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Differential expression of receptors for Shiga and Cholera toxin is regulated by the cell cycle

¹ Max-Planck-Institute of Biophysical Chemistry, Department of Neurobiology, Göttingen, Germany
² Engelhardt Institute of Molecular Biology, Department of Molecular Biology, Vavilova Street 32, Moscow, Russia and The Oslo University Center for Medical Studies at Moscow, Vasilova Street 34/5, Moscow, Russia
³ Medical Institute, Minsk, Belorussia

View larger version (96K): [in a new window]   Fig. 1. Differential binding of Shiga anCd cholera toxin to Vero cells; differences in gene expression in Shiga versus Cholera toxin binding Vero cells. (a) Differential binding of Cy3-CTX (CTX-Cy3) and Cy2-ST (ST-Cy2) to non-synchronized Vero cells. Cells were incubated simultaneously with the two labeled toxins for 10 minutes at 0°C. Unbound toxins were washed away and the live cells observed by fluorescence microscopy. (b) Differential display comparing ST binding cells (S) and CTX binding cells (C). Non-synchonized Vero cells were labeled simultaneously with Cy2-ST and Cy3-CTX as in (a) and separated by FACS. cDNA was synthesized from RNA extracted from ST and CT binding cells. DNA fragments were produced by PCR using different 5' and 3' primers and analyzed by PAGE. The results of the primer pairs P2/T1, P2/T2, P2/T3, and P2/T4 are shown (see Methods section). Note the differentially amplified cDNA fragments marked by (*).

View larger version (39K): [in a new window]   Fig. 2. Cells that can be detached following colchicine treatment bind preferentially to Shiga toxin, but bind again to Shiga and Cholera toxin after desynchronization. (a) Vero cells detached from the culture dish after colchicine treatment bind to Cy2-ST (upper left panel). Cells remaining attached to the dish are larger, have an extended structure (arrow) and bind only to Cy3-CTX (lower right panel) but not Cy2-ST (lower left panel). DIC (differential interference contrast) of the two lower panels. (b) Cells detached after colchicine treatment were analyzed 30 minutes after detachment for binding of Cy2-ST (upper left panel) and Cy3-CTX (upper right panel). When detached cells were cultivated for another 12 hours, they show again exclusive binding (arrows) of either Cy2-ST (lower left panel) or Cy3-CTX (lower right panel) as seen in non-synchronized cell cultures (compare with Fig. 1a).

View larger version (91K): [in a new window]   Fig. 3. Dividing cells bind only to Shiga toxin (Cy3-ST). Cells were partially synchronized by a double-thymidine block and analyzed for toxin binding 6 hours later. The relative number of small round-shaped cells binding preferentially to Cy3-ST (upper left panel) is significantly higher than in non-synchronized cultures. Note that the dividing cells (arrows) bind only to ST. The flat, extended, irregularly shaped cells bind only to Cy2-CTX (upper right panel). Lower left panel: overlay of the two upper panels. Lower right panel: digital interference contrast.

View larger version (72K): [in a new window]   Fig. 4. Cells in S-phase exhibt low binding of both toxins. Cells were pretreated with BrdU, exposed to Cy2-CTX and Cy3-ST and analyzed by fluorescence microscopy as in Fig. 1a. Cells were then fixed, permeabilized and analyzed for BrdU incorporation using anti-BrdU-antibodies. Cells strongly stained for BrdU (S-phase) show little or no binding of CTX (CTX) or ST (ST).

View larger version (78K): [in a new window]   Fig. 5. Cells in G2 (cyclin B1 positive) and anaphase bind preferentially to Shiga toxin. (a,b) Non-synchronized Vero cells were exposed to Cy2-ST and Cy3-CTX and analyzed by fluorescence microscopy as in Fig. 1a. Cells were then fixed, permeabilized and immunostained for cyclin B1 (see Materals and Methods section). (a) and (b) represent different experiments of the same kind. Overlay represents the overlay of CTX, ST and cyclin B1. (c) FACS-separated cells, binding either ST (lane 1) or CTX (lane 2) were extracted and identical amounts of proteins separated by SDS-PAGE. Immunoblots were performed using anti-cyclin-B1- and anti-protein disulfide isomerase antibodies. Lane 3 represents extracts from the same cells but before FACS separation. The amount of protein in lane 3 was about four times that applied in lanes 1 and 2. (d) A cell in anaphase shows a significant binding and uptake of ST but not of CTX. Chromatin staining with DAPI. The cells were fixed about 10 minutes after start of uptake of the toxins. At this time point significant amounts of the toxins have already entered the cells.

View larger version (59K): [in a new window]   Fig. 6. Vero cells that do not bind Shiga or Cholera toxin also lack internal toxin receptors. (a) CTX-binding cells. The left panels (two different experiments) show cells strongly binding Cy3-CTX to the cell surface. The same cells also exhibit a strong binding of Cy5-CTX to internal binding sites after permeabilization, whereas cells that bind little Cy3-CTX to the surface lack internal binding sites for Cy5-CTX. (b) (Upper panels) A cell that strongly binds to Cy2-ST also expresses internal binding sites for Cy5-ST. (Lower panels) Two cells that exhibit strong binding of Cy2-ST to the cell surface also show internal receptors for Cy5-ST after cell permeabilization. Note that surrounding cells that possess almost no internal binding sites fail also to bind ST to the cell surface.

View larger version (46K): [in a new window]   Fig. 10. A schematic presentation of cell-cycle-dependent differential synthesis of receptors for Cholera and Shiga toxin.

View larger version (72K): [in a new window]   Fig. 7. Vero 317 cells in S-phase do not bind to Cholera toxin. The experimental conditions used were the same as in Fig. 4. Cells bind CTX (upper right panel) but not ST (upper left panel). Cells in S-phase (BrdU) do not bind Cy3-CTX (lower left panel). Cy3-CTX-binding cells do not incorporate BrdU.

View larger version (47K): [in a new window]   Fig. 8. PC12 cells also show cell-cycle-dependent expression of receptors for CTX. (a-c) represent identical cells. No binding of Cy2-ST is observed. Cy3-CTX binds almost exclusively to the large peripheral cells; (c) differential interference contrast picture. (d-g) NGF-differentiated cells show increased binding and uptake of Cy3-CTX (d) and (f), whereas undifferentiated cells characterized by their round shape and lack of axonal protrusions (small arrows in (d) and (e)) show little CTX-binding. (h) and (i) PC12 cells in S-phase as characterized by BrdU incorporation (i) do not bind CTX (h).

View larger version (80K): [in a new window]   Fig. 9. Differential binding of Cy2-labeled Shiga toxin and Cy3-labeled Cholera toxin to fetal mouse astrocytes and binding of Cy3-labeled CTX to hippocampal neurons. (a) Binding of Cy3-CTX to astrocytes. Binding conditions were the same as in Fig. 1a. During inspection of the live cells under the fluorescence microscope, some CTX is taken up and accumulates in the Golgi. (b,c) Astrocytes that exhibit significant BrdU incoporation (blue-white nuclei in (c)) show only weak binding of ST (b) or CTX (c). (d,e) Binding and uptake of Cy3-CTX by hippocampal neurons. All axons and dendrites exhibit a strong binding of the toxin. In (e), several neuronal cells on top of a Cy3-CTX-binding astrocyte are depicted.

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