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doi: 10.1242/10.1242/jcs.00130

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The scaffolding domain of caveolin 2 is responsible for its Golgi localization in Caco-2 cells

Lionel Breuza1, Séverine Corby1, Jean-Pierre Arsanto1, Marie-Hélène Delgrossi1, Peter Scheiffele2 and André Le Bivic1,*

1 Laboratoire de Neurogenèse et Morphogenèse au cours du Développement et chez l'Adulte (NMDA), UMR 6156, Institut de Biologie du Développement de Marseille, Faculté des Sciences de Luminy, case 907, Université de la Méditerranée, 13288, Marseille Cedex 09, France
2 Columbia University, New-York, NY 10032, USA



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  		Fig. 1. Analysis of Cav-2 expression in Caco-2 cells. Microsomal fractions of Caco-2 and Huvec (endothelial) cells were analyzed by SDS-PAGE and western blotting. Cav-2 was undetectable in Caco-2 cells but strongly expressed in endothelial cells using an anti-human Cav-2. A molecular mass marker is indicated on the left.

 


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  		Fig. 7. Expression and localization of chimeras in Caco-2 cells. (A) Stable clones of Caco-2 cells expressing Cav-1 (1), Cav-2 (2) or chimeras I, II, III or IV were lysed and 50 µg of each homogenate was analyzed by SDS-PAGE and immunoblotting with the polyclonal antibody against Cav-1 (N-20). Chimeras I, II, III and IV showed a slower migration because of the addition of the Myc epitope. The molecular mass markers are indicated on the left in kDa. (B) Subcellular localization of chimeras by confocal analysis of Caco-2 cells. Cells expressing Chimeras I, II, III and IV were double-labeled with rabbit polyclonal anti-Cav-1 (N20) (red) and mouse monoclonal antibody (green) against Giantin (a Golgi marker) (a,c,e,g) or Ag525 (an endogenous basolateral marker) (b,d,f,h). CH-I (I) and II (II) can be observed at the periphery of cells marked by arrows while CH-III and IV show colocalization with Giantin indicated by arrowheads. Bar, 5 µm.

 


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  		Fig. 2. Subcellular localization of Cav-1 and Cav-2 by confocal analysis of Caco-2 cell clones. Cells expressing Cav-1 (a,c,e) or Cav-2 (b d,f) were double-labeled with rabbit polyclonal anti-Cav-1 (N20) or Cav-2 antibodies (red) and mouse monoclonal antibodies (green) to SI (a,b), Ag 525 (c,d) or Giantin (e,f), used as apical, basolateral or Golgi markers, respectively. Z optical sections (a,b) show that neither Cav-1 (a) nor Cav-2 (b) can be observed at the apical membrane stained for SI (arrowheads). Cav-1 (c,e) is detected both in the Golgi complex and at the lateral membrane (arrows) while Cav-2 (d,f) only showed a perinuclear labeling (arrows). Bar, 5 µm.

 


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  		Fig. 3. Immunoelectron microscopic localization of Cav-2 (A), Cav-1 (B) or Cav-1 and Cav-2 (C-G) in transfected Caco-2 cells. Ultrathin cryosections were stained with antibodies to Cav-1 (pAb N20) and/or Cav-2 (mAb65). (A) Cav-2 labeling appeared confined to intracellular structures (arrows). No gold particles were seen at the plasma membrane, neither at the basal (bpm) and lateral (lpm) nor at the apical (apm) plasma membrane. The star indicates apical microvilli. (B) Immunogold labeling for Cav-1 was observed at the basolateral plasma membrane (bpm, lpm). (C-G) Double immunogold labeling of Cav-1 (6 nm gold) and Cav-2 (15 nm gold) in Caco-2 cells co-expressing Cav-1 and Cav-2. (C) The two types of gold particles for Cav-1 and Cav-2 were found distributed on intracellular profiles, but only Cav-1 labeling was also observed along the plasma membrane (pm, arrowheads). (D-E) Gold particles for Cav-1 (small arrows) or Cav-2 (large arrow) were observed on vesicular or tubular membrane profiles. G, putative Golgi complex. (F-G) Immunogold labeling for Cav-1 could be seen here on the plasma membrane (pm, arrowhead), in particular on caveolae (arrows). Bars, 100 nm.

 


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  		Fig. 4. Localization of Cav-2 in Caco-2 cells expressing Cav-1. (a) Confocal sections showed that Cav-1 and Cav-2 overlapped in a perinuclear region (arrowhead) with Cav-1 showing an additional cytoplasmic and lateral labeling (arrowhead). (b) Cav-2 was still restricted to the Golgi complex co-localizing with Giantin (arrows) in Cav-1 expressing Caco-2 cells. (c,d) Localization of GFP-Cav-2 or Cav-2-GFP (green), respectively, in Cav-1 Caco-2 cells labeled with an antibody against Cav-1 (red). No GFP staining of the plasma membrane could be detected (arrows) while accumulation into an intracellular compartment was observed (arrowheads). Bar, 5 µm.

 


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  		Fig. 5. (A) Cav-1 and Cav-2 form high molecular weight complexes in Caco-2 cells. Caco-2 cells expressing Cav-1, Cav-2 or both, were lyzed, loaded on top of a 5-40% sucrose gradient and centrifuged for 16 hours at 100,000 g. Top to bottom fractions were TCA precipitated, analyzed by SDS-PAGE and immunoblotted with polyclonal caveolin-1 antibodies (anti-Cav-1) or monoclonal antimyc to detect myc-tagged Cav-2. When expressed alone, Cav-1 was present in high molecular weight complexes while Cav-2 alone was not. Co-expression of Cav-1 along with Cav-2 recruited some Cav-2 and increased the proportion of Cav-1 in high molecular weight complexes. (B) Cav-1 and Cav-2 co-immunoprecipitation in Caco-2 cells. Cav-2 and Cav-1+2 Caco-2 cells were pulse-labeled with 35S methionine for 3 hours and lyzed in 1% Triton X-100. Lysates were immunoprecipitated with polyclonal Cav-1 antibody (1) or mAb65 anti-Cav-2 (2). Immunoprecipitates were analyzed by SDS-PAGE and fluorography. Cav-1 was detected in Cav-2 immunoprecipitates in Caco-2 cells expressing both proteins.

 


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  		Fig. 6. Cav-1/Cav-2 chimeras. Chimeras (CH-I to IV) between Cav-1 (in shaded boxes) and Cav-2 (open boxes) were made by PCR. A Myc epitope was added at the C-terminal end of Cav-2 and the chimeras. N, amino-domain; SD, scaffolding domain; Mb, membrane domain; C, carboxyl-domain. The numbers of amino acids contained in each domain are indicated.

 


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  		Fig. 8. Oligomerization state of the chimeras in Caco-2 cells. The formation of oligomers by Cav-1, Cav-2 and the chimeras was analyzed as in Fig. 5 and quantified by densitometry after scanning of the western blots using the N-20 (Cav-1 and chimeras) or the anti-Myc antibody (Cav-2). Results are given as a percentage of the amount of protein found in the lower part of the gradients versus the total amount of protein detected into the gradient. (A) Quantification of the percentage of Cav-1 (black bars) and Cav-2 (empty bars) forming high molecular weight oligomers in Cav-1, Cav-2 and double transfected Caco-2 cells (Cav-1 + Cav-2). (B) Quantification of the percentage of chimeras forming high molecular weight oligomers in transfected Caco-2 cells. Cav-2 and the chimeras are mainly found in mono- and dimeric forms as opposed to Cav-1.

 


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  		Fig. 9. Association of Chimeras with rafts. (A) MDCK and Caco-2 cells expressing Cav-1, Cav-2 or Cav-1 + Cav-2 were lyzed in a buffer containing Triton X-100 at 4°C and loaded in sucrose gradients. Raft and soluble fractions were harvested and analyzed by SDS-PAGE and immunoblotting with the N-20 antibody for Cav-1 (black bars) or the anti-Myc antibody for Cav-2 (empty bars) in Caco-2 cells. Blots were quantified by densitometry after scanning and the results are expressed as the percentage of protein found in the raft fraction versus the total amount of protein found in the gradient (n=3). (B) Caco-2 cells expressing CH-I to IV were treated as in A using the N-20 antibody. The results are expressed as the percentage of protein found in the raft fraction versus the total amount of protein found in the gradient (n=3). Only CH-I is found in majority in the raft fraction.

 

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© The Company of Biologists Ltd 2002