20 µm).

View larger version (88K): [in a new window]   Fig. 2. Apical redistribution of TfR does not result from diffusion across disrupted tight junctions. Transmission electron micrographs are shown of control cells (A) and cells pretreated for 15 minutes with 10 µM BFA (B,C) and then incubated basolaterally with 15 µg/ml HRP-Tf in the presence of absence of BFA for another 30 minutes. In all cases, the HRP reaction product is limited to endosomes and to the basolateral membrane (arrowheads), ending abruptly at the tight junction, with no product present at the apical plasma membrane (arrows). Scale bar, 1 µm (A,B); 0.5 µm (C).

View larger version (90K): [in a new window]   Fig. 3. BFA blocks intracellular sorting of Tf from IgA. 3-D images of living, polarized PTR cells were collected in the presence of basolateral Oregon Green-IgA and TxR-Tf after 30 minutes of uptake (A), and following 15 minutes of pretreatment in10 µM BFA and 35 minutes incubation with probes in the continued presence of BFA (B). In control cells, the sorting of IgA from Tf is apparent in the enrichment of IgA (green) in the ARE lacking Tf (red), whereas both IgA and Tf completely codistribute in BFA-treated cells, resulting in a nearly uniform endosome color in each cell. Each field is 40 µm across. (C) Plots of individual pixel intensities from projected image volumes of control and BFA-treated cells, labeled and imaged as described above. Data represent the pooled data of 8 cells from each condition, with data normalized such that the mean IgA and Tf fluorescence are equal. Sorting of IgA is apparent in the enrichment of IgA in the ARE, represented by the population of pixels with a high ratio of IgA/Tf, relative to the common endosomes, whose pixels show a low ratio. In contrast, only a single population of pixels is found in cells treated with BFA.

View larger version (30K): [in a new window]   Fig. 4. BFA blocks intracellular sorting of Tf from IgA. This biochemical sorting assay is fully described in the Materials and Methods. Briefly, cells were incubated basolaterally with HRP-Tf for 30 minutes at 37°C, then placed on ice and incubated with HRP-Tf, 125I-IgA and 125I-Tfn for 60 minutes. Excess ligand was rinsed away, and cells were incubated in HRP-Tf in the absence or presence of 10 µM BFA for 15 minutes, and then warmed to 37°C. At the indicated times cells were treated with DAB and H2O2 and solubilized. (A) SDS-PAGE and autoradiography of 125I-Tf and 125I-IgA present in the detergent-soluble fraction following internalization of ligands for the indicated periods of time in the absence or presence of 10 µM BFA. (B) The percentage of pre-bound 125I-IgA in the detergent-soluble fraction is plotted as a function of time of internalization for control cells (circles), BFA-treated cells (squares) and cells treated with 33 µM nocodazole (triangles). (C) BFA blocks sorting of pre-internalized IgA and Tf. 125I-IgA and HRP-Tf were internalized for 5 minutes at 37°C, then non-internalized 125I-IgA was removed by acid wash on ice. BFA was then added for 15 minutes (with the cells still on ice) and the cells then either kept on ice (0 minutes) or incubated at 37°C for 15 minutes and analyzed.

View larger version (199K): [in a new window]   Fig. 5. BFA induces tubulation of endosomes associated with GFP-Rab25. 3-D stereopair images of living, polarized MDCK cells stably expressing GFP-Rab25 were collected for untreated cells (A) and cells following 40 minutes incubation in BFA (B). In untreated cells GFP-Rab25 labels punctate compartments, particularly at cell apices, whereas in cells treated with BFA, GFP-Rab25 is found on tubular compartments that extend vertically throughout the cells. Panel A is 24 µm in height, Panel B is 36 µm in height.

View larger version (176K): [in a new window]   Fig. 6. Endosomes of the transcytotic pathway remain distinct in BFA-treated cells. 3-D stereopair images of living, polarized PTR cells were collected in the presence of basolateral Tf conjugated to both fluorescein and rhodamine (FR-Tf). The pH-sensitive fluorescence of fluorescein gives this probe a green fluorescence in the neutral environment of probe bound to the plasma membrane (note bases of cells in A), but an orange fluorescence for Tf in acidic endosomes. Whereas Tf is restricted to acidic endosomes in control cells (A), it is found in both acidic medial endosomes and relatively alkaline apical compartments in cells previously incubated with BFA for 15 minutes, and then in BFA with FR-Tf for another 22 minutes (B). Panels A and B are 34 µm in height.

View larger version (98K): [in a new window]   Fig. 7. BFA induces transport of both Tf and IgA to the same alkaline apical endosomes. (A-H) Cells were labeled to steady state and imaged alive in the presence of FR-Tf and Cy5-IgA. An apical optical section of control cells shows that FR-Tf (A) is missing from the AREs in which Cy5-IgA concentrates (arrowheads in A,B). In a lower focal plane of the same cells, the two probes closely colocalize in uniformly acidic endosomes (arrows in E and F). In cells treated with BFA for 15 minutes, and then incubated for 25 minutes in probes and BFA, FR-Tf is delivered to apical, alkaline compartments containing Cy5-IgA (arrows in C and D). A separate field of cells, incubated for 15 minutes with BFA, then with probes and BFA for 45 minutes (G,H) shows the two are closely colocal in medial acidic (orange) compartments as well as in alkaline tubular endosomes (green, at the top of the panel). (I) A field of untreated cells imaged in the presence of FR-IgA shows that IgA is located in both acidic endosomes as well as in relatively alkaline AREs located at the cell apices. These two distinct compartments are apparent also in images of cells incubated with BFA for 15 minutes, then with BFA and FR-IgA for another 20 (J) or 25 (K) minutes, showing IgA present in both medial acidic endosomes and relatively alkaline apical endosomes. Note that because of differences in cell height, single optical sections collect apical planes of some cells and medial planes of others. (L) Sorting endosomes remain distinct after protracted exposure of cells to BFA. 3-D images of living, polarized PTR cells were collected in the presence of basolateral Alexa488-Tf and diI-LDL following a 15 minute pretreatment with BFA and an 85 minute incubation with both BFA and fluorescent ligands. Whereas Tf is largely distributed into extended tubules, LDL, found in both sorting endosomes containing Tf and in late endosomes lacking Tf, is restricted to vesicular compartments. Panel L is 29 µm in height.

View larger version (155K): [in a new window]   Fig. 8. BFA induces transport of Tf to apical endosomes associated with Rab25 and Rab11a. (A,B) Image volumes of living cells stably expressing GFP-Rab25 incubated to steady state with basolateral Alexa568-IgA were collected, and reproduced as extended focus projections. IgA and Rab25 closely colocalize, in particular Rab25 is strongly associated with the ARE in which IgA accumulates (arrowheads). (C,D) Image volumes collected of living cells incubated with Alexa568-Tf show that Tf is absent from AREs, so that the apical GFP-Rab25 concentrations correlate with holes in the projected Tf images (arrowheads). (E,F) Images of an apical plane of cells pretreated with BFA for 20 minutes, then incubated with Alexa568-Tf in the continued presence of BFA for another 30 minutes show that BFA induces redirection of Tf into the apical compartments associated with GFP-Rab25. (G,H) Extended focus images of living cells expressing GFP-Rab11a show that, similar to Rab25, Rab11a closely codistributes with basolaterally internalized Alexa568-IgA, particularly in AREs, which again appear as holes in extended focus images of Tf (I,J). (K,L) An apical plane of GFP-Rab11a-expressing cells pretreated with BFA for 20 minutes, then incubated with Alexa568-Tf in the continued presence of BFA for another 30 minutes shows that BFA induces redirection of Tf to the apical compartments associated with GFP-Rab11a. Scale bar, 15 µm (A,B); 10 µm for the remaining panels.

View larger version (125K): [in a new window]   Fig. 9. -adaptin is associated with IgA-containing endosomes. (A) In a medial plane of polarized cells, immunolocalized -adaptin associates with multiple intracellular compartments. Punctate compartments correspond to endosomes (see below) while larger tubular structures are likely to be elements of the trans-Golgi network. Treatment of cells with BFA both dissociates -adaptin from intracellular compartments, and also tubulates endosomes, as shown in the medial plane of a field of cells pretreated with BFA for 15 minutes, then incubated with Alexa488-Tf for 40 minutes in the continued presence of BFA (B,C). A medial plane of control cells incubated with Alexa488-Tf shows a limited colocalization of Tf with -adaptin (D-F). In contrast, cells incubated with Alexa488-IgA show a close correspondence between the distributions of IgA and -adaptin. This colocalization is particularly apparent in apical planes, where the patterns of the two closely match in AREs and punctate endosomes (G-I), but also in the focal plane of the same cells collected 6 microns lower (J-L), where the two colocalize in medial endosomes. A few examples of IgA-containing endosomes associated with -adaptin are noted with arrows. Scale bar, 10 µm.

View larger version (21K): [in a new window]   Fig. 10. Correlation analysis shows -adaptin is more closely associated with endosomes containing IgA than with those containing Tf. Correlation coefficients were calculated comparing the 3-D distributions of -adaptin and either Tf or IgA (see Materials and Methods). Briefly, cells were labeled to steady state with either Tf or IgA, and then processed for -adaptin immunofluorescence. 3-D image volumes were collected, and 15 cells from each condition selected for correlation analysis. (A) Correlation coefficients, calculated for individual cells, are plotted against the total fluorescence of internalized Tf (closed circles) or IgA (open circles). Although variable, the correlations between IgA and -adaptin were uniformly higher than those between Tf and -adaptin, and largely independent of the levels of Tf or IgA fluorescence. (B) Bar graphs of the pooled data show a mean correlation coefficient of 0.29±0.05 between IgA and -adaptin, and a mean correlation coefficient of 0.14±0.04 between Tf and -adaptin. The potential range of this assay is demonstrated in cells labeled with a combination of Alexa488-Tf and Alexa568-Tf, which codistribute in the same compartments. Analysis of 15 cells yielded a mean correlation of 0.75±0.04. The mean correlation coefficient dropped to 0.01±0.07 when the region of analysis of one image of each pair was rotated 180°.

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