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Clathrin exchange during clathrin-mediated endocytosis.

Wu X, Zhao X, Baylor L, Kaushal S, Eisenberg E, Greene LE - J. Cell Biol. (2001)

Bottom Line: In the present study, we investigated this question by studying clathrin exchange both in vitro and in vivo.We found that in vitro clathrin in CVs and clathrin baskets do not exchange with free clathrin even in the presence of Hsc70 and ATP where partial uncoating occurs.On the other hand, consistent with the in vitro data both potassium depletion and hypertonic sucrose, which have been reported to transform clathrin-coated pits into clathrin cages just below the surface of the plasma membrane, not only block endocytosis but also block exchange of clathrin.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
During clathrin-mediated endocytosis, clathrin-coated pits invaginate to form clathrin-coated vesicles (CVs). Since clathrin-coated pits are planar structures, whereas CVs are spherical, there must be a structural rearrangement of clathrin as invagination occurs. This could occur through simple addition of clathrin triskelions to the edges of growing clathrin-coated pits with very little exchange occurring between clathrin in the pits and free clathrin in the cytosol, or it could occur through large scale exchange of free and bound clathrin. In the present study, we investigated this question by studying clathrin exchange both in vitro and in vivo. We found that in vitro clathrin in CVs and clathrin baskets do not exchange with free clathrin even in the presence of Hsc70 and ATP where partial uncoating occurs. However, surprisingly FRAP studies on clathrin-coated pits labeled with green fluorescent protein-clathrin light chains in HeLa cells show that even when endocytosis is blocked by expression of a dynamin mutant or depletion of cholesterol from the membrane, replacement of photobleached clathrin in coated pits on the membrane occurs at almost the same rate and magnitude as when endocytosis is occurring. Furthermore, very little of this replacement is due to dissolution of old pits and reformation of new ones; rather, it is caused by a rapid ATP-dependent exchange of clathrin in the pits with free clathrin in the cytosol. On the other hand, consistent with the in vitro data both potassium depletion and hypertonic sucrose, which have been reported to transform clathrin-coated pits into clathrin cages just below the surface of the plasma membrane, not only block endocytosis but also block exchange of clathrin. Taken together, these data show that ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits, but not clathrin baskets, and may be involved in a structural rearrangement of clathrin as clathrin-coated pits invaginate.

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Absence of clathrin exchange in cells treated with hypertonic sucrose, depleted of K+, or depleted of ATP. HeLa cells were treated as described in Materials and methods. Cells treated with hypertonic sucrose (A and C); cells depleted of K+ (D–F); cells depleted of ATP (G–I). Cells were imaged at 28°C before photobleaching (A, D, and G), immediately after photobleaching (B, E, and H), and 5 min after photobleaching (C,F,I). The photobleached area is indicated in each figure. Bars, 5 μm.
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fig9: Absence of clathrin exchange in cells treated with hypertonic sucrose, depleted of K+, or depleted of ATP. HeLa cells were treated as described in Materials and methods. Cells treated with hypertonic sucrose (A and C); cells depleted of K+ (D–F); cells depleted of ATP (G–I). Cells were imaged at 28°C before photobleaching (A, D, and G), immediately after photobleaching (B, E, and H), and 5 min after photobleaching (C,F,I). The photobleached area is indicated in each figure. Bars, 5 μm.

Mentions: We found that when cells were treated with either hypertonic sucrose or were potassium depleted, there were still GFP fluorescent spots on the membrane. However, there were somewhat fewer spots and with a broader size distribution, which became more pronounced as we observed the cells over longer time periods. Since it is quite possible that confocal microscopy would not distinguish between GFP-clathrin–coated pits and GFP-clathrin microcages occurring directly under the plasma membrane, we carried out photobleaching experiments on the fluorescent spots to determine if they show the same exchange properties as clathrin-coated pits. We found that in contrast to clathrin-coated pits fluorescent spots in cells treated by either hypertonic sucrose (Fig. 9, A–C) or potassium depletion (Fig. 9, D–F) showed no recovery from photobleaching. Therefore, these treatments dramatically change the properties of the clathrin structures at the plasma membrane, perhaps by converting clathrin-coated pits into clathrin microcages that no longer show clathrin exchange.


Clathrin exchange during clathrin-mediated endocytosis.

Wu X, Zhao X, Baylor L, Kaushal S, Eisenberg E, Greene LE - J. Cell Biol. (2001)

Absence of clathrin exchange in cells treated with hypertonic sucrose, depleted of K+, or depleted of ATP. HeLa cells were treated as described in Materials and methods. Cells treated with hypertonic sucrose (A and C); cells depleted of K+ (D–F); cells depleted of ATP (G–I). Cells were imaged at 28°C before photobleaching (A, D, and G), immediately after photobleaching (B, E, and H), and 5 min after photobleaching (C,F,I). The photobleached area is indicated in each figure. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198830&req=5

fig9: Absence of clathrin exchange in cells treated with hypertonic sucrose, depleted of K+, or depleted of ATP. HeLa cells were treated as described in Materials and methods. Cells treated with hypertonic sucrose (A and C); cells depleted of K+ (D–F); cells depleted of ATP (G–I). Cells were imaged at 28°C before photobleaching (A, D, and G), immediately after photobleaching (B, E, and H), and 5 min after photobleaching (C,F,I). The photobleached area is indicated in each figure. Bars, 5 μm.
Mentions: We found that when cells were treated with either hypertonic sucrose or were potassium depleted, there were still GFP fluorescent spots on the membrane. However, there were somewhat fewer spots and with a broader size distribution, which became more pronounced as we observed the cells over longer time periods. Since it is quite possible that confocal microscopy would not distinguish between GFP-clathrin–coated pits and GFP-clathrin microcages occurring directly under the plasma membrane, we carried out photobleaching experiments on the fluorescent spots to determine if they show the same exchange properties as clathrin-coated pits. We found that in contrast to clathrin-coated pits fluorescent spots in cells treated by either hypertonic sucrose (Fig. 9, A–C) or potassium depletion (Fig. 9, D–F) showed no recovery from photobleaching. Therefore, these treatments dramatically change the properties of the clathrin structures at the plasma membrane, perhaps by converting clathrin-coated pits into clathrin microcages that no longer show clathrin exchange.

Bottom Line: In the present study, we investigated this question by studying clathrin exchange both in vitro and in vivo.We found that in vitro clathrin in CVs and clathrin baskets do not exchange with free clathrin even in the presence of Hsc70 and ATP where partial uncoating occurs.On the other hand, consistent with the in vitro data both potassium depletion and hypertonic sucrose, which have been reported to transform clathrin-coated pits into clathrin cages just below the surface of the plasma membrane, not only block endocytosis but also block exchange of clathrin.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
During clathrin-mediated endocytosis, clathrin-coated pits invaginate to form clathrin-coated vesicles (CVs). Since clathrin-coated pits are planar structures, whereas CVs are spherical, there must be a structural rearrangement of clathrin as invagination occurs. This could occur through simple addition of clathrin triskelions to the edges of growing clathrin-coated pits with very little exchange occurring between clathrin in the pits and free clathrin in the cytosol, or it could occur through large scale exchange of free and bound clathrin. In the present study, we investigated this question by studying clathrin exchange both in vitro and in vivo. We found that in vitro clathrin in CVs and clathrin baskets do not exchange with free clathrin even in the presence of Hsc70 and ATP where partial uncoating occurs. However, surprisingly FRAP studies on clathrin-coated pits labeled with green fluorescent protein-clathrin light chains in HeLa cells show that even when endocytosis is blocked by expression of a dynamin mutant or depletion of cholesterol from the membrane, replacement of photobleached clathrin in coated pits on the membrane occurs at almost the same rate and magnitude as when endocytosis is occurring. Furthermore, very little of this replacement is due to dissolution of old pits and reformation of new ones; rather, it is caused by a rapid ATP-dependent exchange of clathrin in the pits with free clathrin in the cytosol. On the other hand, consistent with the in vitro data both potassium depletion and hypertonic sucrose, which have been reported to transform clathrin-coated pits into clathrin cages just below the surface of the plasma membrane, not only block endocytosis but also block exchange of clathrin. Taken together, these data show that ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits, but not clathrin baskets, and may be involved in a structural rearrangement of clathrin as clathrin-coated pits invaginate.

Show MeSH
Related in: MedlinePlus