Limits...
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.

Show MeSH

Related in: MedlinePlus

Immunofluorescence triple labeling of control and siRNA-treated cells plated together. (a–c and g). Cells treated with μ2-2 and control cells were labeled with mouse anti-α-adaptin (a; blue in g), rabbit anti-clathrin (b; red in g), and goat anti-epsin 1 (c; green in g). α-Adaptin is cytosolic in the μ2-depleted cells. Clathrin is still membrane-associated; however, many of these membranes are intracellular. Epsin spots are reduced in number, but are still present in the μ2-depleted cells. Many of these spots are also positive for clathrin. The two boxed-in regions are shown at higher magnification in the bottom right corner of all the panels in a–c. (d–f and h) Cells treated with chc-2 and control cells were labeled with mouse anti-α-adaptin (d; blue in h), rabbit anti-clathrin (e; red in h), and goat anti-epsin (f; green in h). Clathrin is undetectable in the siRNA-treated cells. α-Adaptin labeling is not markedly different; however, epsin labeling is brighter in the clathrin-depleted cells. Bars, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172830&req=5

fig2: Immunofluorescence triple labeling of control and siRNA-treated cells plated together. (a–c and g). Cells treated with μ2-2 and control cells were labeled with mouse anti-α-adaptin (a; blue in g), rabbit anti-clathrin (b; red in g), and goat anti-epsin 1 (c; green in g). α-Adaptin is cytosolic in the μ2-depleted cells. Clathrin is still membrane-associated; however, many of these membranes are intracellular. Epsin spots are reduced in number, but are still present in the μ2-depleted cells. Many of these spots are also positive for clathrin. The two boxed-in regions are shown at higher magnification in the bottom right corner of all the panels in a–c. (d–f and h) Cells treated with chc-2 and control cells were labeled with mouse anti-α-adaptin (d; blue in h), rabbit anti-clathrin (e; red in h), and goat anti-epsin (f; green in h). Clathrin is undetectable in the siRNA-treated cells. α-Adaptin labeling is not markedly different; however, epsin labeling is brighter in the clathrin-depleted cells. Bars, 20 μm.

Mentions: Fig. 2 shows that normally, the three proteins have overlapping distributions at the plasma membrane, with additional intracellular structures labeled with the clathrin antibody. Knocking down μ2 causes a reduction in the number of epsin spots (c). These spots do not contain any detectable α-adaptin, which is now diffuse and cytosolic (a). However, many of the epsin-positive spots in these cells are also positive for clathrin (b; insets), suggesting that the two proteins are able to co-assemble on the plasma membrane even in the absence of AP-2.


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

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

Immunofluorescence triple labeling of control and siRNA-treated cells plated together. (a–c and g). Cells treated with μ2-2 and control cells were labeled with mouse anti-α-adaptin (a; blue in g), rabbit anti-clathrin (b; red in g), and goat anti-epsin 1 (c; green in g). α-Adaptin is cytosolic in the μ2-depleted cells. Clathrin is still membrane-associated; however, many of these membranes are intracellular. Epsin spots are reduced in number, but are still present in the μ2-depleted cells. Many of these spots are also positive for clathrin. The two boxed-in regions are shown at higher magnification in the bottom right corner of all the panels in a–c. (d–f and h) Cells treated with chc-2 and control cells were labeled with mouse anti-α-adaptin (d; blue in h), rabbit anti-clathrin (e; red in h), and goat anti-epsin (f; green in h). Clathrin is undetectable in the siRNA-treated cells. α-Adaptin labeling is not markedly different; however, epsin labeling is brighter in the clathrin-depleted cells. Bars, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Immunofluorescence triple labeling of control and siRNA-treated cells plated together. (a–c and g). Cells treated with μ2-2 and control cells were labeled with mouse anti-α-adaptin (a; blue in g), rabbit anti-clathrin (b; red in g), and goat anti-epsin 1 (c; green in g). α-Adaptin is cytosolic in the μ2-depleted cells. Clathrin is still membrane-associated; however, many of these membranes are intracellular. Epsin spots are reduced in number, but are still present in the μ2-depleted cells. Many of these spots are also positive for clathrin. The two boxed-in regions are shown at higher magnification in the bottom right corner of all the panels in a–c. (d–f and h) Cells treated with chc-2 and control cells were labeled with mouse anti-α-adaptin (d; blue in h), rabbit anti-clathrin (e; red in h), and goat anti-epsin (f; green in h). Clathrin is undetectable in the siRNA-treated cells. α-Adaptin labeling is not markedly different; however, epsin labeling is brighter in the clathrin-depleted cells. Bars, 20 μm.
Mentions: Fig. 2 shows that normally, the three proteins have overlapping distributions at the plasma membrane, with additional intracellular structures labeled with the clathrin antibody. Knocking down μ2 causes a reduction in the number of epsin spots (c). These spots do not contain any detectable α-adaptin, which is now diffuse and cytosolic (a). However, many of the epsin-positive spots in these cells are also positive for clathrin (b; insets), suggesting that the two proteins are able to co-assemble on the plasma membrane even in the absence of AP-2.

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