Limits...
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.

Show MeSH

Related in: MedlinePlus

Cav1-GFP and -RFP become mobile in stimulated heterokaryons. (A–H) HeLa cells expressing Cav1-GFP (A) were fused with the ones expressing Cav1-RFP (B), or the cells expressing clathrin light chain–GFP (E) were fused with cells expressing clathrin light chain–RFP (F) by 2 min PEG treatment (merge, C and G, respectively). The cells were incubated in the presence of CHX after fusion, fixed after 3 h, and viewed by confocal microscopy. The dashed circles show the positions of the nuclei. Note that in C, the Cav1 spots remain either red or green, and that only few spots have moved across the fusion boundary. In G, even distribution and colocalization of the green and red clathrin is apparent throughout the cell. (D and H) The mixing of Cav1-GFP and -RFP in heterokaryons was enhanced upon stimulation. HeLa cells expressing Cav1-GFP and -RFP were fused and exposed to stimuli. SV40 (MOI 103; D) or 1 mM vanadate (H) was added at 1.5 h after PEG-induced fusion, and cells were incubated for another 1.5 h before fixation. Note extensive mixing of Cav1-GFP and -RFP throughout the cell. Bar, 10 μm. (I) Quantification of caveolar mobility from the experiments in C, D, G, and H. See Fig. S2 for details.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171342&req=5

fig3: Cav1-GFP and -RFP become mobile in stimulated heterokaryons. (A–H) HeLa cells expressing Cav1-GFP (A) were fused with the ones expressing Cav1-RFP (B), or the cells expressing clathrin light chain–GFP (E) were fused with cells expressing clathrin light chain–RFP (F) by 2 min PEG treatment (merge, C and G, respectively). The cells were incubated in the presence of CHX after fusion, fixed after 3 h, and viewed by confocal microscopy. The dashed circles show the positions of the nuclei. Note that in C, the Cav1 spots remain either red or green, and that only few spots have moved across the fusion boundary. In G, even distribution and colocalization of the green and red clathrin is apparent throughout the cell. (D and H) The mixing of Cav1-GFP and -RFP in heterokaryons was enhanced upon stimulation. HeLa cells expressing Cav1-GFP and -RFP were fused and exposed to stimuli. SV40 (MOI 103; D) or 1 mM vanadate (H) was added at 1.5 h after PEG-induced fusion, and cells were incubated for another 1.5 h before fixation. Note extensive mixing of Cav1-GFP and -RFP throughout the cell. Bar, 10 μm. (I) Quantification of caveolar mobility from the experiments in C, D, G, and H. See Fig. S2 for details.

Mentions: Two important results were immediately apparent. First, the red and green caveolar spots crossed the former boundaries between the fused cells slowly (Fig. 3, A–C). After 3 h, 64% (n = 33) of the heterokaryons still displayed steep color gradients across the fusion boundary (Fig. 3, C and I; and Fig. S2 available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1). In many cells, the boundary was clearly visible up to 6 h. This confirmed the relatively immobile character of most Cav1-containing structures in unstimulated cells.


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-GFP and -RFP become mobile in stimulated heterokaryons. (A–H) HeLa cells expressing Cav1-GFP (A) were fused with the ones expressing Cav1-RFP (B), or the cells expressing clathrin light chain–GFP (E) were fused with cells expressing clathrin light chain–RFP (F) by 2 min PEG treatment (merge, C and G, respectively). The cells were incubated in the presence of CHX after fusion, fixed after 3 h, and viewed by confocal microscopy. The dashed circles show the positions of the nuclei. Note that in C, the Cav1 spots remain either red or green, and that only few spots have moved across the fusion boundary. In G, even distribution and colocalization of the green and red clathrin is apparent throughout the cell. (D and H) The mixing of Cav1-GFP and -RFP in heterokaryons was enhanced upon stimulation. HeLa cells expressing Cav1-GFP and -RFP were fused and exposed to stimuli. SV40 (MOI 103; D) or 1 mM vanadate (H) was added at 1.5 h after PEG-induced fusion, and cells were incubated for another 1.5 h before fixation. Note extensive mixing of Cav1-GFP and -RFP throughout the cell. Bar, 10 μm. (I) Quantification of caveolar mobility from the experiments in C, D, G, and H. See Fig. S2 for details.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Cav1-GFP and -RFP become mobile in stimulated heterokaryons. (A–H) HeLa cells expressing Cav1-GFP (A) were fused with the ones expressing Cav1-RFP (B), or the cells expressing clathrin light chain–GFP (E) were fused with cells expressing clathrin light chain–RFP (F) by 2 min PEG treatment (merge, C and G, respectively). The cells were incubated in the presence of CHX after fusion, fixed after 3 h, and viewed by confocal microscopy. The dashed circles show the positions of the nuclei. Note that in C, the Cav1 spots remain either red or green, and that only few spots have moved across the fusion boundary. In G, even distribution and colocalization of the green and red clathrin is apparent throughout the cell. (D and H) The mixing of Cav1-GFP and -RFP in heterokaryons was enhanced upon stimulation. HeLa cells expressing Cav1-GFP and -RFP were fused and exposed to stimuli. SV40 (MOI 103; D) or 1 mM vanadate (H) was added at 1.5 h after PEG-induced fusion, and cells were incubated for another 1.5 h before fixation. Note extensive mixing of Cav1-GFP and -RFP throughout the cell. Bar, 10 μm. (I) Quantification of caveolar mobility from the experiments in C, D, G, and H. See Fig. S2 for details.
Mentions: Two important results were immediately apparent. First, the red and green caveolar spots crossed the former boundaries between the fused cells slowly (Fig. 3, A–C). After 3 h, 64% (n = 33) of the heterokaryons still displayed steep color gradients across the fusion boundary (Fig. 3, C and I; and Fig. S2 available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1). In many cells, the boundary was clearly visible up to 6 h. This confirmed the relatively immobile character of most Cav1-containing structures in unstimulated cells.

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