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Clathrin-mediated endocytosis in AP-2-depleted cells.

Motley A, Bright NA, Seaman MN, Robinson MS - J. Cell Biol. (2003)

Bottom Line: Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2-depleted cells.These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor.Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge, Department of Clinical Biochemistry, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK.

ABSTRACT
We have used RNA interference to knock down the AP-2 mu2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2-depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2-depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2-depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

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Kinetics of ligand uptake in control and siRNA-treated cells. (a) Cells were allowed to bind 125I-labeled transferrin at 4°C, and were then shifted to 37°C for the indicated length of time, after which the medium was harvested, ligand remaining on the cell surface was released with an acid wash, the cells were solubilized with NaOH, and all three fractions were quantified and expressed as a percentage of total counts/m. The graph shows percentage of total counts recovered in the NaOH extract (i.e., internalized transferrin). Each point is derived from at least three separate experiments; the error bars show the SEM. Both μ2- and clathrin-depleted cells are strongly impaired in their ability to internalize transferrin. (b) Cells were treated exactly as in a, but using 125I-labeled EGF. Uptake is strongly inhibited in the clathrin-depleted cells, but it is not significantly different from control in the μ2-depleted cells. (c) Cells expressing a chimera consisting of the CD8 extracellular and lumenal domains fused to the LDL receptor cytoplasmic tail were incubated at 4°C with an mAb against CD8 followed by 125I-labeled protein A, and were then shifted to 37°C and treated as in a and b. Again, uptake is strongly inhibited in the clathrin-depleted cells, but similar to controls in the μ2-depleted cells. Virtually identical results were obtained using α-depleted cells, except that the effect on transferrin uptake was not quite so profound, presumably because the knockdown was less complete (not depicted).
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fig5: Kinetics of ligand uptake in control and siRNA-treated cells. (a) Cells were allowed to bind 125I-labeled transferrin at 4°C, and were then shifted to 37°C for the indicated length of time, after which the medium was harvested, ligand remaining on the cell surface was released with an acid wash, the cells were solubilized with NaOH, and all three fractions were quantified and expressed as a percentage of total counts/m. The graph shows percentage of total counts recovered in the NaOH extract (i.e., internalized transferrin). Each point is derived from at least three separate experiments; the error bars show the SEM. Both μ2- and clathrin-depleted cells are strongly impaired in their ability to internalize transferrin. (b) Cells were treated exactly as in a, but using 125I-labeled EGF. Uptake is strongly inhibited in the clathrin-depleted cells, but it is not significantly different from control in the μ2-depleted cells. (c) Cells expressing a chimera consisting of the CD8 extracellular and lumenal domains fused to the LDL receptor cytoplasmic tail were incubated at 4°C with an mAb against CD8 followed by 125I-labeled protein A, and were then shifted to 37°C and treated as in a and b. Again, uptake is strongly inhibited in the clathrin-depleted cells, but similar to controls in the μ2-depleted cells. Virtually identical results were obtained using α-depleted cells, except that the effect on transferrin uptake was not quite so profound, presumably because the knockdown was less complete (not depicted).

Mentions: Fig. 5 a shows the effects of AP-2 and clathrin knockdown on transferrin receptor endocytosis. For these experiments, the cells were incubated at 4°C with 125I-labeled transferrin to allow binding (but not internalization) to occur, and then were warmed to 37°C for various lengths of time. At the end of the incubation, the medium was harvested, surface-bound ligand was stripped off at low pH, the cells were solubilized in 1 M NaOH, and the label in all three fractions was quantified. The percentage of counts in the NaOH extract (i.e., intracellular counts) is shown in the graph. In control cells, >50% of the prebound transferrin is internalized within 5 min after warm-up, but after 10 min, the amount of intracellular transferrin starts to go down, as it gets recycled back into the medium. Knocking down both AP-2 and clathrin causes a profound inhibition of transferrin uptake. In both cases, the transferrin never accumulates inside the cells, but remains primarily on the cell surface, where it slowly dissociates into the medium. Even after 30 min, <10% of the transferrin is recovered in the intracellular fraction. Thus, as expected, both AP-2 and clathrin are required for efficient internalization of the transferrin receptor.


Clathrin-mediated endocytosis in AP-2-depleted cells.

Motley A, Bright NA, Seaman MN, Robinson MS - J. Cell Biol. (2003)

Kinetics of ligand uptake in control and siRNA-treated cells. (a) Cells were allowed to bind 125I-labeled transferrin at 4°C, and were then shifted to 37°C for the indicated length of time, after which the medium was harvested, ligand remaining on the cell surface was released with an acid wash, the cells were solubilized with NaOH, and all three fractions were quantified and expressed as a percentage of total counts/m. The graph shows percentage of total counts recovered in the NaOH extract (i.e., internalized transferrin). Each point is derived from at least three separate experiments; the error bars show the SEM. Both μ2- and clathrin-depleted cells are strongly impaired in their ability to internalize transferrin. (b) Cells were treated exactly as in a, but using 125I-labeled EGF. Uptake is strongly inhibited in the clathrin-depleted cells, but it is not significantly different from control in the μ2-depleted cells. (c) Cells expressing a chimera consisting of the CD8 extracellular and lumenal domains fused to the LDL receptor cytoplasmic tail were incubated at 4°C with an mAb against CD8 followed by 125I-labeled protein A, and were then shifted to 37°C and treated as in a and b. Again, uptake is strongly inhibited in the clathrin-depleted cells, but similar to controls in the μ2-depleted cells. Virtually identical results were obtained using α-depleted cells, except that the effect on transferrin uptake was not quite so profound, presumably because the knockdown was less complete (not depicted).
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fig5: Kinetics of ligand uptake in control and siRNA-treated cells. (a) Cells were allowed to bind 125I-labeled transferrin at 4°C, and were then shifted to 37°C for the indicated length of time, after which the medium was harvested, ligand remaining on the cell surface was released with an acid wash, the cells were solubilized with NaOH, and all three fractions were quantified and expressed as a percentage of total counts/m. The graph shows percentage of total counts recovered in the NaOH extract (i.e., internalized transferrin). Each point is derived from at least three separate experiments; the error bars show the SEM. Both μ2- and clathrin-depleted cells are strongly impaired in their ability to internalize transferrin. (b) Cells were treated exactly as in a, but using 125I-labeled EGF. Uptake is strongly inhibited in the clathrin-depleted cells, but it is not significantly different from control in the μ2-depleted cells. (c) Cells expressing a chimera consisting of the CD8 extracellular and lumenal domains fused to the LDL receptor cytoplasmic tail were incubated at 4°C with an mAb against CD8 followed by 125I-labeled protein A, and were then shifted to 37°C and treated as in a and b. Again, uptake is strongly inhibited in the clathrin-depleted cells, but similar to controls in the μ2-depleted cells. Virtually identical results were obtained using α-depleted cells, except that the effect on transferrin uptake was not quite so profound, presumably because the knockdown was less complete (not depicted).
Mentions: Fig. 5 a shows the effects of AP-2 and clathrin knockdown on transferrin receptor endocytosis. For these experiments, the cells were incubated at 4°C with 125I-labeled transferrin to allow binding (but not internalization) to occur, and then were warmed to 37°C for various lengths of time. At the end of the incubation, the medium was harvested, surface-bound ligand was stripped off at low pH, the cells were solubilized in 1 M NaOH, and the label in all three fractions was quantified. The percentage of counts in the NaOH extract (i.e., intracellular counts) is shown in the graph. In control cells, >50% of the prebound transferrin is internalized within 5 min after warm-up, but after 10 min, the amount of intracellular transferrin starts to go down, as it gets recycled back into the medium. Knocking down both AP-2 and clathrin causes a profound inhibition of transferrin uptake. In both cases, the transferrin never accumulates inside the cells, but remains primarily on the cell surface, where it slowly dissociates into the medium. Even after 30 min, <10% of the transferrin is recovered in the intracellular fraction. Thus, as expected, both AP-2 and clathrin are required for efficient internalization of the transferrin receptor.

Bottom Line: Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2-depleted cells.These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor.Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge, Department of Clinical Biochemistry, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK.

ABSTRACT
We have used RNA interference to knock down the AP-2 mu2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2-depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2-depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2-depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

Show MeSH
Related in: MedlinePlus