We began our study by investigating whether fluorescently tagged AP2 colocalizes with clathrin in clathrin-coated pits on the plasma membrane of HeLa cells. We constructed and transfected HeLa cells with a plasmid encoding the rat AP2 α chain with GFP attached at its amino terminus. Immunofluorescence studies using both anti-clathrin and anti-AP2 antibodies showed that most of the GFP-AP2 colocalized with the clathrin-coated pits. We also found that expression of GFP-AP2 had no effect on transferrin uptake, suggesting that labeling the α chain of AP2 with GFP did not affect the function of the AP2 (our unpublished data). Figure , A–C, shows that, as expected, essentially all of the GFP-AP2 colocalizes with cyan fluorescent protein (CFP)-clathrin. Many of the GFP-AP2 pits at the cell periphery were ~0.3 μm or smaller in diameter, about the same size we obtained for the pits with CFP-clathrin (Wu et al., 2001
). However, whether we used CFP-clathrin or GFP-AP2 to visualize the pits, the cells always had areas where pits were clustered (Figure , arrow); these areas may represent hot spots of pit formation (Gaidarov et al., 1999
Figure 1 Colocalization of clathrin and AP2 on the plasma membrane. Cells were imaged using CFP-clathrin and GFP-AP2 by using the 63× objective. (A) CFP-clathrin. (B) GFP-AP2. (C) Overlap between clathrin (red) and AP2 (green). The arrow points to an area (more ...)
We next determined whether the fluorescence of GFP-AP2 in clathrin-coated pits recovered after photobleaching. Figure , A–C, shows that after photobleaching a small region of the plasma membrane of control cells at 37°C there was an immediate decrease in the fluorescence of GFP-AP2 followed by nearly full fluorescence after 2 min. Of course, because clathrin-mediated endocytosis was occurring in these cells, the fluorescence recovery was at least partly due to old pits invaginating and new pits forming. Although it seems that the new pits reformed at the same position as the old pits (Figure C), this is because to avoid damage in the FRAP studies we used a 40× objective rather than a 63× objective so that clusters of pits tended to be brighter than individual pits. This was much less of problem when we imaged GFP-clathrin because the individual pits were brighter than with GFP-AP2. When we used a 63× objective to follow the fluorescence of GFP-AP2 it was clear that old pits disappeared and new pits formed because there was little overlap of fluorescent pits over a period of 1 min (our unpublished data).
Figure 2 Fluorescence recovery after photobleaching GFP-AP2 in HeLa cells at 37°C under different conditions. (A–C) Control HeLa cells. (D–F) HeLa depleted of cholesterol. (G–I) HeLa cells expressing K44A-dynamin. (A, D, and G) (more ...)
To determine whether replacement of AP2 occurred in clathrin-coated pits even when endocytosis was blocked we carried out FRAP experiments on GFP-AP2 in cells either depleted of cholesterol or expressing the dynamin mutant K44A, treatments that block endocytosis but do not significantly affect the structure of clathrin-coated pits (Damke et al., 1994
; Rodal et al., 1999
; Subtil et al., 1999
). We found that with both treatments, GFP-AP2 fluorescence recovered after photobleaching (Figure , D–I). Thus, as occurred with clathrin, even though endocytosis is blocked the bleached GFP-AP2 in the pits is replaced by unbleached GFP-AP2.
Figure shows the time course of recovery of GFP-AP2 fluorescence after photobleaching both in control cells and in cells expressing K44A dynamin or depleted of cholesterol to block endocytosis. As summarized in Table , our data show that blocking clathrin-mediated endocytosis had little effect on the rate or extent of fluorescence recovery, demonstrating that GFP-AP2 exchange occurs at a rapid rate even when clathrin-mediated endocytosis is blocked. Another method of blocking clathrin-mediated endocytosis is to decrease the temperature to 15°C, which we previously found greatly reduced the rate of transferrin uptake so that over a period of 5 min almost no uptake occurred (Wu et al., 2001
). Over the same period of time almost complete recovery of AP2 fluorescence occurred (Table ), and the time course of this recovery in all cases was essentially the same as that previously found for clathrin at the plasma membrane (Wu et al., 2001
). Therefore, consistent with our previous observations (Wu et al., 2001
), rapid replacement of bleached GFP-AP2 occurs in clathrin-coated pits even when clathrin-mediated endocytosis is blocked.
Figure 3 Kinetics of GFP-AP2 recovery after photobleaching at 37°C. HeLa control cells (triangles). HeLa cells depleted of cholesterol (circles). HeLa cells expressing K44A-dynamin (diamonds). Cells were photobleached at 10 s and then scanned at low laser (more ...)
Fluorescence recovery after photobleaching
Our data with AP2 at the plasma membrane show that clathrin-coated pits are dynamic structures in which both clathrin and AP2 rapidly exchange during clathrin-mediated endocytosis. However, clathrin and adaptors are present not only in clathrin-coated pits at the plasma membrane but also at the TGN where AP1 is a major clathrin adaptor. Because we were interested in examining the fluorescence recovery during clathrin budding at the TGN of AP1 we constructed a GFP-AP1. Immunofluorescence studies using antibodies showed that the GFP-AP1 colocalized with AP1 and clathrin at the TGN. We also found that, after treatment with brefeldin A, GFP-AP1 dissociated from the TGN with the same time course as native AP1 (our unpublished data), suggesting that labeling the γ chain of AP1 with GFP does not affect AP1 function. AP1 has also been labeled with YFP on its μ1A chain to study vesicle movement form the TGN (Huang et al., 2001
). Figure , A–F, shows the colocalization of GFP-AP1 and CFP-clathrin at the TGN as well as their recovery after photobleaching at 37°C. It should be noted that although much of the perinuclear clathrin and AP1 is undoubtedly present in the TGN, some may also be present in endosome, which we cannot distinguish from the TGN in the 2-min period of these studies. As expected, in control cells, after bleaching of both CFP-clathrin and GFP-AP1, essentially complete replacement of the bleached clathrin and AP1 occurred at 37°C. Because CFP-constructs cause more UV damage to proteins upon photobleaching than GFP, this result was confirmed using GFP-clathrin, and we found that, as with CFP-clathrin, after photobleaching the fluorescence of the GFP-clathrin at the TGN recovered over a short period of time (Table ).
Figure 4 Fluorescence recover after photobleaching of CFP-clathrin and GFP-AP1 at the trans-Golgi network at 37 and 20°C. HeLa cells at 37°C (A–F) and 20°C (G–L) were imaged for CFP-clathrin (A–C and G–I) (more ...)
We next determined whether this same recovery of fluorescence occurs at 20°C, a temperature that blocks transport from the TGN (Griffiths et al., 1985
). As shown in Figure , G–L, the fluorescence of both the bleached CFP-clathrin and the bleached GFP-AP1 recovered over a period of 5 min, demonstrating that replacement of both AP1 and clathrin occur at the TGN under conditions where clathrin budding is prevented. Figure and Table show the time course of the fluorescence recovery of GFP-clathrin and GFP-AP1 at the TGN both at 37 and 20°C. At both temperatures, particularly at 20°C, the time course of recovery of GFP-AP1 is faster than that of GFP-clathrin. Perhaps because the GFP-clathrin on the TGN was unusually sensitive to damage by photobleaching at 20°C, we observed only ~55% recovery of the GFP-clathrin fluorescence after photobleaching at 20°C but fluorescence loss in photobleaching experiments confirmed that all of the bound clathrin was freely exchangeable at this temperature (our unpublished data). We conclude that replacement of clathrin and adaptors during budding of clathrin-coated pits is a general phenomenon that occurs at both the plasma membrane and the TGN. Of course, although we could observe individual clathrin-coated pits at the plasma membrane and, therefore, conclude that replacement was due to clathrin exchange (Wu et al., 2001
), at the TGN we could not observe individual pits and therefore this replacement could have been caused by either exchange or dissolution and replacement of whole clathrin-coated pits.
Kinetics of recovery after photobleaching of CFP-clathrin and GFP-AP1 at the TGN at 37°C (closed symbols) and 20°C (open symbols). Time course of recovery are shown for GFP-clathrin (circles) and GFP-AP1 (triangles).
The observation that at 20°C AP1 exchange is somewhat faster than clathrin exchange at the TGN suggests that adaptor exchange may not always be linked with clathrin exchange. Another way of approaching this question is to treat the cells with hypertonic sucrose or by K+
depletion. Although cholesterol depletion and expression of K44A block endocytosis without significantly affecting the structure of the clathrin-coated pits, hypertonic sucrose and K+
depletion also block endocytosis but they do so by altering the structure of the clathrin-coated pits. It has been reported that both hypertonic sucrose and K+
depletion reduce the number of clathrin-coated pits at the plasma membrane and at the same time induce the formation of clathrin microcages near the remaining clathrin latices on the plasma membrane (Heuser and Anderson, 1989
). Hansen et al. (1993)
, however, using ultrastructural immunogold microscopy, did not detect either clathrin on the plasma membrane or clathrin microcages in HEp-2 cells in hypertonic sucrose and K+
depletion. In our previous study on clathrin exchange, we found that both hypertonic sucrose and K+
depletion not only blocked endocytosis but also almost completely blocked clathrin exchange at the plasma membrane. Therefore, we were interested in determining whether AP2 exchange was also blocked by these treatments.
As we showed previously, these treatments caused a marked alteration in clathrin distribution (Figure , A and B). Both treatments tended to cause formation of clusters of clathrin structures some of which were not colocalized with AP2 (arrows) and therefore may represent the the clathrin microcages observed by freeze-fracture electron microscopy. In addition, based on MetaMorph (Universal Imaging, Downingtown, PA) analysis we found that ~50% of the AP2 colocalized with both clusters of clathrin structures and with smaller clathrin fluorescent spots. Therefore, these structures may represent clusters of clathrin-coated pits and individual clathrin-coated pits, respectively. In support of this view, electron microscopy of the treated samples indeed showed that there were clathrin-coated pits present after treatment with either hypertonic sucrose or K+
depletion (our unpublished data). Finally we observed AP2 structures devoid of clathrin similar to the structures that were observed by Brown et al., 1999
. Therefore, although clathrin microbaskets without AP2 and AP2 pits without clathrin are present, considerable clathrin-coated pits also occur in the treated samples.
Figure 6 Colocalization of CFP-clathrin and GFP-AP2 in cells treated with hypertonic sucrose or depleted of K+. HeLa cells were treated as described in MATERIALS OF METHODS to block clathrin exchange at the coated pits. HeLa cells were then imaged at 63× (more ...)
This allowed us to test whether, after treatment of the cells to hypertonic sucrose or K+
depletion, clathrin and AP2 exchange occurred in the fluorescent structures on the plasma membrane that contained both clathrin and AP2. Table shows that, in contrast to the previous results we obtained in which clathrin exchange was completely blocked (Wu et al., 2001
), more than 90% of the GFP-AP2 fluorescence recovered after photobleaching, and this AP2 exchange occurred at essentially the same rate as occurs in untreated cells. Furthermore, we found that this AP2 exchange specifically occurred in structures where AP2 and clathrin were colocalized (Figure ). Because our fluorescence studies do not reveal whether AP2 is present in any of the clathrin microcages, we obviously cannot determine whether AP2 exchange occurs in the microcages. However, because all of the clathrin we observe on the plasma membrane shows no exchange, whereas 90% of the AP2 we observe does show exchange, including the AP2 colocalized with clathrin, it seems that AP2 is exchanging in clathrin-coated pits on the plasma membrane where clathrin exchange is blocked.
Figure 7 Fluorescence recovery after photobleaching GFP-AP2 in HeLa cells in cells depleted of K+ (B–D) or treated with hypertonic sucrose (F–H) at 37°C. (A and E) Image of CFP-clathrin and GFP-AP2 in treated HeLa cells. (B and (more ...)
We next investigated how hypertonic sucrose and K+
depletion affect clathrin and AP1 dynamics at the TGN. Hypertonic sucrose and K+
depletion were previously found to markedly decrease the number of clathrin buds at the TGN as well as at the plasma membrane in HEp-2 cells (Hansen et al., 1993
). However, we observed no significant decrease in fluorescence of the GFP clathrin on the plasma membrane and TGN in HeLa cells after treatment with hypertonic sucrose or K+
depletion. Furthermore, we found that recovery of clathrin fluorescence at the TGN was completely blocked by both K+
depletion and hypertonic sucrose just as we observed at the plasma membrane (Figures B and 9, A–C and G–I). In addition, like AP2 at the plasma membrane, AP1 at the TGN showed significant fluorescence recovery (Figures B and , D–F and J–L), although the rate of AP1 fluorescence recovery was slightly slower than in control cells (Table ). Therefore, AP1 bound to the TGN is in equilibrium with free AP1 in the cytosol even when clathrin is immobilized on the TGN by treatment with hypertonic sucrose or K+
Figure 8 Time course of fluorescence recovery after photobleaching of adaptors and clathrin in cells depleted of K+ (open symbols) or treated with hypertonic sucrose at 37°C filled symbols) (A) Time course of recovery of GFP-AP2 (triangles) and (more ...)
Figure 9 Fluorescence recovery after photobleaching of CFP-clathrin and GFP-AP1 at the trans-Golgi network of cells depleted of K+ or treated with hypertonic sucrose at 37°C. HeLa cells were imaged for CFP-clathrin (A–C and G–I) (more ...)
Fluorescence recovery after photobleaching of GFP-GGA1, a recently discovered AP (Dell'Angelica et al.
, 2000; Hirst et al., 2000
; Costaguta et al., 2001
) at the TGN, also occurred at about the same rate as clathrin (Figure ; Table ). Furthermore in HeLa cells depleted of K+
or treated with hypertonic sucrose, after photobleaching GFP-GGA1 showed ~70% fluorescence recovery at a rate slightly slower than the rate of GFP-AP1 fluorescence recovery under the same conditions (Figure ; Table ). Therefore, when clathrin exchange at the TGN is blocked by treatment with hypertonic sucrose or K+
depletion, like AP1, GGA1 is still able to detach from and reattach to the TGN. It should be noted that treatment of the TGN with hypertonic sucrose or K+
depletion caused fragmentation (our unpublished data). Nevertheless, even after fragmentation occurred, clathrin exchange was still blocked while both AP1 and GGA1 continued to exchange.
Figure 10 Fluorescence recovery after photobleaching of GFP-GGA1 at the TGN of cells under different conditions at 37°C. HeLa cells were imaged for GFP-GGA1 before photobleaching (A, D, and G), immediately after photobleaching (B, E, and H), and 2 min (C, (more ...)