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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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Cholesterol depletion and repletion allow mixing of Cav1-GFP and -RFP. (A–D) Distribution of Cav1-GFP and -RFP in heterokaryons of HeLa cells expressing Cav1-GFP and -RFP, respectively. (A) An intracellular view of a heterokaryon in untreated control imaged by confocal microscopy. Fused cells were incubated in the presence of CHX, and were imaged 5 h after fusion. (B) An intracellular view of a heterokaryon after cholesterol depletion and repletion imaged by confocal microscopy. The cells were fused, recovered for 1 h, treated with cholesterol-depleting drugs nystatin (25 μg/ml) and progesterone (10 μg/ml) for 2 h, repleted with cholesterol by addition of 10% FCS for 2 h, and imaged 5 h after fusion. Bar, 2 μm. (C) A surface view of untreated heterokaryon (same condition as in A) was imaged by TIR-FM 5 h after fusion. (D) A surface view of a heterokaryon after cholesterol depletion and repletion (same condition as in B) was imaged by TIR-FM 5 h after fusion. Note in B, there are caveosomes that reassembled into completely yellow caveosomes, and in D, yellow surface caveolae that presumably transported from intracellular pool to surface are visible. Bar, 2 μm.
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fig5: Cholesterol depletion and repletion allow mixing of Cav1-GFP and -RFP. (A–D) Distribution of Cav1-GFP and -RFP in heterokaryons of HeLa cells expressing Cav1-GFP and -RFP, respectively. (A) An intracellular view of a heterokaryon in untreated control imaged by confocal microscopy. Fused cells were incubated in the presence of CHX, and were imaged 5 h after fusion. (B) An intracellular view of a heterokaryon after cholesterol depletion and repletion imaged by confocal microscopy. The cells were fused, recovered for 1 h, treated with cholesterol-depleting drugs nystatin (25 μg/ml) and progesterone (10 μg/ml) for 2 h, repleted with cholesterol by addition of 10% FCS for 2 h, and imaged 5 h after fusion. Bar, 2 μm. (C) A surface view of untreated heterokaryon (same condition as in A) was imaged by TIR-FM 5 h after fusion. (D) A surface view of a heterokaryon after cholesterol depletion and repletion (same condition as in B) was imaged by TIR-FM 5 h after fusion. Note in B, there are caveosomes that reassembled into completely yellow caveosomes, and in D, yellow surface caveolae that presumably transported from intracellular pool to surface are visible. Bar, 2 μm.

Mentions: Although the mixing of Cav1-GFP and -RFP was by no means complete, it was clear that the number of yellow spots was higher in heterokaryons in which the cholesterol level had been transiently lowered (Fig. 5 B). Of the spots in caveosomes, 21% showed overlap between GFP and RFP (Fig. 5 B) compared with 7% in control heterokaryons (Fig. 5 A). In the PM, as viewed by TIR-FM, the colocalization was 22% and 5% (Fig. 5, D and C, respectively). The increased fraction of yellow spots indicated that cholesterol does indeed play a role in stabilizing caveolae. The reformation of caveolae indicated that Cav1 devoid of cholesterol remained assembly competent upon readdition of cholesterol.


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)

Cholesterol depletion and repletion allow mixing of Cav1-GFP and -RFP. (A–D) Distribution of Cav1-GFP and -RFP in heterokaryons of HeLa cells expressing Cav1-GFP and -RFP, respectively. (A) An intracellular view of a heterokaryon in untreated control imaged by confocal microscopy. Fused cells were incubated in the presence of CHX, and were imaged 5 h after fusion. (B) An intracellular view of a heterokaryon after cholesterol depletion and repletion imaged by confocal microscopy. The cells were fused, recovered for 1 h, treated with cholesterol-depleting drugs nystatin (25 μg/ml) and progesterone (10 μg/ml) for 2 h, repleted with cholesterol by addition of 10% FCS for 2 h, and imaged 5 h after fusion. Bar, 2 μm. (C) A surface view of untreated heterokaryon (same condition as in A) was imaged by TIR-FM 5 h after fusion. (D) A surface view of a heterokaryon after cholesterol depletion and repletion (same condition as in B) was imaged by TIR-FM 5 h after fusion. Note in B, there are caveosomes that reassembled into completely yellow caveosomes, and in D, yellow surface caveolae that presumably transported from intracellular pool to surface are visible. Bar, 2 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171342&req=5

fig5: Cholesterol depletion and repletion allow mixing of Cav1-GFP and -RFP. (A–D) Distribution of Cav1-GFP and -RFP in heterokaryons of HeLa cells expressing Cav1-GFP and -RFP, respectively. (A) An intracellular view of a heterokaryon in untreated control imaged by confocal microscopy. Fused cells were incubated in the presence of CHX, and were imaged 5 h after fusion. (B) An intracellular view of a heterokaryon after cholesterol depletion and repletion imaged by confocal microscopy. The cells were fused, recovered for 1 h, treated with cholesterol-depleting drugs nystatin (25 μg/ml) and progesterone (10 μg/ml) for 2 h, repleted with cholesterol by addition of 10% FCS for 2 h, and imaged 5 h after fusion. Bar, 2 μm. (C) A surface view of untreated heterokaryon (same condition as in A) was imaged by TIR-FM 5 h after fusion. (D) A surface view of a heterokaryon after cholesterol depletion and repletion (same condition as in B) was imaged by TIR-FM 5 h after fusion. Note in B, there are caveosomes that reassembled into completely yellow caveosomes, and in D, yellow surface caveolae that presumably transported from intracellular pool to surface are visible. Bar, 2 μm.
Mentions: Although the mixing of Cav1-GFP and -RFP was by no means complete, it was clear that the number of yellow spots was higher in heterokaryons in which the cholesterol level had been transiently lowered (Fig. 5 B). Of the spots in caveosomes, 21% showed overlap between GFP and RFP (Fig. 5 B) compared with 7% in control heterokaryons (Fig. 5 A). In the PM, as viewed by TIR-FM, the colocalization was 22% and 5% (Fig. 5, D and C, respectively). The increased fraction of yellow spots indicated that cholesterol does indeed play a role in stabilizing caveolae. The reformation of caveolae indicated that Cav1 devoid of cholesterol remained assembly competent upon readdition of cholesterol.

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