Figure 5.
Figure 5. Dynamin inhibition or silencing does not affect αBTX-induced AChR endocytosis. (A–D) CHO-K1/A5 cells were transiently transfected with GFP-K44A dynamin (outlined cells in A and C), and, 12 h later, cells were labeled with Cy3αBTX (A) or b-Cy3αBTX (C) and incubated at 37°C for an additional 6-h period. At the end of the incubation, Cy5-Tf was added to the cells in A to assess the effect of dynamin K44A on inhibition of clathrin-dependent endocytosis. The histogram in B shows the extent of inhibition of Tf endocytosis relative to untransfected controls. KI quenching (KI) and Cy5-SA accessibility (SA) were used to quantify the effect of dynamin K44A in transfected cells with respect to untransfected (control) cells as described in Fig. 4 (A and B), and the resultant data are shown as histograms in D. Bars represent the accessibility of b-Cy3-αBTX–labeled AChR to Cy5-SA and KI in control and untransfected cells normalized to 0 h. Weighted mean and errors from the mean were obtained from four independent experiments, each consisting of two coverslip dishes from which at least 30 cells per dish were analyzed. Error bars represent SD. *, P < 0.005; **, P > 0.1. (E) Cells transfected with a dominant-negative eps15 construct, EH21-GFP, for 14 h were incubated with Cy3-αBTX at 4°C for 1 h and chased for 6 h at 37°C. During the last 20 min of chase, Cy5-Tf was added to the cells. Cells were washed, and surface Tf was stripped off, fixed, and imaged. Note that cells expressing EH21 show reduction in Tf uptake as compared with surrounding cells, whereas internalized αBTX remains unaffected. In each experiment, at least 15 cells were analyzed, and 40% of cells showed a reduction in Tf uptake, whereas αBTX internalization is unaffected in that population. (F–J) CHO-K1/A5 cells were labeled with mAb 35 on ice and were either directly incubated with secondary antibody before (0 h) or after chase for 6 h at 37°C (F). Surface levels of AChR in cells treated with transfected pGSUPER-based dynamin shRNA-expressing constructs targeted against human (h) or mouse (m) dynamin 2 were quantified by measuring the level of labeled secondary antibody staining against mAb 35 remaining at the cell surface in transfected or untransfected cells, respectively, and were normalized to the level of secondary antibody staining at 0 h. Cells transfected with shRNA plasmids were identified by GFP expression from pGSUPER vector (corresponding images are not depicted). The histogram in G represents the weighted mean of average values obtained from at least 60 cells per dish in each case. *, P < 0.0001; **, P > 0.1. CHO-K1/A5 cells were transfected with dynamin shRNA-expressing constructs and, 68–72 h later, were either monitored for Tf uptake (H) or for dynamin levels (I and J). The histogram in H represents Tf-AlexaFluor546 uptake (ratio of internalized/surface Tf fluorescence) in cells transfected with shRNA constructs, and the ratio was further normalized to uptake in control. Each bar in the histogram represents weighted mean of averages ± SEM (error bars). *, P < 0.001. To assess dynamin levels, CHO-K1/A5 cells were transfected with shRNA vectors, harvested 72 h after transfection, and lysed, and levels of dynamin were detected on Western blots (I). Blots represent dynamin levels, and bottom lanes represent loading controls. The histogram in J shows quantification of dynamin levels in CHO-K1/A5 cells. Each bar represents the mean of dynamin levels normalized to actin levels per lane taken from two independent experiments, further normalized to the control in each case ± SD (error bars). Bars: (C) 20 μm; (F) 10 μm.

Dynamin inhibition or silencing does not affect αBTX-induced AChR endocytosis. (A–D) CHO-K1/A5 cells were transiently transfected with GFP-K44A dynamin (outlined cells in A and C), and, 12 h later, cells were labeled with Cy3αBTX (A) or b-Cy3αBTX (C) and incubated at 37°C for an additional 6-h period. At the end of the incubation, Cy5-Tf was added to the cells in A to assess the effect of dynamin K44A on inhibition of clathrin-dependent endocytosis. The histogram in B shows the extent of inhibition of Tf endocytosis relative to untransfected controls. KI quenching (KI) and Cy5-SA accessibility (SA) were used to quantify the effect of dynamin K44A in transfected cells with respect to untransfected (control) cells as described in Fig. 4 (A and B), and the resultant data are shown as histograms in D. Bars represent the accessibility of b-Cy3-αBTX–labeled AChR to Cy5-SA and KI in control and untransfected cells normalized to 0 h. Weighted mean and errors from the mean were obtained from four independent experiments, each consisting of two coverslip dishes from which at least 30 cells per dish were analyzed. Error bars represent SD. *, P < 0.005; **, P > 0.1. (E) Cells transfected with a dominant-negative eps15 construct, EH21-GFP, for 14 h were incubated with Cy3-αBTX at 4°C for 1 h and chased for 6 h at 37°C. During the last 20 min of chase, Cy5-Tf was added to the cells. Cells were washed, and surface Tf was stripped off, fixed, and imaged. Note that cells expressing EH21 show reduction in Tf uptake as compared with surrounding cells, whereas internalized αBTX remains unaffected. In each experiment, at least 15 cells were analyzed, and 40% of cells showed a reduction in Tf uptake, whereas αBTX internalization is unaffected in that population. (F–J) CHO-K1/A5 cells were labeled with mAb 35 on ice and were either directly incubated with secondary antibody before (0 h) or after chase for 6 h at 37°C (F). Surface levels of AChR in cells treated with transfected pGSUPER-based dynamin shRNA-expressing constructs targeted against human (h) or mouse (m) dynamin 2 were quantified by measuring the level of labeled secondary antibody staining against mAb 35 remaining at the cell surface in transfected or untransfected cells, respectively, and were normalized to the level of secondary antibody staining at 0 h. Cells transfected with shRNA plasmids were identified by GFP expression from pGSUPER vector (corresponding images are not depicted). The histogram in G represents the weighted mean of average values obtained from at least 60 cells per dish in each case. *, P < 0.0001; **, P > 0.1. CHO-K1/A5 cells were transfected with dynamin shRNA-expressing constructs and, 68–72 h later, were either monitored for Tf uptake (H) or for dynamin levels (I and J). The histogram in H represents Tf-AlexaFluor546 uptake (ratio of internalized/surface Tf fluorescence) in cells transfected with shRNA constructs, and the ratio was further normalized to uptake in control. Each bar in the histogram represents weighted mean of averages ± SEM (error bars). *, P < 0.001. To assess dynamin levels, CHO-K1/A5 cells were transfected with shRNA vectors, harvested 72 h after transfection, and lysed, and levels of dynamin were detected on Western blots (I). Blots represent dynamin levels, and bottom lanes represent loading controls. The histogram in J shows quantification of dynamin levels in CHO-K1/A5 cells. Each bar represents the mean of dynamin levels normalized to actin levels per lane taken from two independent experiments, further normalized to the control in each case ± SD (error bars). Bars: (C) 20 μm; (F) 10 μm.

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