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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 does not exchange between caveolar domains. (A) Dual-color TIR-FM images at the cell surface of HeLa heterokaryons expressing Cav1-GFP and -RFP, in the presence of stimuli. SV40 (MOI 103, left) or vanadate (1 mM, right) were added 1.5 h after fusion, and cells were incubated for another 1.5 h before fixation. Bars, 2 μm. (B–E) Distribution of Cav1--GFP and -RFP (B–D) or clathrin light chain–GFP and –RFP (E) was imaged on confocal microscope 3 h after fusion. In the absence (B) and presence of SV40 (C; MOI 103, added at 1.5 h), Cav1-GFP and -RFP coexpressed (no fusion, D), or clathrin light chain–GFP and –RFP at 3 h after fusion (E). Note the individual red and green spots in the absence or presence of SV40 (B and C; and Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1), and the yellow spots indicating complete colocalization after coexpression of Cav1-GFP and -RFP (D), and in fusion of clathrin light chain–GFP and –RFP (E). Note a caveosome with a mosaic of red, green, and yellow regions in C (arrowhead), whereas in B, there are either red or green caveosomes and very few caveosomes containing both colors. Bars, 2 μm. (F–I) Zoomed-in images of caveosomes (upper panels) prepared as in B–E above, and corresponding fluorescence intensity curves of GFP and RFP (graphs below). In the absence (F) and presence of SV40 (G; and Video 8), Cav1-GFP and -RFP coexpressed (no fusion, H) or clathrin light chain–GFP and –RFP at 3 h after fusion (I). Fluorescence intensity of GFP and RFP along the line across the caveosome structures were measured (RI, relative fluorescence intensity) and directly plotted against the distance (red line, RI of RFP; green line, RI of GFP).
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fig4: Cav1 does not exchange between caveolar domains. (A) Dual-color TIR-FM images at the cell surface of HeLa heterokaryons expressing Cav1-GFP and -RFP, in the presence of stimuli. SV40 (MOI 103, left) or vanadate (1 mM, right) were added 1.5 h after fusion, and cells were incubated for another 1.5 h before fixation. Bars, 2 μm. (B–E) Distribution of Cav1--GFP and -RFP (B–D) or clathrin light chain–GFP and –RFP (E) was imaged on confocal microscope 3 h after fusion. In the absence (B) and presence of SV40 (C; MOI 103, added at 1.5 h), Cav1-GFP and -RFP coexpressed (no fusion, D), or clathrin light chain–GFP and –RFP at 3 h after fusion (E). Note the individual red and green spots in the absence or presence of SV40 (B and C; and Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1), and the yellow spots indicating complete colocalization after coexpression of Cav1-GFP and -RFP (D), and in fusion of clathrin light chain–GFP and –RFP (E). Note a caveosome with a mosaic of red, green, and yellow regions in C (arrowhead), whereas in B, there are either red or green caveosomes and very few caveosomes containing both colors. Bars, 2 μm. (F–I) Zoomed-in images of caveosomes (upper panels) prepared as in B–E above, and corresponding fluorescence intensity curves of GFP and RFP (graphs below). In the absence (F) and presence of SV40 (G; and Video 8), Cav1-GFP and -RFP coexpressed (no fusion, H) or clathrin light chain–GFP and –RFP at 3 h after fusion (I). Fluorescence intensity of GFP and RFP along the line across the caveosome structures were measured (RI, relative fluorescence intensity) and directly plotted against the distance (red line, RI of RFP; green line, RI of GFP).

Mentions: The second observation was that individual Cav1-GFP– and -RFP–labeled spots remained either green or red whether they had crossed the boundary or not. The persistence of color segregation is best seen in the zoomed-in views in Fig. 4, B and F. This indicated that once formed, Cav1 domains were stable. There was no detectable exchange of Cav1 between them. The experiment was repeated with Cav1-YFP and -CFP, with the same results (unpublished data).


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 does not exchange between caveolar domains. (A) Dual-color TIR-FM images at the cell surface of HeLa heterokaryons expressing Cav1-GFP and -RFP, in the presence of stimuli. SV40 (MOI 103, left) or vanadate (1 mM, right) were added 1.5 h after fusion, and cells were incubated for another 1.5 h before fixation. Bars, 2 μm. (B–E) Distribution of Cav1--GFP and -RFP (B–D) or clathrin light chain–GFP and –RFP (E) was imaged on confocal microscope 3 h after fusion. In the absence (B) and presence of SV40 (C; MOI 103, added at 1.5 h), Cav1-GFP and -RFP coexpressed (no fusion, D), or clathrin light chain–GFP and –RFP at 3 h after fusion (E). Note the individual red and green spots in the absence or presence of SV40 (B and C; and Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1), and the yellow spots indicating complete colocalization after coexpression of Cav1-GFP and -RFP (D), and in fusion of clathrin light chain–GFP and –RFP (E). Note a caveosome with a mosaic of red, green, and yellow regions in C (arrowhead), whereas in B, there are either red or green caveosomes and very few caveosomes containing both colors. Bars, 2 μm. (F–I) Zoomed-in images of caveosomes (upper panels) prepared as in B–E above, and corresponding fluorescence intensity curves of GFP and RFP (graphs below). In the absence (F) and presence of SV40 (G; and Video 8), Cav1-GFP and -RFP coexpressed (no fusion, H) or clathrin light chain–GFP and –RFP at 3 h after fusion (I). Fluorescence intensity of GFP and RFP along the line across the caveosome structures were measured (RI, relative fluorescence intensity) and directly plotted against the distance (red line, RI of RFP; green line, RI of GFP).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171342&req=5

fig4: Cav1 does not exchange between caveolar domains. (A) Dual-color TIR-FM images at the cell surface of HeLa heterokaryons expressing Cav1-GFP and -RFP, in the presence of stimuli. SV40 (MOI 103, left) or vanadate (1 mM, right) were added 1.5 h after fusion, and cells were incubated for another 1.5 h before fixation. Bars, 2 μm. (B–E) Distribution of Cav1--GFP and -RFP (B–D) or clathrin light chain–GFP and –RFP (E) was imaged on confocal microscope 3 h after fusion. In the absence (B) and presence of SV40 (C; MOI 103, added at 1.5 h), Cav1-GFP and -RFP coexpressed (no fusion, D), or clathrin light chain–GFP and –RFP at 3 h after fusion (E). Note the individual red and green spots in the absence or presence of SV40 (B and C; and Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1), and the yellow spots indicating complete colocalization after coexpression of Cav1-GFP and -RFP (D), and in fusion of clathrin light chain–GFP and –RFP (E). Note a caveosome with a mosaic of red, green, and yellow regions in C (arrowhead), whereas in B, there are either red or green caveosomes and very few caveosomes containing both colors. Bars, 2 μm. (F–I) Zoomed-in images of caveosomes (upper panels) prepared as in B–E above, and corresponding fluorescence intensity curves of GFP and RFP (graphs below). In the absence (F) and presence of SV40 (G; and Video 8), Cav1-GFP and -RFP coexpressed (no fusion, H) or clathrin light chain–GFP and –RFP at 3 h after fusion (I). Fluorescence intensity of GFP and RFP along the line across the caveosome structures were measured (RI, relative fluorescence intensity) and directly plotted against the distance (red line, RI of RFP; green line, RI of GFP).
Mentions: The second observation was that individual Cav1-GFP– and -RFP–labeled spots remained either green or red whether they had crossed the boundary or not. The persistence of color segregation is best seen in the zoomed-in views in Fig. 4, B and F. This indicated that once formed, Cav1 domains were stable. There was no detectable exchange of Cav1 between them. The experiment was repeated with Cav1-YFP and -CFP, with the same results (unpublished data).

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