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Caveolin transfection results in caveolae formation but not apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins in epithelial cells.

Lipardi C, Mora R, Colomer V, Paladino S, Nitsch L, Rodriguez-Boulan E, Zurzolo C - J. Cell Biol. (1998)

Bottom Line: Biol.However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts.Alternatively, cav1 and caveolae may not be directly involved in these processes.

View Article: PubMed Central - PubMed

Affiliation: Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli Federico II, 80131 Napoli, Italy.

ABSTRACT
Most epithelial cells sort glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface. The "raft" hypothesis, based on data mainly obtained in the prototype cell line MDCK, postulates that apical sorting depends on the incorporation of apical proteins into cholesterol/glycosphingolipid (GSL) rafts, rich in the cholesterol binding protein caveolin/VIP21, in the Golgi apparatus. Fischer rat thyroid (FRT) cells constitute an ideal model to test this hypothesis, since they missort both endogenous and transfected GPI-anchored proteins to the basolateral plasma membrane and fail to incorporate them into cholesterol/glycosphingolipid clusters. Because FRT cells lack caveolin, a major component of the caveolar coat that has been proposed to have a role in apical sorting of GPI-anchored proteins (Zurzolo, C., W. Van't Hoff, G. van Meer, and E. Rodriguez-Boulan. 1994. EMBO [Eur. Mol. Biol. Organ.] J. 13:42-53.), we carried out experiments to determine whether the lack of caveolin accounted for the sorting/clustering defect of GPI-anchored proteins. We report here that FRT cells lack morphological caveolae, but, upon stable transfection of the caveolin1 gene (cav1), form typical flask-shaped caveolae. However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts. Our results demonstrate that the absence of caveolin1 and morphologically identifiable caveolae cannot explain the inability of FRT cells to sort GPI-anchored proteins to the apical domain. Thus, FRT cells may lack additional factors required for apical sorting or for the clustering with GSLs of GPI-anchored proteins, or express factors that inhibit these events. Alternatively, cav1 and caveolae may not be directly involved in these processes.

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Localization by double immunofluorescence of cav1  and gD1–DAF in transfected FRT cells. A stable FRT clonal line  expressing cav1 and gD1–DAF was grown to confluence on glass  coverslips. Cells were fixed with paraformaldehyde and permeabilized with PBS CM containing 0.2% gelatin and 0.075% saponin, and then stained using a polyclonal antibody against caveolin  (A) and a monoclonal antibody against gD1–DAF (B). Primary  antibodies were visualized using anti–mouse TRITC-conjugated  and anti-rabbit FITC-conjugated antibodies. Cav1 gave a punctate staining localized both at the plasma membrane and, less intensely, at the Golgi apparatus, whereas gD1–DAF was enriched  mainly at the basolateral surface. Bar, 50 mm.
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Figure 1: Localization by double immunofluorescence of cav1 and gD1–DAF in transfected FRT cells. A stable FRT clonal line expressing cav1 and gD1–DAF was grown to confluence on glass coverslips. Cells were fixed with paraformaldehyde and permeabilized with PBS CM containing 0.2% gelatin and 0.075% saponin, and then stained using a polyclonal antibody against caveolin (A) and a monoclonal antibody against gD1–DAF (B). Primary antibodies were visualized using anti–mouse TRITC-conjugated and anti-rabbit FITC-conjugated antibodies. Cav1 gave a punctate staining localized both at the plasma membrane and, less intensely, at the Golgi apparatus, whereas gD1–DAF was enriched mainly at the basolateral surface. Bar, 50 mm.

Mentions: To determine whether caveolin was involved in either the apical sorting of gD1–DAF or in its clustering with GSLs, we stably transfected FRT cells with a cDNA encoding the cav1 protein. After initial attempts to transfect cav1 into FRT cells already expressing gD1–DAF failed, we simultaneously transfected a plasmid containing the gD1–DAF gene under control of the RSV promoter (Lisanti et al., 1989), and then a plasmid containing the cav1 coding sequence fused to the cytomegalovirus (CMV) promoter and the gene conferring resistance to neomycin (G418). By selection with G418, we isolated different clones expressing both proteins. Among many clones heterogeneous in their expression of caveolin and gD1–DAF proteins, as visualized by double immunofluorescence experiments (data not shown), we selected two clones (FRTcl1 and FRTcl2) that displayed a homogeneous distribution of both markers within all cells (Fig. 1, A and B). Both clones displayed caveolin as a punctate pattern at the plasma membrane and within the Golgi apparatus (Fig. 1 A). gD1– DAF was similarly enriched at the plasma membrane, albeit with a different, more diffuse, pattern (Fig. 1 B), but was also found intracellularly, as previously shown (Zurzolo et al., 1993).


Caveolin transfection results in caveolae formation but not apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins in epithelial cells.

Lipardi C, Mora R, Colomer V, Paladino S, Nitsch L, Rodriguez-Boulan E, Zurzolo C - J. Cell Biol. (1998)

Localization by double immunofluorescence of cav1  and gD1–DAF in transfected FRT cells. A stable FRT clonal line  expressing cav1 and gD1–DAF was grown to confluence on glass  coverslips. Cells were fixed with paraformaldehyde and permeabilized with PBS CM containing 0.2% gelatin and 0.075% saponin, and then stained using a polyclonal antibody against caveolin  (A) and a monoclonal antibody against gD1–DAF (B). Primary  antibodies were visualized using anti–mouse TRITC-conjugated  and anti-rabbit FITC-conjugated antibodies. Cav1 gave a punctate staining localized both at the plasma membrane and, less intensely, at the Golgi apparatus, whereas gD1–DAF was enriched  mainly at the basolateral surface. Bar, 50 mm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140173&req=5

Figure 1: Localization by double immunofluorescence of cav1 and gD1–DAF in transfected FRT cells. A stable FRT clonal line expressing cav1 and gD1–DAF was grown to confluence on glass coverslips. Cells were fixed with paraformaldehyde and permeabilized with PBS CM containing 0.2% gelatin and 0.075% saponin, and then stained using a polyclonal antibody against caveolin (A) and a monoclonal antibody against gD1–DAF (B). Primary antibodies were visualized using anti–mouse TRITC-conjugated and anti-rabbit FITC-conjugated antibodies. Cav1 gave a punctate staining localized both at the plasma membrane and, less intensely, at the Golgi apparatus, whereas gD1–DAF was enriched mainly at the basolateral surface. Bar, 50 mm.
Mentions: To determine whether caveolin was involved in either the apical sorting of gD1–DAF or in its clustering with GSLs, we stably transfected FRT cells with a cDNA encoding the cav1 protein. After initial attempts to transfect cav1 into FRT cells already expressing gD1–DAF failed, we simultaneously transfected a plasmid containing the gD1–DAF gene under control of the RSV promoter (Lisanti et al., 1989), and then a plasmid containing the cav1 coding sequence fused to the cytomegalovirus (CMV) promoter and the gene conferring resistance to neomycin (G418). By selection with G418, we isolated different clones expressing both proteins. Among many clones heterogeneous in their expression of caveolin and gD1–DAF proteins, as visualized by double immunofluorescence experiments (data not shown), we selected two clones (FRTcl1 and FRTcl2) that displayed a homogeneous distribution of both markers within all cells (Fig. 1, A and B). Both clones displayed caveolin as a punctate pattern at the plasma membrane and within the Golgi apparatus (Fig. 1 A). gD1– DAF was similarly enriched at the plasma membrane, albeit with a different, more diffuse, pattern (Fig. 1 B), but was also found intracellularly, as previously shown (Zurzolo et al., 1993).

Bottom Line: Biol.However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts.Alternatively, cav1 and caveolae may not be directly involved in these processes.

View Article: PubMed Central - PubMed

Affiliation: Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli Federico II, 80131 Napoli, Italy.

ABSTRACT
Most epithelial cells sort glycosylphosphatidylinositol (GPI)-anchored proteins to the apical surface. The "raft" hypothesis, based on data mainly obtained in the prototype cell line MDCK, postulates that apical sorting depends on the incorporation of apical proteins into cholesterol/glycosphingolipid (GSL) rafts, rich in the cholesterol binding protein caveolin/VIP21, in the Golgi apparatus. Fischer rat thyroid (FRT) cells constitute an ideal model to test this hypothesis, since they missort both endogenous and transfected GPI-anchored proteins to the basolateral plasma membrane and fail to incorporate them into cholesterol/glycosphingolipid clusters. Because FRT cells lack caveolin, a major component of the caveolar coat that has been proposed to have a role in apical sorting of GPI-anchored proteins (Zurzolo, C., W. Van't Hoff, G. van Meer, and E. Rodriguez-Boulan. 1994. EMBO [Eur. Mol. Biol. Organ.] J. 13:42-53.), we carried out experiments to determine whether the lack of caveolin accounted for the sorting/clustering defect of GPI-anchored proteins. We report here that FRT cells lack morphological caveolae, but, upon stable transfection of the caveolin1 gene (cav1), form typical flask-shaped caveolae. However, cav1 expression did not redistribute GPI-anchored proteins to the apical surface, nor promote their inclusion into cholesterol/GSL rafts. Our results demonstrate that the absence of caveolin1 and morphologically identifiable caveolae cannot explain the inability of FRT cells to sort GPI-anchored proteins to the apical domain. Thus, FRT cells may lack additional factors required for apical sorting or for the clustering with GSLs of GPI-anchored proteins, or express factors that inhibit these events. Alternatively, cav1 and caveolae may not be directly involved in these processes.

Show MeSH
Related in: MedlinePlus