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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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Newly assembled caveolar domains in the Golgi complex and in transit to the PM. (A) Appearance of yellow Cav1 in the heterokaryons of HeLa cells expressing Cav1-GFP and -RFP, respectively, was detected on the PM (surface) by TIR-FM as well as in the Golgi complex by confocal microscopy in the absence of CHX, 3 h after fusion. Cav1-GFP, -RFP, merged image of Cav1-GFP and -RFP (merge), and anti-Giantin are shown. Bars, 5 μm. (B) Kinetics of Cav1 appearance on the PM in CV-1 cells expressing Cav1-GFP observed by TIR-FM. The number of spots in three homogenously illuminated areas per cell was counted, normalized to the average total visible cell surface (671 ± 228 μm2, disregarding indentations and non-flat areas of the PM), and plotted against time. Red, blue, and green lines represent three independent experiments, and the error bars are SDs at each time point within each set of experiment. (C) Spinning disc confocal images of Cav1 structures leaving from the Golgi complex in CV-1 cells expressing Cav1-GFP (arrowheads). Images taken at 2 Hz (0–4 s) (Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1) and a projected image over the 4 s (projection) are shown. Bar, 5 μm. (D) Cav1 structures leaving from the Golgi complex to the PM in CV-1 cells expressing Cav1-GFP were imaged at 1 Hz for 300 frames on the TIR-FM, but with illumination such that cell surface and part of the Golgi complex were visible simultaneously (Video 10). Consecutive frames were subtracted from each other to yield a stack of images with only moving objects. The frames were projected, and trajectories generated this way are shown. Note that the Cav1 spots in the trajectories do not change their appearance (arrowheads). Bar, 10 μm. (E) Cav1 structures leaving from the Golgi complex area (cloudy staining in the background) and arriving on the cell surface (arrowheads) in CV-1 cells expressing Cav1-GFP. Images were taken as in D (Video 10), and selected frames (0, 3, 7, 9, 19, and 46 s) are shown. Bar, 2 μm.
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fig6: Newly assembled caveolar domains in the Golgi complex and in transit to the PM. (A) Appearance of yellow Cav1 in the heterokaryons of HeLa cells expressing Cav1-GFP and -RFP, respectively, was detected on the PM (surface) by TIR-FM as well as in the Golgi complex by confocal microscopy in the absence of CHX, 3 h after fusion. Cav1-GFP, -RFP, merged image of Cav1-GFP and -RFP (merge), and anti-Giantin are shown. Bars, 5 μm. (B) Kinetics of Cav1 appearance on the PM in CV-1 cells expressing Cav1-GFP observed by TIR-FM. The number of spots in three homogenously illuminated areas per cell was counted, normalized to the average total visible cell surface (671 ± 228 μm2, disregarding indentations and non-flat areas of the PM), and plotted against time. Red, blue, and green lines represent three independent experiments, and the error bars are SDs at each time point within each set of experiment. (C) Spinning disc confocal images of Cav1 structures leaving from the Golgi complex in CV-1 cells expressing Cav1-GFP (arrowheads). Images taken at 2 Hz (0–4 s) (Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1) and a projected image over the 4 s (projection) are shown. Bar, 5 μm. (D) Cav1 structures leaving from the Golgi complex to the PM in CV-1 cells expressing Cav1-GFP were imaged at 1 Hz for 300 frames on the TIR-FM, but with illumination such that cell surface and part of the Golgi complex were visible simultaneously (Video 10). Consecutive frames were subtracted from each other to yield a stack of images with only moving objects. The frames were projected, and trajectories generated this way are shown. Note that the Cav1 spots in the trajectories do not change their appearance (arrowheads). Bar, 10 μm. (E) Cav1 structures leaving from the Golgi complex area (cloudy staining in the background) and arriving on the cell surface (arrowheads) in CV-1 cells expressing Cav1-GFP. Images were taken as in D (Video 10), and selected frames (0, 3, 7, 9, 19, and 46 s) are shown. Bar, 2 μm.

Mentions: When CHX was omitted, heterokaryons of HeLa cells expressing Cav1-GFP and -RFP did display discrete yellow spots on the cell surface visible by TIR-FM in addition to the preexisting red and the green spots (Fig. 6 A, surface). The yellow spots appeared within the first 2–3 h after fusion (16 ± 4% yellow spots of total at 3 h).


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)

Newly assembled caveolar domains in the Golgi complex and in transit to the PM. (A) Appearance of yellow Cav1 in the heterokaryons of HeLa cells expressing Cav1-GFP and -RFP, respectively, was detected on the PM (surface) by TIR-FM as well as in the Golgi complex by confocal microscopy in the absence of CHX, 3 h after fusion. Cav1-GFP, -RFP, merged image of Cav1-GFP and -RFP (merge), and anti-Giantin are shown. Bars, 5 μm. (B) Kinetics of Cav1 appearance on the PM in CV-1 cells expressing Cav1-GFP observed by TIR-FM. The number of spots in three homogenously illuminated areas per cell was counted, normalized to the average total visible cell surface (671 ± 228 μm2, disregarding indentations and non-flat areas of the PM), and plotted against time. Red, blue, and green lines represent three independent experiments, and the error bars are SDs at each time point within each set of experiment. (C) Spinning disc confocal images of Cav1 structures leaving from the Golgi complex in CV-1 cells expressing Cav1-GFP (arrowheads). Images taken at 2 Hz (0–4 s) (Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1) and a projected image over the 4 s (projection) are shown. Bar, 5 μm. (D) Cav1 structures leaving from the Golgi complex to the PM in CV-1 cells expressing Cav1-GFP were imaged at 1 Hz for 300 frames on the TIR-FM, but with illumination such that cell surface and part of the Golgi complex were visible simultaneously (Video 10). Consecutive frames were subtracted from each other to yield a stack of images with only moving objects. The frames were projected, and trajectories generated this way are shown. Note that the Cav1 spots in the trajectories do not change their appearance (arrowheads). Bar, 10 μm. (E) Cav1 structures leaving from the Golgi complex area (cloudy staining in the background) and arriving on the cell surface (arrowheads) in CV-1 cells expressing Cav1-GFP. Images were taken as in D (Video 10), and selected frames (0, 3, 7, 9, 19, and 46 s) are shown. Bar, 2 μm.
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Related In: Results  -  Collection

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fig6: Newly assembled caveolar domains in the Golgi complex and in transit to the PM. (A) Appearance of yellow Cav1 in the heterokaryons of HeLa cells expressing Cav1-GFP and -RFP, respectively, was detected on the PM (surface) by TIR-FM as well as in the Golgi complex by confocal microscopy in the absence of CHX, 3 h after fusion. Cav1-GFP, -RFP, merged image of Cav1-GFP and -RFP (merge), and anti-Giantin are shown. Bars, 5 μm. (B) Kinetics of Cav1 appearance on the PM in CV-1 cells expressing Cav1-GFP observed by TIR-FM. The number of spots in three homogenously illuminated areas per cell was counted, normalized to the average total visible cell surface (671 ± 228 μm2, disregarding indentations and non-flat areas of the PM), and plotted against time. Red, blue, and green lines represent three independent experiments, and the error bars are SDs at each time point within each set of experiment. (C) Spinning disc confocal images of Cav1 structures leaving from the Golgi complex in CV-1 cells expressing Cav1-GFP (arrowheads). Images taken at 2 Hz (0–4 s) (Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1) and a projected image over the 4 s (projection) are shown. Bar, 5 μm. (D) Cav1 structures leaving from the Golgi complex to the PM in CV-1 cells expressing Cav1-GFP were imaged at 1 Hz for 300 frames on the TIR-FM, but with illumination such that cell surface and part of the Golgi complex were visible simultaneously (Video 10). Consecutive frames were subtracted from each other to yield a stack of images with only moving objects. The frames were projected, and trajectories generated this way are shown. Note that the Cav1 spots in the trajectories do not change their appearance (arrowheads). Bar, 10 μm. (E) Cav1 structures leaving from the Golgi complex area (cloudy staining in the background) and arriving on the cell surface (arrowheads) in CV-1 cells expressing Cav1-GFP. Images were taken as in D (Video 10), and selected frames (0, 3, 7, 9, 19, and 46 s) are shown. Bar, 2 μm.
Mentions: When CHX was omitted, heterokaryons of HeLa cells expressing Cav1-GFP and -RFP did display discrete yellow spots on the cell surface visible by TIR-FM in addition to the preexisting red and the green spots (Fig. 6 A, surface). The yellow spots appeared within the first 2–3 h after fusion (16 ± 4% yellow spots of total at 3 h).

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