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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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CFP-clathrin–coated pit pattern is not changed significantly in cells undergoing exchange when endocytosis is blocked. Control HeLa (A); HeLa expressing WT dynamin (B); HeLa expressing K44A dynamin (C); cholesterol depleted HeLa (D); HeLa cells incubated at 15°C (E). Cells were imaged with a 63×,1.4 NA objective as described in Materials and methods, and images collected were overlayed at 0 and 90 s in A–D and 0 and 270 s in E. The inset magnifies the boxed area (length of box is 8 μm) to show the distribution of clathrin-coated pits at the two different times. Bar, 5 μm.
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fig8: CFP-clathrin–coated pit pattern is not changed significantly in cells undergoing exchange when endocytosis is blocked. Control HeLa (A); HeLa expressing WT dynamin (B); HeLa expressing K44A dynamin (C); cholesterol depleted HeLa (D); HeLa cells incubated at 15°C (E). Cells were imaged with a 63×,1.4 NA objective as described in Materials and methods, and images collected were overlayed at 0 and 90 s in A–D and 0 and 270 s in E. The inset magnifies the boxed area (length of box is 8 μm) to show the distribution of clathrin-coated pits at the two different times. Bar, 5 μm.

Mentions: There are two mechanisms which could account for replacement of bleached clathrin with unbleached clathrin of cells where clathrin-mediated endocytosis is blocked. First, even though endocytosis is not occurring there may be dissolution and replacement of whole clathrin-coated pits. Alternatively, individual clathrin molecules in the pits may exchange. To investigate this question, we followed the fate of individual pits for 90 s to determine if whole pits were replaced with new pits at a different location or whether the clathrin in preexisting pits gradually exchanged. We found that in control HeLa cells a considerable fraction of clathrin replacement occurred because photobleached pits were replaced by new pits (Fig. 8 A). The same result was obtained in cells expressing WT dynamin (Fig. 8 B) as expected since endocytosis is occurring in these cells.


Clathrin exchange during clathrin-mediated endocytosis.

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

CFP-clathrin–coated pit pattern is not changed significantly in cells undergoing exchange when endocytosis is blocked. Control HeLa (A); HeLa expressing WT dynamin (B); HeLa expressing K44A dynamin (C); cholesterol depleted HeLa (D); HeLa cells incubated at 15°C (E). Cells were imaged with a 63×,1.4 NA objective as described in Materials and methods, and images collected were overlayed at 0 and 90 s in A–D and 0 and 270 s in E. The inset magnifies the boxed area (length of box is 8 μm) to show the distribution of clathrin-coated pits at the two different times. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: CFP-clathrin–coated pit pattern is not changed significantly in cells undergoing exchange when endocytosis is blocked. Control HeLa (A); HeLa expressing WT dynamin (B); HeLa expressing K44A dynamin (C); cholesterol depleted HeLa (D); HeLa cells incubated at 15°C (E). Cells were imaged with a 63×,1.4 NA objective as described in Materials and methods, and images collected were overlayed at 0 and 90 s in A–D and 0 and 270 s in E. The inset magnifies the boxed area (length of box is 8 μm) to show the distribution of clathrin-coated pits at the two different times. Bar, 5 μm.
Mentions: There are two mechanisms which could account for replacement of bleached clathrin with unbleached clathrin of cells where clathrin-mediated endocytosis is blocked. First, even though endocytosis is not occurring there may be dissolution and replacement of whole clathrin-coated pits. Alternatively, individual clathrin molecules in the pits may exchange. To investigate this question, we followed the fate of individual pits for 90 s to determine if whole pits were replaced with new pits at a different location or whether the clathrin in preexisting pits gradually exchanged. We found that in control HeLa cells a considerable fraction of clathrin replacement occurred because photobleached pits were replaced by new pits (Fig. 8 A). The same result was obtained in cells expressing WT dynamin (Fig. 8 B) as expected since endocytosis is occurring in these cells.

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