Figure 1.
The giant vacuoles induced by Bin possess characteristics of late endocytotic and lysosomal compartments.
(A-E) MDCK-Cpm1 cells were transfected with GFP-Rab4, GFP-Rab5, GFP-Rab7, GFP-Lamp1 or YFP-Golgi and treated with BinA and BinB for 6 h. LysoTracker (1 µM) was added to the medium 5 min before the end of the treatment. (F and G) MDCK-Cpm1 cells were co-transfected with DsRed2-Endoplasmic Reticulum or DsRed2-Mitochondria and GFP-Lamp1 before intoxication. Bin-induced vacuoles stained with LysoTracker or GFP-Lamp1 do not colocalize with marker of Golgi apparatus, ER and mitochondria. Bars, 10 µm.
Figure 2.
Bin-induced vacuoles originate from autolysosomes.
(A) MDCK-Cpm1 cells transfected with GFP-LC3 and grown in complete medium (CM) or in nutrient-free medium (EBSS). Diffuse GFP-LC3 pattern seen in CM-grown cells is representative of non-autophagic cells. In contrast, GFP-LC3 punctated pattern seen in EBSS-grown cells is characteristic of autophagic cells. (B) Localization of the autophagic marker GFP-LC3 in cells treated with Bin for 6 h. To allow the detection of GFP-LC3 associated with autolysosomes NH4Cl (5 mM, final concentration) was added to the culture medium during the last hour of intoxication. A similar treatment with NH4Cl in the absence of toxin had no toxic effect on the cells and did not induce any change in GFP-LC3 pattern. (C) Bin-treated cells were processed for EM analysis as described in Experimental procedures. Vacuoles induced by Bin are electron-clear matrices enclosed by a single membrane (black arrows). In contrast, autophagosomes are double membrane vesicles (white arrows). Bin-induced vacuoles often contained vesicle-like structure (arrow-head) and partially degraded material (large black arrows). Bars, 5 µm (A and B); 2 µm (C).
Figure 3.
Dynamics of Bin-induced vacuolation.
(A) Bin-treated (for 24 h) subconfluent MDCK-Cpm1 cells were submitted to time-lapse phase contrast video microscopy, as exemplified in video S1. At the indicated period of time the percentage of MDCK-Cpm1 cells displaying at least one vacuole was determined. Mean values ± SD, n = 3. In the same videos, the percentage of cells that present post-mitotic vacuolation among the cells that display at least one vacuole, as illustrated in (B) was determined. Mean values ± SD, n = 3. (B) Bin-treated cells displaying the post-mitotic vacuolation phenomenon; images selected from video S2. The selection starts 15h after intoxication, intervals in hours. Black and white arrows pinpoint two dividing cells. Bars, 10 µm.
Figure 4.
Autophagy is enhanced in Bin treated cells and contributes to vacuolation reversion.
(A) MDCK-Cpm1 cells were grown for 6 h in complete medium in the absence (CM) or the presence of Bin (CM + Bin), or in nutrient-free medium in the absence of the toxin (EBSS), and then processed for EM analysis. Pictures from randomly chosen EM sections were analyzed for the presence of autophagosomes as defined in figure 2. The results are expressed as the number of autophagosomes per cell. Mean values ± SD, n = 3. Different letters indicate statistically significant differences; P<0.05. (B) MDCK-Cpm1 cells expressing GFP-LC3 were grown under the conditions described above. At the indicated time, the number of GFP-LC3 positive cells was scored. Mean values ± SD, n = 3. Different letters indicate statistically significant differences; P<0.05. (C) Cells were grown in complete medium or in nutrient-free medium in the presence of toxin. At the indicated period of time, the percentage of vacuolating cells was determined. Mean values ± SD, n = 3. Asterisks indicate statistically significant differences; P<0.05. (D) Images selected from time lapse videomicroscopy of Bin-treated cells in nutrient-free medium displaying the post-mitotic vacuolation phenomenon, the selection starts 20 h after intoxication (Video S3). Black and white arrows pinpoint two dividing cells that will display this phenomenon. Bars, 10 µm. (E) MDCK-Cpm1 cells were grown in complete medium for 3 h (Δ, ⧫, •). Then the toxin was washed out with either complete medium (•), or nutrient-free medium (Δ, ⧫). Two hours after washing, the cells were returned to CM (⧫). Mean values ± SD, n = 3. (F) Representative pictures illustrating the various patterns of vacuolation corresponding to each medium condition.
Figure 5.
Internalized Bin is associated with its receptor but is not bound to vacuoles.
(A, C and E) MDCK-Cpm1 cells were incubated with BinA labeled with Alexa 488 (BinA-Al488) and BinB labelled with Alexa 543 (BinB-Al543) at 37°C for 10 min (A), 30 min (C) or 6h (E) and processed for confocal microscopy analysis. At any time point, BinA-Al488 and BinB-Al543 were found colocalized in multiple intracellular vesicles. (B) Cells transfected with GPI-GFP and treated for 10 min with unlabelled BinA and BinB-Al543. Vesicles positive for BinB-Al543 were also stained by GPI-GFP. (D) Immunodetection of Cpm1 in MDCK-Cpm1 cells treated for 30 min with unlabelled BinA and BinB-Al543. Cpm1 signal and BinB-Al543 labeled vesicles were found colocalized. (E) BinA-Al488 and BinB-Al543-containing vesicles were found clustered between the vacuoles but not decorating the membrane of these vacuoles nor inside. Bars, 5 µm.
Figure 6.
After internalization, Bin reaches recycling endosomes.
MDCK-Cpm1 cells were transfected with markers of early and late endocytotic compartments, intoxicated with a mixture of unlabelled BinA and BinB-Al543 for 30 min and processed for confocal microscopy analysis. (A) BinB-Al543 fluorescent signal colocalized with GFP-Rab4, a typical marker of recycling compartments. (B) A significant colocalization was detected with TfR-myc that marks both endocytotic and recycling vesicles. (C - E) No colocalization was found with GFP-Rab7 and GFP-Lamp1 that marks late-endocytotic/lysosomal compartments and GFP-LC3 a marker of autophagic vesicles. Bars, 10 µm.
Figure 7.
Fate of internalized toxin in dividing cells.
(A) Immunodetection of β-tubulin and DNA staining were performed in MDCK-Cpm1 cells treated with unlabelled BinA and BinB-Al543 for 6 hours. (B) Cells were transfected with GFP-Rab4 then intoxicated as described above, fixed, and stained for DNA. Cells in the process of cell division were analyzed for the localization of the toxin. (C) Time lapse videomicroscopy analysis was performed on MDCK-Cpm1 cells treated with BinA-Al488 and BinB-Al543 for 24 h. The panel selected from video S4 shows a cell displaying post-mitotic vacuolation, the selection starts 16 hours after intoxication. While the cell was progressing through mitosis, internalized toxins were colocalized and partitioned in the daughter cells. Bars, 10 µm.