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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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Related in: MedlinePlus

Morphometric analysis of the EM data, using 128 images of each condition. For the control cells, 7,630 plasma membrane intersections were scored, of which 45 were coated pit intersections (0.6%). For the μ2-2–treated cells, 11,361 plasma membrane intersections were scored, of which six were coated pit intersections (0.05%). For the chc-2–treated cells, 7,585 plasma membrane intersections were scored, of which none were coated pit intersections.
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fig4: Morphometric analysis of the EM data, using 128 images of each condition. For the control cells, 7,630 plasma membrane intersections were scored, of which 45 were coated pit intersections (0.6%). For the μ2-2–treated cells, 11,361 plasma membrane intersections were scored, of which six were coated pit intersections (0.05%). For the chc-2–treated cells, 7,585 plasma membrane intersections were scored, of which none were coated pit intersections.

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)

Morphometric analysis of the EM data, using 128 images of each condition. For the control cells, 7,630 plasma membrane intersections were scored, of which 45 were coated pit intersections (0.6%). For the μ2-2–treated cells, 11,361 plasma membrane intersections were scored, of which six were coated pit intersections (0.05%). For the chc-2–treated cells, 7,585 plasma membrane intersections were scored, of which none were coated pit intersections.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172830&req=5

fig4: Morphometric analysis of the EM data, using 128 images of each condition. For the control cells, 7,630 plasma membrane intersections were scored, of which 45 were coated pit intersections (0.6%). For the μ2-2–treated cells, 11,361 plasma membrane intersections were scored, of which six were coated pit intersections (0.05%). For the chc-2–treated cells, 7,585 plasma membrane intersections were scored, of which none were coated pit intersections.
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