cells bsc1 monkey kidney epithelial cells  (ATCC)

Journal:

Article Title: Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways D⃞V⃞

doi: 10.1091/mbc.E04-04-0283

Figure Lengend Snippet: Vesicles and tubules internalize CT to a perinuclear endosomal compartment. (A) CT traffics in vesicles (arrows) and tubules (arrowheads) from the plasma membrane toward the perinuclear endosomal/Golgi area (Movie 1). BSC1 cells were incubated with 20 nM nontoxic mutant CT(E112D) labeled with Alexa594 (red) at 37°C for 45 min. (B) CT enters BSC1 cells via both vesicles and tubules (arrowheads). BSC1 cells were incubated with 20 nM CT(E112D) at 4°C for 30 min, shifted to 37°C for 2 min, and then the time course of toxin internalization was recorded (inset and Movie 3). Nucleus was stained with Hoechst (blue). (C) Endocytic tubules containing CT (arrowhead) extend from the cell surface toward a perinuclear endosomal compartment in close contact with the Golgi complex (GalT-EGFP, green). BSC1 cells were incubated as described in B. Selected snapshots of time course of CT internalization are shown. The plot shows the time course of CT appearance in endosomes (red line) within the indicated area (red stippled circle); after this brief uptake period, almost no CT reaches the Golgi, including the regions in proximity with the tubules (* in image and green line in plot). Colocalization of CT with the Golgi region was calculated by r analysis as explained in MATERIALS AND METHODS. (D) CT is first transported to endosomes before reaching the Golgi complex. CT(E112D) was added to BSC1/GalT-EGFP cells at 37°C during recording of time lapse. Selected snapshots are shown after 0, 15, and 30 min of CT addition. Colocalization of CT with the Golgi region (stippled box) was calculated as described in C. The plot shows the continuous accumulation of CT in endosomes (red line) and Golgi complex (green line). Bars, 20 μm (whole cell) and 10 μm (cropped areas).

Article Snippet: Cells BSC1 monkey kidney epithelial cells (ATCC CCL-26), and Y-1 mouse adrenal cells (ATCC CCL-79) were grown in DMEM or RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nonessential amino acids (0.1 mM).

Techniques: Clinical Proteomics, Membrane, Incubation, Mutagenesis, Labeling, Staining

Journal:

Article Title: Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways D⃞V⃞

doi: 10.1091/mbc.E04-04-0283

Figure Lengend Snippet: Cholera toxin colocalizes with markers of the clathrin-, caveolin-, and Arf6-dependent endocytic pathways. BSC1 cells were incubated with 20 nM CT(E112D) and 50 μg/ml Alexa 647-hTf for 15 min (top two rows) or 45 min at 37°C and fixed. Colocalization between CT and AP-2, EEA1, Arf6, or MHC-I was established by immunofluorescence. Caveolin 1 and EHD1 were tagged with EGFP and transiently expressed in BSC1 cells. Arrow-heads indicate colocalization of CT with AP-2 (presumably in clathrin-coated pits and vesicles), with EEA1 (early endosomes) together with internalized Tf. Representative confocal images from ventral (top row) or middle sections of cells (all others) are shown. Selected regions (stippled-line boxes) were cropped, enlarged, and displayed using an inverted monochrome color scale to aid visualization. Bar, 20 μm.

Article Snippet: Cells BSC1 monkey kidney epithelial cells (ATCC CCL-26), and Y-1 mouse adrenal cells (ATCC CCL-79) were grown in DMEM or RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nonessential amino acids (0.1 mM).

Techniques: Incubation, Immunofluorescence

Journal:

Article Title: Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways D⃞V⃞

doi: 10.1091/mbc.E04-04-0283

Figure Lengend Snippet: Clathrin and caveolin-dependent pathways mediate CT internalization. (A) BSC1 cells stably expressing EYFP-LCA cells were incubated with 20 nM CT(E112D) for 30 min at 37°C, washed, and processed for time-lapse recording. A clathrin-coated pit-like structure, selected from the dorsal cell surface (image 1, stippled-line box, and enlarged in images 2 and 3), containing CT was analyzed with the aid of a kymograph plot for clathrin and toxin content over time (images 4–6). (B) CT(E112D) was added during the time-lapse acquisition to BSC1 cells transiently expressing Cav1-EGFP. The time-lapse series (stippled-line box) shows entry of CT into a caveolin 1-containing tubulo-vesicular structure (Movie 7, a and b). The initial short tubular invagination stretches and eventually disconnects from the cell surface. Bars, 20 μm (whole cell) and 5 and 3 μm (cropped regions).

Article Snippet: Cells BSC1 monkey kidney epithelial cells (ATCC CCL-26), and Y-1 mouse adrenal cells (ATCC CCL-79) were grown in DMEM or RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nonessential amino acids (0.1 mM).

Techniques: Stable Transfection, Expressing, Incubation

Journal:

Article Title: Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways D⃞V⃞

doi: 10.1091/mbc.E04-04-0283

Figure Lengend Snippet: Combined inhibition of the clathrin-, caveolin-, and Arf6-dependent pathways strongly inhibits CT entry. (A) Inhibition of clathrin-, caveolin-, or Arf6-dependent pathways has minimal effects on CT entry. Representative images of BSC1 cells transiently expressing EGFP-Eps15Δ95-295, Cav1S80E-myc, Arf6Q67L-HA, Arf6T27N-HA for 24–48 h or treated with 100 nM PMA for 30 min (treatment known to disrupt caveolae) are shown. The block in Tf uptake assessed the inhibition of clathrin-mediated endocytosis; inhibition of caveolin-mediated uptake resulted in a 25% reduction in simian virus 40 infectivity (our unpublished data); and interference with the Arf6-dependent pathway by overexpression of Arf6Q67L resulted in the expected vacuolization of the endosomal compartment (Naslavsky et al., 2003 ). (B) Combined inhibition of clathrin- and caveolin-dependent pathways partially inhibits CT entry. Representative images of BSC1 cells treated with 10 mM methyl-β-cyclodextrin for 30 min or overexpressing Dyn1K44A-HA for 48 h are shown. Under both conditions, Tf uptake was strongly inhibited. Although the retention of CT at the cell surface increased upon cholesterol depletion, and in less degree by interference with dynamin-dependent pathways, its internalization was partially prevented. The number of tubules containing CT increased upon expression of dynamin 1 or 2 K44A mutants, which now contain Tf, Cav1-EGFP, Arf6, EGFP-EHD1, and EGFP-MHC-I (arrowheads and Fig. s2 in Supplement). (C) Coexpression of Dyn2K44A and Arf6Q67L or Arf6T27N strongly prevents CT entry and abolished tubule formation. (A–C) Representative confocal images from middle sections of cells (n = 40–60) displayed using an inverted monochrome scale to aid visualization. Transfected cells were identified by imaging EGFP or by staining for the myc or HA epitopes fused to the corresponding overexpressed proteins. In coexpression of Arf6-HA mutants with DynK44A, we used Dyn2K44A-EGFP. Bars, 20 μm.

Article Snippet: Cells BSC1 monkey kidney epithelial cells (ATCC CCL-26), and Y-1 mouse adrenal cells (ATCC CCL-79) were grown in DMEM or RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nonessential amino acids (0.1 mM).

Techniques: Inhibition, Expressing, Blocking Assay, Virus, Infection, Over Expression, Transfection, Imaging, Staining

Journal:

Article Title: Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways D⃞V⃞

doi: 10.1091/mbc.E04-04-0283

Figure Lengend Snippet: Decrease of cholesterol or interference with dynamin function inhibits CT transport to the TGN and the ER. Transport of CT to the TGN (A) and ER (B) in cells subjected to different conditions that perturb endocytosis was assessed by colocalization of the toxin with TGN46 or PDI, respectively. BSC1 cells either expressing the indicated dominant proteins for 24–48 h or treated with nocodazole or methyl-β-cyclodextrin for 30 min were incubated with CT(E112D) for 45 min (A) or 90 min (B) at 37°C. Transfected cells were identified as described in Figure 4. For each condition, representative confocal images are shown (out of at least 50 cells analyzed). Selected regions (stippled-line boxes) of the TGN and ER nuclear envelope were cropped, enlarged, and displayed using an inverted monochrome color scale to facilitate inspection of colocalization. Expression of Dyn1K44A or depletion of cholesterol (by treatment with 10 mM methyl-β-cyclodextrin) significantly reduced CT transport to the TGN and ER, whereas inhibition of single endocytic pathways had no effect. In contrast, microtubule depolymerization (by treatment with 10 μM nocodazole) only inhibited retrograde transport the ER. Bars, 20 μm.

Article Snippet: Cells BSC1 monkey kidney epithelial cells (ATCC CCL-26), and Y-1 mouse adrenal cells (ATCC CCL-79) were grown in DMEM or RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nonessential amino acids (0.1 mM).

Techniques: Expressing, Incubation, Transfection, Inhibition

Journal:

Article Title: Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways D⃞V⃞

doi: 10.1091/mbc.E04-04-0283

Figure Lengend Snippet: Vesicles and tubules internalize CT to a perinuclear endosomal compartment. (A) CT traffics in vesicles (arrows) and tubules (arrowheads) from the plasma membrane toward the perinuclear endosomal/Golgi area (Movie 1). BSC1 cells were incubated with 20 nM nontoxic mutant CT(E112D) labeled with Alexa594 (red) at 37°C for 45 min. (B) CT enters BSC1 cells via both vesicles and tubules (arrowheads). BSC1 cells were incubated with 20 nM CT(E112D) at 4°C for 30 min, shifted to 37°C for 2 min, and then the time course of toxin internalization was recorded (inset and Movie 3). Nucleus was stained with Hoechst (blue). (C) Endocytic tubules containing CT (arrowhead) extend from the cell surface toward a perinuclear endosomal compartment in close contact with the Golgi complex (GalT-EGFP, green). BSC1 cells were incubated as described in B. Selected snapshots of time course of CT internalization are shown. The plot shows the time course of CT appearance in endosomes (red line) within the indicated area (red stippled circle); after this brief uptake period, almost no CT reaches the Golgi, including the regions in proximity with the tubules (* in image and green line in plot). Colocalization of CT with the Golgi region was calculated by r analysis as explained in MATERIALS AND METHODS. (D) CT is first transported to endosomes before reaching the Golgi complex. CT(E112D) was added to BSC1/GalT-EGFP cells at 37°C during recording of time lapse. Selected snapshots are shown after 0, 15, and 30 min of CT addition. Colocalization of CT with the Golgi region (stippled box) was calculated as described in C. The plot shows the continuous accumulation of CT in endosomes (red line) and Golgi complex (green line). Bars, 20 μm (whole cell) and 10 μm (cropped areas).

Article Snippet: BSC1 monkey kidney epithelial cells (ATCC CCL-26), and Y-1 mouse adrenal cells (ATCC CCL-79) were grown in DMEM or RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nonessential amino acids (0.1 mM).

Techniques: Clinical Proteomics, Membrane, Incubation, Mutagenesis, Labeling, Staining

Journal:

Article Title: Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways D⃞V⃞

doi: 10.1091/mbc.E04-04-0283

Figure Lengend Snippet: Cholera toxin colocalizes with markers of the clathrin-, caveolin-, and Arf6-dependent endocytic pathways. BSC1 cells were incubated with 20 nM CT(E112D) and 50 μg/ml Alexa 647-hTf for 15 min (top two rows) or 45 min at 37°C and fixed. Colocalization between CT and AP-2, EEA1, Arf6, or MHC-I was established by immunofluorescence. Caveolin 1 and EHD1 were tagged with EGFP and transiently expressed in BSC1 cells. Arrow-heads indicate colocalization of CT with AP-2 (presumably in clathrin-coated pits and vesicles), with EEA1 (early endosomes) together with internalized Tf. Representative confocal images from ventral (top row) or middle sections of cells (all others) are shown. Selected regions (stippled-line boxes) were cropped, enlarged, and displayed using an inverted monochrome color scale to aid visualization. Bar, 20 μm.

Article Snippet: BSC1 monkey kidney epithelial cells (ATCC CCL-26), and Y-1 mouse adrenal cells (ATCC CCL-79) were grown in DMEM or RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nonessential amino acids (0.1 mM).

Techniques: Incubation, Immunofluorescence

Journal:

Article Title: Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways D⃞V⃞

doi: 10.1091/mbc.E04-04-0283

Figure Lengend Snippet: Clathrin and caveolin-dependent pathways mediate CT internalization. (A) BSC1 cells stably expressing EYFP-LCA cells were incubated with 20 nM CT(E112D) for 30 min at 37°C, washed, and processed for time-lapse recording. A clathrin-coated pit-like structure, selected from the dorsal cell surface (image 1, stippled-line box, and enlarged in images 2 and 3), containing CT was analyzed with the aid of a kymograph plot for clathrin and toxin content over time (images 4–6). (B) CT(E112D) was added during the time-lapse acquisition to BSC1 cells transiently expressing Cav1-EGFP. The time-lapse series (stippled-line box) shows entry of CT into a caveolin 1-containing tubulo-vesicular structure (Movie 7, a and b). The initial short tubular invagination stretches and eventually disconnects from the cell surface. Bars, 20 μm (whole cell) and 5 and 3 μm (cropped regions).

Article Snippet: BSC1 monkey kidney epithelial cells (ATCC CCL-26), and Y-1 mouse adrenal cells (ATCC CCL-79) were grown in DMEM or RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nonessential amino acids (0.1 mM).

Techniques: Stable Transfection, Expressing, Incubation

Journal:

Article Title: Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways D⃞V⃞

doi: 10.1091/mbc.E04-04-0283

Figure Lengend Snippet: Combined inhibition of the clathrin-, caveolin-, and Arf6-dependent pathways strongly inhibits CT entry. (A) Inhibition of clathrin-, caveolin-, or Arf6-dependent pathways has minimal effects on CT entry. Representative images of BSC1 cells transiently expressing EGFP-Eps15Δ95-295, Cav1S80E-myc, Arf6Q67L-HA, Arf6T27N-HA for 24–48 h or treated with 100 nM PMA for 30 min (treatment known to disrupt caveolae) are shown. The block in Tf uptake assessed the inhibition of clathrin-mediated endocytosis; inhibition of caveolin-mediated uptake resulted in a 25% reduction in simian virus 40 infectivity (our unpublished data); and interference with the Arf6-dependent pathway by overexpression of Arf6Q67L resulted in the expected vacuolization of the endosomal compartment (Naslavsky et al., 2003 ). (B) Combined inhibition of clathrin- and caveolin-dependent pathways partially inhibits CT entry. Representative images of BSC1 cells treated with 10 mM methyl-β-cyclodextrin for 30 min or overexpressing Dyn1K44A-HA for 48 h are shown. Under both conditions, Tf uptake was strongly inhibited. Although the retention of CT at the cell surface increased upon cholesterol depletion, and in less degree by interference with dynamin-dependent pathways, its internalization was partially prevented. The number of tubules containing CT increased upon expression of dynamin 1 or 2 K44A mutants, which now contain Tf, Cav1-EGFP, Arf6, EGFP-EHD1, and EGFP-MHC-I (arrowheads and Fig. s2 in Supplement). (C) Coexpression of Dyn2K44A and Arf6Q67L or Arf6T27N strongly prevents CT entry and abolished tubule formation. (A–C) Representative confocal images from middle sections of cells (n = 40–60) displayed using an inverted monochrome scale to aid visualization. Transfected cells were identified by imaging EGFP or by staining for the myc or HA epitopes fused to the corresponding overexpressed proteins. In coexpression of Arf6-HA mutants with DynK44A, we used Dyn2K44A-EGFP. Bars, 20 μm.

Article Snippet: BSC1 monkey kidney epithelial cells (ATCC CCL-26), and Y-1 mouse adrenal cells (ATCC CCL-79) were grown in DMEM or RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nonessential amino acids (0.1 mM).

Techniques: Inhibition, Expressing, Blocking Assay, Virus, Infection, Over Expression, Transfection, Imaging, Staining

Journal:

Article Title: Cholera Toxin Toxicity Does Not Require Functional Arf6- and Dynamin-dependent Endocytic Pathways D⃞V⃞

doi: 10.1091/mbc.E04-04-0283

Figure Lengend Snippet: Decrease of cholesterol or interference with dynamin function inhibits CT transport to the TGN and the ER. Transport of CT to the TGN (A) and ER (B) in cells subjected to different conditions that perturb endocytosis was assessed by colocalization of the toxin with TGN46 or PDI, respectively. BSC1 cells either expressing the indicated dominant proteins for 24–48 h or treated with nocodazole or methyl-β-cyclodextrin for 30 min were incubated with CT(E112D) for 45 min (A) or 90 min (B) at 37°C. Transfected cells were identified as described in Figure 4. For each condition, representative confocal images are shown (out of at least 50 cells analyzed). Selected regions (stippled-line boxes) of the TGN and ER nuclear envelope were cropped, enlarged, and displayed using an inverted monochrome color scale to facilitate inspection of colocalization. Expression of Dyn1K44A or depletion of cholesterol (by treatment with 10 mM methyl-β-cyclodextrin) significantly reduced CT transport to the TGN and ER, whereas inhibition of single endocytic pathways had no effect. In contrast, microtubule depolymerization (by treatment with 10 μM nocodazole) only inhibited retrograde transport the ER. Bars, 20 μm.

Article Snippet: BSC1 monkey kidney epithelial cells (ATCC CCL-26), and Y-1 mouse adrenal cells (ATCC CCL-79) were grown in DMEM or RPMI 1640 medium, respectively, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nonessential amino acids (0.1 mM).

Techniques: Expressing, Incubation, Transfection, Inhibition