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Assembly and trafficking of caveolar domains in the cell: caveolae as stable, cargo-triggered, vesicular transporters.

Tagawa A, Mezzacasa A, Hayer A, Longatti A, Pelkmans L, Helenius A - J. Cell Biol. (2005)

Bottom Line: Activation also resulted in increased microtubule (MT)-dependent, long-range movement of caveolar vesicles.Thus, in contrast to clathrin-, or other types of coated transport vesicles, caveolae constitute stable, cholesterol-dependent membrane domains that can serve as fixed containers through vesicle traffic.Finally, we identified the Golgi complex as the site where newly assembled caveolar domains appeared first.

View Article: PubMed Central - PubMed

Affiliation: Swiss Federal Institute of Technology (ETH) Zürich, ETH-Hönggerberg, 8093 Zürich, Switzerland.

ABSTRACT
Using total internal reflection fluorescence microscopy (TIR-FM), fluorescence recovery after photobleaching (FRAP), and other light microscopy techniques, we analyzed the dynamics, the activation, and the assembly of caveolae labeled with fluorescently tagged caveolin-1 (Cav1). We found that when activated by simian virus 40 (SV40), a non-enveloped DNA virus that uses caveolae for cell entry, the fraction of mobile caveolae was dramatically enhanced both in the plasma membrane (PM) and in the caveosome, an intracellular organelle that functions as an intermediate station in caveolar endocytosis. Activation also resulted in increased microtubule (MT)-dependent, long-range movement of caveolar vesicles. We generated heterokaryons that contained GFP- and RFP-tagged caveolae by fusing cells expressing Cav1-GFP and -RFP, respectively, and showed that even when activated, individual caveolar domains underwent little exchange of Cav1. Only when the cells were subjected to transient cholesterol depletion, did the caveolae domain exchange Cav1. Thus, in contrast to clathrin-, or other types of coated transport vesicles, caveolae constitute stable, cholesterol-dependent membrane domains that can serve as fixed containers through vesicle traffic. Finally, we identified the Golgi complex as the site where newly assembled caveolar domains appeared first.

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Cav1-containing structures move long distances in activated cells. (A) Large peripheral areas of CV-1 cells expressing Cav1-GFP were bleached (marked areas in bleach panels), and the movement of Cav1-GFP into the bleached area was monitored omitting the 5-μm region closest to the bleach boundary (marked in 15 min panels). The experiment was performed in the absence (untreated, upper panels) and the presence of SV40 (1 h incubation, MOI 60; +SV40, lower panels). Before (Prebleach), immediately after (Bleach), and 15 min after (15 min) bleaching are shown. Note the increase in long-distance movement in the presence of SV40 (see Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1). Bars, 10 μm. (B) Recovery of fluorescence due to the long-distance movement of Cav1-GFP in CV-1 cells increases after addition of SV40, vanadate, or latA. The CV-1 cells expressing Cav1-GFP were either untreated, exposed to SV40 (MOI 60) for 1 h, to 1 mM vanadate for 1 h, to 5 μM nocodazole for 30 min, to 5 μM nocodazole for 30 min and then 1 h to SV40 (MOI 60), or to 0.8 μM latA for 10 min before the FRAP experiments. The fluorescence recovery was quantified in the bleached area omitting the 5 μm region closest to the bleach boundary (marked in 15 min panels in A) after 15 min. Recovery was calculated by measuring the fluorescence intensity in the defined area before and 15 min after bleaching (see Videos 5–7). The error bars indicate standard deviations of five independent experiments.
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fig2: Cav1-containing structures move long distances in activated cells. (A) Large peripheral areas of CV-1 cells expressing Cav1-GFP were bleached (marked areas in bleach panels), and the movement of Cav1-GFP into the bleached area was monitored omitting the 5-μm region closest to the bleach boundary (marked in 15 min panels). The experiment was performed in the absence (untreated, upper panels) and the presence of SV40 (1 h incubation, MOI 60; +SV40, lower panels). Before (Prebleach), immediately after (Bleach), and 15 min after (15 min) bleaching are shown. Note the increase in long-distance movement in the presence of SV40 (see Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1). Bars, 10 μm. (B) Recovery of fluorescence due to the long-distance movement of Cav1-GFP in CV-1 cells increases after addition of SV40, vanadate, or latA. The CV-1 cells expressing Cav1-GFP were either untreated, exposed to SV40 (MOI 60) for 1 h, to 1 mM vanadate for 1 h, to 5 μM nocodazole for 30 min, to 5 μM nocodazole for 30 min and then 1 h to SV40 (MOI 60), or to 0.8 μM latA for 10 min before the FRAP experiments. The fluorescence recovery was quantified in the bleached area omitting the 5 μm region closest to the bleach boundary (marked in 15 min panels in A) after 15 min. Recovery was calculated by measuring the fluorescence intensity in the defined area before and 15 min after bleaching (see Videos 5–7). The error bars indicate standard deviations of five independent experiments.

Mentions: To examine whether the mobilization of PM and caveosomal pools of Cav1 represented movement between the two sites, we quantified the movement of Cav1 over distances of 5 μm or longer using FRAP analysis. We bleached peripheral segments of CV-1 cells expressing Cav1-GFP and followed the movement of Cav1-GFP over time into the bleached section by confocal microscopy. The results are shown in Fig. 2, A and B, and the corresponding videos in the supplemental material (Videos 5–7 available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1).


Assembly and trafficking of caveolar domains in the cell: caveolae as stable, cargo-triggered, vesicular transporters.

Tagawa A, Mezzacasa A, Hayer A, Longatti A, Pelkmans L, Helenius A - J. Cell Biol. (2005)

Cav1-containing structures move long distances in activated cells. (A) Large peripheral areas of CV-1 cells expressing Cav1-GFP were bleached (marked areas in bleach panels), and the movement of Cav1-GFP into the bleached area was monitored omitting the 5-μm region closest to the bleach boundary (marked in 15 min panels). The experiment was performed in the absence (untreated, upper panels) and the presence of SV40 (1 h incubation, MOI 60; +SV40, lower panels). Before (Prebleach), immediately after (Bleach), and 15 min after (15 min) bleaching are shown. Note the increase in long-distance movement in the presence of SV40 (see Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1). Bars, 10 μm. (B) Recovery of fluorescence due to the long-distance movement of Cav1-GFP in CV-1 cells increases after addition of SV40, vanadate, or latA. The CV-1 cells expressing Cav1-GFP were either untreated, exposed to SV40 (MOI 60) for 1 h, to 1 mM vanadate for 1 h, to 5 μM nocodazole for 30 min, to 5 μM nocodazole for 30 min and then 1 h to SV40 (MOI 60), or to 0.8 μM latA for 10 min before the FRAP experiments. The fluorescence recovery was quantified in the bleached area omitting the 5 μm region closest to the bleach boundary (marked in 15 min panels in A) after 15 min. Recovery was calculated by measuring the fluorescence intensity in the defined area before and 15 min after bleaching (see Videos 5–7). The error bars indicate standard deviations of five independent experiments.
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fig2: Cav1-containing structures move long distances in activated cells. (A) Large peripheral areas of CV-1 cells expressing Cav1-GFP were bleached (marked areas in bleach panels), and the movement of Cav1-GFP into the bleached area was monitored omitting the 5-μm region closest to the bleach boundary (marked in 15 min panels). The experiment was performed in the absence (untreated, upper panels) and the presence of SV40 (1 h incubation, MOI 60; +SV40, lower panels). Before (Prebleach), immediately after (Bleach), and 15 min after (15 min) bleaching are shown. Note the increase in long-distance movement in the presence of SV40 (see Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1). Bars, 10 μm. (B) Recovery of fluorescence due to the long-distance movement of Cav1-GFP in CV-1 cells increases after addition of SV40, vanadate, or latA. The CV-1 cells expressing Cav1-GFP were either untreated, exposed to SV40 (MOI 60) for 1 h, to 1 mM vanadate for 1 h, to 5 μM nocodazole for 30 min, to 5 μM nocodazole for 30 min and then 1 h to SV40 (MOI 60), or to 0.8 μM latA for 10 min before the FRAP experiments. The fluorescence recovery was quantified in the bleached area omitting the 5 μm region closest to the bleach boundary (marked in 15 min panels in A) after 15 min. Recovery was calculated by measuring the fluorescence intensity in the defined area before and 15 min after bleaching (see Videos 5–7). The error bars indicate standard deviations of five independent experiments.
Mentions: To examine whether the mobilization of PM and caveosomal pools of Cav1 represented movement between the two sites, we quantified the movement of Cav1 over distances of 5 μm or longer using FRAP analysis. We bleached peripheral segments of CV-1 cells expressing Cav1-GFP and followed the movement of Cav1-GFP over time into the bleached section by confocal microscopy. The results are shown in Fig. 2, A and B, and the corresponding videos in the supplemental material (Videos 5–7 available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1).

Bottom Line: Activation also resulted in increased microtubule (MT)-dependent, long-range movement of caveolar vesicles.Thus, in contrast to clathrin-, or other types of coated transport vesicles, caveolae constitute stable, cholesterol-dependent membrane domains that can serve as fixed containers through vesicle traffic.Finally, we identified the Golgi complex as the site where newly assembled caveolar domains appeared first.

View Article: PubMed Central - PubMed

Affiliation: Swiss Federal Institute of Technology (ETH) Zürich, ETH-Hönggerberg, 8093 Zürich, Switzerland.

ABSTRACT
Using total internal reflection fluorescence microscopy (TIR-FM), fluorescence recovery after photobleaching (FRAP), and other light microscopy techniques, we analyzed the dynamics, the activation, and the assembly of caveolae labeled with fluorescently tagged caveolin-1 (Cav1). We found that when activated by simian virus 40 (SV40), a non-enveloped DNA virus that uses caveolae for cell entry, the fraction of mobile caveolae was dramatically enhanced both in the plasma membrane (PM) and in the caveosome, an intracellular organelle that functions as an intermediate station in caveolar endocytosis. Activation also resulted in increased microtubule (MT)-dependent, long-range movement of caveolar vesicles. We generated heterokaryons that contained GFP- and RFP-tagged caveolae by fusing cells expressing Cav1-GFP and -RFP, respectively, and showed that even when activated, individual caveolar domains underwent little exchange of Cav1. Only when the cells were subjected to transient cholesterol depletion, did the caveolae domain exchange Cav1. Thus, in contrast to clathrin-, or other types of coated transport vesicles, caveolae constitute stable, cholesterol-dependent membrane domains that can serve as fixed containers through vesicle traffic. Finally, we identified the Golgi complex as the site where newly assembled caveolar domains appeared first.

Show MeSH
Related in: MedlinePlus