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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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Mobility of surface and caveosomal Cav1-GFP can be activated. (A and B) Dynamics of surface Cav1-GFP recorded by TIR-FM in untreated CV-1 cells expressing Cav1-GFP (A), or 1 h after addition of SV40 (MOI 103; B). The images shown are snapshots from Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1). The video was recorded for 2 min at 4 hertz. Red and blue arrowheads indicate appearing and disappearing vesicles, respectively. Note that an increased number of docking and leaving vesicles are observed in the presence of SV40 (see Video 1). Bars, 1 μm. (C) The fraction of mobile Cav1 vesicles at the surface recorded by TIR-FM doubles upon addition of SV40. Experiments were performed as described in A and B, and the fraction of Cav1-GFP vesicles that underwent docking or lateral movement at the cell surface was determined as described in Fig. S1. The mean of three independent experimental sets is shown. Error bars indicate standard deviations of the three experiments. (D and E) Individual caveosomes were bleached in CV-1 cells expressing Cav1-GFP. The cells were (D) untreated or (E) bleaching was performed 1 h after addition of SV40 (MOI 60). From left to right, before (Prebleach), immediately after (Bleach), and 1, 2, and 10 min after bleaching are shown. The circles show the bleached areas. Note that little recovery occurs in the absence of SV40 (see Video 2). Bars, 2 μm. (F) FRAP curves for individual caveosomes in CV-1 cells expressing Cav1-GFP. The cells were either untreated, exposed to SV40 (MOI 60) for 1 h, to 100 μM genistein for 30 min and then 1 h to SV40 (MOI 60), or to 1 mM vanadate for 1 h before the FRAP experiments. Fluorescence recovery was recorded every 10 s for 10 min. Error bars indicate standard deviations of five experiments for untreated, +SV40, vanadate, and three for +SV40/+genistein. Note that in the presence of SV40 or vanadate, the recovery rate is high, and that preincubation with genistein brings the recovery rate back to the slow rate seen in untreated cells. (G) Cav1-GFP does not diffuse laterally in caveosomes. A part of a caveosome (dashed rectangle) was bleached in a CV-1 cell expressing Cav1-GFP. Recovery of fluorescence was recorded every 6 s for 6 min. Before (Prebleach), immediately after (Bleach), and 2, 4, and 6 min after bleaching are shown. Note the slow and incomplete fluorescence recovery, and the lack of lateral redistribution of Cav1-GFP–labeled domains (see Video 3). Bar, 2 μm. (H) A small portion of an endosome (dashed rectangle) was bleached in a CV-1 cell expressing Rab 7-GFP and recovery was recorded as in G. Note that despite the small area bleached, fluorescence in the entire endosome was eliminated, and that recovery was rapid and complete (see Video 4). Bar, 2 μm.
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fig1: Mobility of surface and caveosomal Cav1-GFP can be activated. (A and B) Dynamics of surface Cav1-GFP recorded by TIR-FM in untreated CV-1 cells expressing Cav1-GFP (A), or 1 h after addition of SV40 (MOI 103; B). The images shown are snapshots from Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1). The video was recorded for 2 min at 4 hertz. Red and blue arrowheads indicate appearing and disappearing vesicles, respectively. Note that an increased number of docking and leaving vesicles are observed in the presence of SV40 (see Video 1). Bars, 1 μm. (C) The fraction of mobile Cav1 vesicles at the surface recorded by TIR-FM doubles upon addition of SV40. Experiments were performed as described in A and B, and the fraction of Cav1-GFP vesicles that underwent docking or lateral movement at the cell surface was determined as described in Fig. S1. The mean of three independent experimental sets is shown. Error bars indicate standard deviations of the three experiments. (D and E) Individual caveosomes were bleached in CV-1 cells expressing Cav1-GFP. The cells were (D) untreated or (E) bleaching was performed 1 h after addition of SV40 (MOI 60). From left to right, before (Prebleach), immediately after (Bleach), and 1, 2, and 10 min after bleaching are shown. The circles show the bleached areas. Note that little recovery occurs in the absence of SV40 (see Video 2). Bars, 2 μm. (F) FRAP curves for individual caveosomes in CV-1 cells expressing Cav1-GFP. The cells were either untreated, exposed to SV40 (MOI 60) for 1 h, to 100 μM genistein for 30 min and then 1 h to SV40 (MOI 60), or to 1 mM vanadate for 1 h before the FRAP experiments. Fluorescence recovery was recorded every 10 s for 10 min. Error bars indicate standard deviations of five experiments for untreated, +SV40, vanadate, and three for +SV40/+genistein. Note that in the presence of SV40 or vanadate, the recovery rate is high, and that preincubation with genistein brings the recovery rate back to the slow rate seen in untreated cells. (G) Cav1-GFP does not diffuse laterally in caveosomes. A part of a caveosome (dashed rectangle) was bleached in a CV-1 cell expressing Cav1-GFP. Recovery of fluorescence was recorded every 6 s for 6 min. Before (Prebleach), immediately after (Bleach), and 2, 4, and 6 min after bleaching are shown. Note the slow and incomplete fluorescence recovery, and the lack of lateral redistribution of Cav1-GFP–labeled domains (see Video 3). Bar, 2 μm. (H) A small portion of an endosome (dashed rectangle) was bleached in a CV-1 cell expressing Rab 7-GFP and recovery was recorded as in G. Note that despite the small area bleached, fluorescence in the entire endosome was eliminated, and that recovery was rapid and complete (see Video 4). Bar, 2 μm.

Mentions: By TIR-FM, the majority of the Cav1-GFP–labeled caveolae were stationary. We found that 32 ± 1% of them either appeared or underwent rapid lateral movement during a 2-min recording (Fig. 1, A and C; and Video 1, untreated, and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1). Interestingly, the fraction of mobile spots increased to 66 ± 7% when cells were recorded between 45 and 75 min after addition of SV40 at a multiplicity of infection (MOI) of 103 (Fig. 1, B and C; and Video 1, +SV40). It was evident that the virus induced a dramatic elevation in caveolar dynamics on the cell surface.


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)

Mobility of surface and caveosomal Cav1-GFP can be activated. (A and B) Dynamics of surface Cav1-GFP recorded by TIR-FM in untreated CV-1 cells expressing Cav1-GFP (A), or 1 h after addition of SV40 (MOI 103; B). The images shown are snapshots from Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1). The video was recorded for 2 min at 4 hertz. Red and blue arrowheads indicate appearing and disappearing vesicles, respectively. Note that an increased number of docking and leaving vesicles are observed in the presence of SV40 (see Video 1). Bars, 1 μm. (C) The fraction of mobile Cav1 vesicles at the surface recorded by TIR-FM doubles upon addition of SV40. Experiments were performed as described in A and B, and the fraction of Cav1-GFP vesicles that underwent docking or lateral movement at the cell surface was determined as described in Fig. S1. The mean of three independent experimental sets is shown. Error bars indicate standard deviations of the three experiments. (D and E) Individual caveosomes were bleached in CV-1 cells expressing Cav1-GFP. The cells were (D) untreated or (E) bleaching was performed 1 h after addition of SV40 (MOI 60). From left to right, before (Prebleach), immediately after (Bleach), and 1, 2, and 10 min after bleaching are shown. The circles show the bleached areas. Note that little recovery occurs in the absence of SV40 (see Video 2). Bars, 2 μm. (F) FRAP curves for individual caveosomes in CV-1 cells expressing Cav1-GFP. The cells were either untreated, exposed to SV40 (MOI 60) for 1 h, to 100 μM genistein for 30 min and then 1 h to SV40 (MOI 60), or to 1 mM vanadate for 1 h before the FRAP experiments. Fluorescence recovery was recorded every 10 s for 10 min. Error bars indicate standard deviations of five experiments for untreated, +SV40, vanadate, and three for +SV40/+genistein. Note that in the presence of SV40 or vanadate, the recovery rate is high, and that preincubation with genistein brings the recovery rate back to the slow rate seen in untreated cells. (G) Cav1-GFP does not diffuse laterally in caveosomes. A part of a caveosome (dashed rectangle) was bleached in a CV-1 cell expressing Cav1-GFP. Recovery of fluorescence was recorded every 6 s for 6 min. Before (Prebleach), immediately after (Bleach), and 2, 4, and 6 min after bleaching are shown. Note the slow and incomplete fluorescence recovery, and the lack of lateral redistribution of Cav1-GFP–labeled domains (see Video 3). Bar, 2 μm. (H) A small portion of an endosome (dashed rectangle) was bleached in a CV-1 cell expressing Rab 7-GFP and recovery was recorded as in G. Note that despite the small area bleached, fluorescence in the entire endosome was eliminated, and that recovery was rapid and complete (see Video 4). Bar, 2 μm.
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fig1: Mobility of surface and caveosomal Cav1-GFP can be activated. (A and B) Dynamics of surface Cav1-GFP recorded by TIR-FM in untreated CV-1 cells expressing Cav1-GFP (A), or 1 h after addition of SV40 (MOI 103; B). The images shown are snapshots from Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1). The video was recorded for 2 min at 4 hertz. Red and blue arrowheads indicate appearing and disappearing vesicles, respectively. Note that an increased number of docking and leaving vesicles are observed in the presence of SV40 (see Video 1). Bars, 1 μm. (C) The fraction of mobile Cav1 vesicles at the surface recorded by TIR-FM doubles upon addition of SV40. Experiments were performed as described in A and B, and the fraction of Cav1-GFP vesicles that underwent docking or lateral movement at the cell surface was determined as described in Fig. S1. The mean of three independent experimental sets is shown. Error bars indicate standard deviations of the three experiments. (D and E) Individual caveosomes were bleached in CV-1 cells expressing Cav1-GFP. The cells were (D) untreated or (E) bleaching was performed 1 h after addition of SV40 (MOI 60). From left to right, before (Prebleach), immediately after (Bleach), and 1, 2, and 10 min after bleaching are shown. The circles show the bleached areas. Note that little recovery occurs in the absence of SV40 (see Video 2). Bars, 2 μm. (F) FRAP curves for individual caveosomes in CV-1 cells expressing Cav1-GFP. The cells were either untreated, exposed to SV40 (MOI 60) for 1 h, to 100 μM genistein for 30 min and then 1 h to SV40 (MOI 60), or to 1 mM vanadate for 1 h before the FRAP experiments. Fluorescence recovery was recorded every 10 s for 10 min. Error bars indicate standard deviations of five experiments for untreated, +SV40, vanadate, and three for +SV40/+genistein. Note that in the presence of SV40 or vanadate, the recovery rate is high, and that preincubation with genistein brings the recovery rate back to the slow rate seen in untreated cells. (G) Cav1-GFP does not diffuse laterally in caveosomes. A part of a caveosome (dashed rectangle) was bleached in a CV-1 cell expressing Cav1-GFP. Recovery of fluorescence was recorded every 6 s for 6 min. Before (Prebleach), immediately after (Bleach), and 2, 4, and 6 min after bleaching are shown. Note the slow and incomplete fluorescence recovery, and the lack of lateral redistribution of Cav1-GFP–labeled domains (see Video 3). Bar, 2 μm. (H) A small portion of an endosome (dashed rectangle) was bleached in a CV-1 cell expressing Rab 7-GFP and recovery was recorded as in G. Note that despite the small area bleached, fluorescence in the entire endosome was eliminated, and that recovery was rapid and complete (see Video 4). Bar, 2 μm.
Mentions: By TIR-FM, the majority of the Cav1-GFP–labeled caveolae were stationary. We found that 32 ± 1% of them either appeared or underwent rapid lateral movement during a 2-min recording (Fig. 1, A and C; and Video 1, untreated, and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200506103/DC1). Interestingly, the fraction of mobile spots increased to 66 ± 7% when cells were recorded between 45 and 75 min after addition of SV40 at a multiplicity of infection (MOI) of 103 (Fig. 1, B and C; and Video 1, +SV40). It was evident that the virus induced a dramatic elevation in caveolar dynamics on the cell surface.

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