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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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Effects of depleting AP-2 and clathrin heavy chain from HeLaM cells. (a) Equal protein loadings of homogenates of either control cells or cells treated with siRNAs directed against clathrin heavy chain, α-adaptin, or μ2 were subjected to SDS-PAGE, and Western blots were probed with antibodies against the indicated protein. Clathrin heavy chain and the AP-2 μ2 subunit were both undetectable after knockdown, whereas a weak signal (<5% of control) was detected after α knockdown. (b) Equal protein loadings of homogenates from control and siRNA-treated cells were subjected to SDS-PAGE, and Western blots were cut in four and probed with the indicated antibody. Anti-actin was included as a loading control. As well as affecting its target, the α siRNA causes a depletion in μ2, and the μ2 siRNA causes a depletion in α. (c) Homogenates of control and μ2-depleted cells were centrifuged at high speed, and supernatants and pellets were probed with anti-α. Knocking down μ2 increases the percentage of α in the supernatant. (d–f) Phase-contrast micrographs of cells treated with a control (nonfunctional) siRNA (d), cells treated with μ2 siRNA (e), and cells treated with clathrin heavy chain siRNA (f). The μ2 siRNA-treated cells look essentially normal. However, many of the clathrin heavy chain siRNA-treated cells are vacuolated, and nearly half are multinucleated. (g–k) Control cells (g), cells treated once with α (h) and μ2 (i) siRNAs, and cells treated twice with α (j) and μ2 (k) siRNAs were labeled with an antibody against the AP-2 α subunit. The labeling becomes patchy after one hit, and after both hits there is little or no label associated with the plasma membrane. (l–n) Control cells (l) and cells treated once (m) or twice (n) with a clathrin heavy chain siRNA were labeled with an antibody against clathrin. The signal disappears more uniformly than the AP-2 signal, again becoming undetectable on membranes after two hits. Bars, 20 μm.
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fig1: Effects of depleting AP-2 and clathrin heavy chain from HeLaM cells. (a) Equal protein loadings of homogenates of either control cells or cells treated with siRNAs directed against clathrin heavy chain, α-adaptin, or μ2 were subjected to SDS-PAGE, and Western blots were probed with antibodies against the indicated protein. Clathrin heavy chain and the AP-2 μ2 subunit were both undetectable after knockdown, whereas a weak signal (<5% of control) was detected after α knockdown. (b) Equal protein loadings of homogenates from control and siRNA-treated cells were subjected to SDS-PAGE, and Western blots were cut in four and probed with the indicated antibody. Anti-actin was included as a loading control. As well as affecting its target, the α siRNA causes a depletion in μ2, and the μ2 siRNA causes a depletion in α. (c) Homogenates of control and μ2-depleted cells were centrifuged at high speed, and supernatants and pellets were probed with anti-α. Knocking down μ2 increases the percentage of α in the supernatant. (d–f) Phase-contrast micrographs of cells treated with a control (nonfunctional) siRNA (d), cells treated with μ2 siRNA (e), and cells treated with clathrin heavy chain siRNA (f). The μ2 siRNA-treated cells look essentially normal. However, many of the clathrin heavy chain siRNA-treated cells are vacuolated, and nearly half are multinucleated. (g–k) Control cells (g), cells treated once with α (h) and μ2 (i) siRNAs, and cells treated twice with α (j) and μ2 (k) siRNAs were labeled with an antibody against the AP-2 α subunit. The labeling becomes patchy after one hit, and after both hits there is little or no label associated with the plasma membrane. (l–n) Control cells (l) and cells treated once (m) or twice (n) with a clathrin heavy chain siRNA were labeled with an antibody against clathrin. The signal disappears more uniformly than the AP-2 signal, again becoming undetectable on membranes after two hits. Bars, 20 μm.

Mentions: To find the most effective conditions for depleting AP-2 and clathrin, a number of siRNAs were synthesized, and both single and double transfections were performed. We found that siRNAs μ2-2 and chc-2 (see Materials and methods), directed against the AP-2 μ2 subunit and clathrin heavy chain, respectively, produced the most complete disruption, and that in both cases it was best to transfect the cells twice, with a 48-h interval in between. An siRNA directed against the AP-2 α subunit (α-2) also worked well, but not quite as effectively as μ2-2 or chc-2. Fig. 1 a shows Western blots of equal protein loadings of control and siRNA-treated cells two days after the second transfection. The signals from both μ2 and clathrin heavy chain are undetectable after knockdown, whereas a weak signal (<5% of control) could be detected in the α-2–treated cells.


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

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

Effects of depleting AP-2 and clathrin heavy chain from HeLaM cells. (a) Equal protein loadings of homogenates of either control cells or cells treated with siRNAs directed against clathrin heavy chain, α-adaptin, or μ2 were subjected to SDS-PAGE, and Western blots were probed with antibodies against the indicated protein. Clathrin heavy chain and the AP-2 μ2 subunit were both undetectable after knockdown, whereas a weak signal (<5% of control) was detected after α knockdown. (b) Equal protein loadings of homogenates from control and siRNA-treated cells were subjected to SDS-PAGE, and Western blots were cut in four and probed with the indicated antibody. Anti-actin was included as a loading control. As well as affecting its target, the α siRNA causes a depletion in μ2, and the μ2 siRNA causes a depletion in α. (c) Homogenates of control and μ2-depleted cells were centrifuged at high speed, and supernatants and pellets were probed with anti-α. Knocking down μ2 increases the percentage of α in the supernatant. (d–f) Phase-contrast micrographs of cells treated with a control (nonfunctional) siRNA (d), cells treated with μ2 siRNA (e), and cells treated with clathrin heavy chain siRNA (f). The μ2 siRNA-treated cells look essentially normal. However, many of the clathrin heavy chain siRNA-treated cells are vacuolated, and nearly half are multinucleated. (g–k) Control cells (g), cells treated once with α (h) and μ2 (i) siRNAs, and cells treated twice with α (j) and μ2 (k) siRNAs were labeled with an antibody against the AP-2 α subunit. The labeling becomes patchy after one hit, and after both hits there is little or no label associated with the plasma membrane. (l–n) Control cells (l) and cells treated once (m) or twice (n) with a clathrin heavy chain siRNA were labeled with an antibody against clathrin. The signal disappears more uniformly than the AP-2 signal, again becoming undetectable on membranes after two hits. Bars, 20 μm.
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Related In: Results  -  Collection

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fig1: Effects of depleting AP-2 and clathrin heavy chain from HeLaM cells. (a) Equal protein loadings of homogenates of either control cells or cells treated with siRNAs directed against clathrin heavy chain, α-adaptin, or μ2 were subjected to SDS-PAGE, and Western blots were probed with antibodies against the indicated protein. Clathrin heavy chain and the AP-2 μ2 subunit were both undetectable after knockdown, whereas a weak signal (<5% of control) was detected after α knockdown. (b) Equal protein loadings of homogenates from control and siRNA-treated cells were subjected to SDS-PAGE, and Western blots were cut in four and probed with the indicated antibody. Anti-actin was included as a loading control. As well as affecting its target, the α siRNA causes a depletion in μ2, and the μ2 siRNA causes a depletion in α. (c) Homogenates of control and μ2-depleted cells were centrifuged at high speed, and supernatants and pellets were probed with anti-α. Knocking down μ2 increases the percentage of α in the supernatant. (d–f) Phase-contrast micrographs of cells treated with a control (nonfunctional) siRNA (d), cells treated with μ2 siRNA (e), and cells treated with clathrin heavy chain siRNA (f). The μ2 siRNA-treated cells look essentially normal. However, many of the clathrin heavy chain siRNA-treated cells are vacuolated, and nearly half are multinucleated. (g–k) Control cells (g), cells treated once with α (h) and μ2 (i) siRNAs, and cells treated twice with α (j) and μ2 (k) siRNAs were labeled with an antibody against the AP-2 α subunit. The labeling becomes patchy after one hit, and after both hits there is little or no label associated with the plasma membrane. (l–n) Control cells (l) and cells treated once (m) or twice (n) with a clathrin heavy chain siRNA were labeled with an antibody against clathrin. The signal disappears more uniformly than the AP-2 signal, again becoming undetectable on membranes after two hits. Bars, 20 μm.
Mentions: To find the most effective conditions for depleting AP-2 and clathrin, a number of siRNAs were synthesized, and both single and double transfections were performed. We found that siRNAs μ2-2 and chc-2 (see Materials and methods), directed against the AP-2 μ2 subunit and clathrin heavy chain, respectively, produced the most complete disruption, and that in both cases it was best to transfect the cells twice, with a 48-h interval in between. An siRNA directed against the AP-2 α subunit (α-2) also worked well, but not quite as effectively as μ2-2 or chc-2. Fig. 1 a shows Western blots of equal protein loadings of control and siRNA-treated cells two days after the second transfection. The signals from both μ2 and clathrin heavy chain are undetectable after knockdown, whereas a weak signal (<5% of control) could be detected in the α-2–treated cells.

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