Adaptor and clathrin exchange at the plasma membrane and trans-Golgi network - PubMed (original) (raw)

Adaptor and clathrin exchange at the plasma membrane and trans-Golgi network

Xufeng Wu et al. Mol Biol Cell. 2003 Feb.

Abstract

We previously demonstrated, using fluorescence recovery after photobleaching, that clathrin in clathrin-coated pits at the plasma membrane exchanges with free clathrin in the cytosol, suggesting that clathrin-coated pits are dynamic structures. We now investigated whether clathrin at the trans-Golgi network as well as the clathrin adaptors AP2 and AP1 in clathrin-coated pits at the plasma membrane and trans-Golgi network, respectively, also exchange with free proteins in the cytosol. We found that when the budding of clathrin-coated vesicle is blocked without significantly affecting the structure of clathrin-coated pits, both clathrin and AP2 at the plasma membrane and clathrin and AP1 at the trans-Golgi network exchange rapidly with free proteins in the cytosol. In contrast, when budding of clathrin-coated vesicles was blocked at the plasma membrane or trans-Golgi network by hypertonic sucrose or K(+) depletion, conditions that markedly affect the structure of clathrin-coated pits, clathrin exchange was blocked but AP2 at the plasma membrane and both AP1 and the GGA1 adaptor at the trans-Golgi network continue to rapidly exchange. We conclude that clathrin-coated pits are dynamic structures with rapid exchange of both clathrin and adaptors and that adaptors are able to exchange independently of clathrin when clathrin exchange is blocked.

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Figures

Figure 1

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 of clustered pits. The insets show the distribution of clathrin, AP2, and overlap between clathrin and AP2. Bar, 9 μm.

Figure 2

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) Images obtained directly before being photobleached. (B, E, and H) Images immediately after photobleaching. (C, F, and I) Images 2 min after photobleaching. The photobleached area is indicated in each figure. Bars, A–C, 6.5 μm; D–F; 9.5 μm; and G–I, 9 μm.

Figure 3

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 power.

Figure 4

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) and GFP-AP1 (D–F and J–L) before photobleaching (A, D, G, and J), immediately after photobleaching (B, E, H, and K), and either 2 min (C and F) or 5 min (I and L) after photobleaching. The photobleached area is indicated in each figure. Bars, A–F, 11 μm; G–L, 13 μm.

Figure 5

Figure 5

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).

Figure 6

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× to examine clathrin and AP2 in K+-depleted cells (A) or cells treated with hypertonic sucrose (B). The inset shows the distribution of clathrin, AP2, and overlap between clathrin and AP2. The arrows point to the clathrin clusters. Bar, 5 μm.

Figure 7

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 F) AP2 image obtained directly before being photobleached. (C and G) Image immediately after photobleaching. (D and H) Image 2 min after photobleaching. The photobleached area is indicated in each figure. Bars, 5 μm.

Figure 8

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 GFP-clathrin (circles) at the plasma membrane. (B) Time course of recovery of GFP-AP1 (triangles) and GFP-clathrin (circles) at the TGN.

Figure 9

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) and GFP-AP1 (D–F and J–L) before photobleaching (A, D, G, and J), immediately after photobleaching (B, E, H, K), and 2 min (C, F, I, L) after photobleaching in cells K+ depleted (A–F) or treated with hypertonic sucrose (G–L). The photobleached area is indicated in each figure. Bar, A–F, 7.5 μm; G–L, 5.5 μm.

Figure 10

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, F, and I) after photobleaching in control cells (A–C), in cells K+ depleted (D–F) or treated with hypertonic sucrose (G–I). The photobleached area is indicated in each figure. Bar, 12 μm.

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