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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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Electron micrographs of control and siRNA-treated cells. The cells in these experiments were incubated with an antibody against the transferrin receptor coupled to 8-nm gold before fixation to monitor the efficiency of transferrin receptor endocytosis. (a and b) Control cells have abundant clathrin-coated pits (large arrowheads) associated with the plasma membrane. Gold particles (small arrowheads) can be seen in endosomes. (c–f) Cells were treated with μ2-2 siRNA. These cells still have clathrin-coated pits associated with the plasma membrane, but when the pits were quantified by morphometry in control and AP-2–depleted cells, they were found to be 12-fold less abundant in the AP-2–depleted cells. Morphologically, the coated pits are similar to those in control cells, but they tend to be smaller. Gold particles (small arrowheads) remain on the cell surface. A clathrin-coated bud or vesicle in the Golgi region (G) is indicated with the arrow in f. (g) Clathrin-depleted cells have no recognizable clathrin-coated pits or vesicles, either at the plasma membrane or on intracellular membranes, although COP-coated vesicles can still be seen on the cis side of the Golgi stack. In addition, membranes on the trans side of the Golgi stack are swollen. Bar, 500 nm.
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fig3: Electron micrographs of control and siRNA-treated cells. The cells in these experiments were incubated with an antibody against the transferrin receptor coupled to 8-nm gold before fixation to monitor the efficiency of transferrin receptor endocytosis. (a and b) Control cells have abundant clathrin-coated pits (large arrowheads) associated with the plasma membrane. Gold particles (small arrowheads) can be seen in endosomes. (c–f) Cells were treated with μ2-2 siRNA. These cells still have clathrin-coated pits associated with the plasma membrane, but when the pits were quantified by morphometry in control and AP-2–depleted cells, they were found to be 12-fold less abundant in the AP-2–depleted cells. Morphologically, the coated pits are similar to those in control cells, but they tend to be smaller. Gold particles (small arrowheads) remain on the cell surface. A clathrin-coated bud or vesicle in the Golgi region (G) is indicated with the arrow in f. (g) Clathrin-depleted cells have no recognizable clathrin-coated pits or vesicles, either at the plasma membrane or on intracellular membranes, although COP-coated vesicles can still be seen on the cis side of the Golgi stack. In addition, membranes on the trans side of the Golgi stack are swollen. Bar, 500 nm.

Mentions: To observe the phenotype of AP-2– and clathrin-depleted cells at the ultrastructural level, EM was performed. Fig. 3 (a and b) shows that control cells contain numerous clathrin-coated pits associated with the plasma membrane, indicated with the large arrowheads. A morphometric analysis (Fig. 4) showed that in these cells, 0.6% of the cell surface is occupied by clathrin-coated pits. In the AP-2–depleted cells (c–e), clathrin-coated pits were found to be 12-fold less abundant, occupying only 0.05% of the cell surface. The morphology of the coat appears to be identical in control and AP-2–depleted cells; however, the coated pits tend to be smaller in the AP-2–depleted cells. In the clathrin-depleted cells, clathrin-coated pits were undetectable, and nearly all of the budding profiles that could be observed at the plasma membrane had the characteristic appearance of caveolae.


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

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

Electron micrographs of control and siRNA-treated cells. The cells in these experiments were incubated with an antibody against the transferrin receptor coupled to 8-nm gold before fixation to monitor the efficiency of transferrin receptor endocytosis. (a and b) Control cells have abundant clathrin-coated pits (large arrowheads) associated with the plasma membrane. Gold particles (small arrowheads) can be seen in endosomes. (c–f) Cells were treated with μ2-2 siRNA. These cells still have clathrin-coated pits associated with the plasma membrane, but when the pits were quantified by morphometry in control and AP-2–depleted cells, they were found to be 12-fold less abundant in the AP-2–depleted cells. Morphologically, the coated pits are similar to those in control cells, but they tend to be smaller. Gold particles (small arrowheads) remain on the cell surface. A clathrin-coated bud or vesicle in the Golgi region (G) is indicated with the arrow in f. (g) Clathrin-depleted cells have no recognizable clathrin-coated pits or vesicles, either at the plasma membrane or on intracellular membranes, although COP-coated vesicles can still be seen on the cis side of the Golgi stack. In addition, membranes on the trans side of the Golgi stack are swollen. Bar, 500 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172830&req=5

fig3: Electron micrographs of control and siRNA-treated cells. The cells in these experiments were incubated with an antibody against the transferrin receptor coupled to 8-nm gold before fixation to monitor the efficiency of transferrin receptor endocytosis. (a and b) Control cells have abundant clathrin-coated pits (large arrowheads) associated with the plasma membrane. Gold particles (small arrowheads) can be seen in endosomes. (c–f) Cells were treated with μ2-2 siRNA. These cells still have clathrin-coated pits associated with the plasma membrane, but when the pits were quantified by morphometry in control and AP-2–depleted cells, they were found to be 12-fold less abundant in the AP-2–depleted cells. Morphologically, the coated pits are similar to those in control cells, but they tend to be smaller. Gold particles (small arrowheads) remain on the cell surface. A clathrin-coated bud or vesicle in the Golgi region (G) is indicated with the arrow in f. (g) Clathrin-depleted cells have no recognizable clathrin-coated pits or vesicles, either at the plasma membrane or on intracellular membranes, although COP-coated vesicles can still be seen on the cis side of the Golgi stack. In addition, membranes on the trans side of the Golgi stack are swollen. Bar, 500 nm.
Mentions: To observe the phenotype of AP-2– and clathrin-depleted cells at the ultrastructural level, EM was performed. Fig. 3 (a and b) shows that control cells contain numerous clathrin-coated pits associated with the plasma membrane, indicated with the large arrowheads. A morphometric analysis (Fig. 4) showed that in these cells, 0.6% of the cell surface is occupied by clathrin-coated pits. In the AP-2–depleted cells (c–e), clathrin-coated pits were found to be 12-fold less abundant, occupying only 0.05% of the cell surface. The morphology of the coat appears to be identical in control and AP-2–depleted cells; however, the coated pits tend to be smaller in the AP-2–depleted cells. In the clathrin-depleted cells, clathrin-coated pits were undetectable, and nearly all of the budding profiles that could be observed at the plasma membrane had the characteristic appearance of caveolae.

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