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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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Kinetics of GFP-clathrin recovery after photobleaching at 28°C. HeLa cells (•), HeLa cells expressing K44A-dynamin (▵), and HeLa cells depleted of cholesterol (○) were photobleached at 15 s. After photobleaching, a time series of scanning were performed at low laser power.
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fig6: Kinetics of GFP-clathrin recovery after photobleaching at 28°C. HeLa cells (•), HeLa cells expressing K44A-dynamin (▵), and HeLa cells depleted of cholesterol (○) were photobleached at 15 s. After photobleaching, a time series of scanning were performed at low laser power.

Mentions: However, interestingly when we measured the rate of fluorescence recovery at 28°C (2.3 × 10−2 s−1) (Table I) we found that it was ∼30 times faster than the rate obtained for transferrin uptake (8.1 ± 1.4 × 10−4 s−1; n = 8) at the same temperature (and about threefold faster than the rate of disappearance of fluorescence pits) because as the temperature is decreased from 37 to 28°C the rate of transferrin uptake decreased by a factor of four, whereas the rate of clathrin replacement decreased only by a factor of two. Since it is unlikely that only 3% of the transferrin on the membrane is located in clathrin-coated pits at 28°C, these data suggest that at this temperature a considerable part of the clathrin replacement is indeed due to clathrin exchange rather than endocytosis. In confirmation, as we observed at 37°C, at 28°C both the rate and magnitude of clathrin replacement after photobleaching is nearly the same in cells when endocytosis is blocked by either cholesterol depletion or expression of the K44A dynamin mutant as in control or WT dynamin-expressing cells (Fig. 6 and Table I). Therefore, as the temperature decreases replacement of bleached clathrin becomes dominated by clathrin exchange rather than endocytosis, whereas at 37°C the rates of endocytosis and clathrin exchange appear to be similar. Of course, if the rates of endocytosis and clathrin exchange were identical at 37°C the rate of fluorescence recovery in control cells should be about twice the rate in cells where endocytosis is blocked, since both endocytosis and clathrin exchange occur in WT cells. In fact, we found that the rates of fluorescence recovery are about equal in control and blocked cells at 37°C, but this could easily be due to variability in the measurement or to a twofold faster rate of clathrin exchange occurring in blocked cells compared with control cells.


Clathrin exchange during clathrin-mediated endocytosis.

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

Kinetics of GFP-clathrin recovery after photobleaching at 28°C. HeLa cells (•), HeLa cells expressing K44A-dynamin (▵), and HeLa cells depleted of cholesterol (○) were photobleached at 15 s. After photobleaching, a time series of scanning were performed at low laser power.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Kinetics of GFP-clathrin recovery after photobleaching at 28°C. HeLa cells (•), HeLa cells expressing K44A-dynamin (▵), and HeLa cells depleted of cholesterol (○) were photobleached at 15 s. After photobleaching, a time series of scanning were performed at low laser power.
Mentions: However, interestingly when we measured the rate of fluorescence recovery at 28°C (2.3 × 10−2 s−1) (Table I) we found that it was ∼30 times faster than the rate obtained for transferrin uptake (8.1 ± 1.4 × 10−4 s−1; n = 8) at the same temperature (and about threefold faster than the rate of disappearance of fluorescence pits) because as the temperature is decreased from 37 to 28°C the rate of transferrin uptake decreased by a factor of four, whereas the rate of clathrin replacement decreased only by a factor of two. Since it is unlikely that only 3% of the transferrin on the membrane is located in clathrin-coated pits at 28°C, these data suggest that at this temperature a considerable part of the clathrin replacement is indeed due to clathrin exchange rather than endocytosis. In confirmation, as we observed at 37°C, at 28°C both the rate and magnitude of clathrin replacement after photobleaching is nearly the same in cells when endocytosis is blocked by either cholesterol depletion or expression of the K44A dynamin mutant as in control or WT dynamin-expressing cells (Fig. 6 and Table I). Therefore, as the temperature decreases replacement of bleached clathrin becomes dominated by clathrin exchange rather than endocytosis, whereas at 37°C the rates of endocytosis and clathrin exchange appear to be similar. Of course, if the rates of endocytosis and clathrin exchange were identical at 37°C the rate of fluorescence recovery in control cells should be about twice the rate in cells where endocytosis is blocked, since both endocytosis and clathrin exchange occur in WT cells. In fact, we found that the rates of fluorescence recovery are about equal in control and blocked cells at 37°C, but this could easily be due to variability in the measurement or to a twofold faster rate of clathrin exchange occurring in blocked cells compared with control 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