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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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Electron micrographs of wild-type and  caveolin-expressing FRT cells. Cav1-transfected  and nontransfected FRT cells were grown on filters for 5 d, and then fixed and treated for EM as  described in Materials and Methods. Apical side  (A) and basal side (B) of nontransfected FRT  cells. Note the absence of plasmalemmal caveolae on both surfaces. FRT cells transfected with  cav1 contain caveolae both on the apical (C) and  basolateral membranes (D and E) characterized  as coat-free flask-shaped plasmalemmal invaginations with the diaphragm at the neck (small arrows). Note on the basal side (E) the characteristic cluster of caveolae into racemose structures  (small arrow). cp indicates coated pit; n indicates  nucleus; arrowheads show the edge of the filter.
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Figure 8: Electron micrographs of wild-type and caveolin-expressing FRT cells. Cav1-transfected and nontransfected FRT cells were grown on filters for 5 d, and then fixed and treated for EM as described in Materials and Methods. Apical side (A) and basal side (B) of nontransfected FRT cells. Note the absence of plasmalemmal caveolae on both surfaces. FRT cells transfected with cav1 contain caveolae both on the apical (C) and basolateral membranes (D and E) characterized as coat-free flask-shaped plasmalemmal invaginations with the diaphragm at the neck (small arrows). Note on the basal side (E) the characteristic cluster of caveolae into racemose structures (small arrow). cp indicates coated pit; n indicates nucleus; arrowheads show the edge of the filter.

Mentions: Previous experiments showed that transient expression of VIP21/cav1 using a Semliki Forest virus vector in lymphocytes lacking caveolin promoted the formation of plasmalemmal caveolae (Fra et al., 1995). Furthermore, it was suggested that the formation of large caveolin oligomers might be required for caveolar assembly (Parton and Simons, 1995; Sargiacomo et al., 1995). To definitively address the role of caveolin in caveolar formation, we assayed for the presence of caveolae in the stably transfected and nontransfected FRT cells. Using 0.1% tannic acid to enhance visualization of caveolar invaginations by EM (Palade and Bruns, 1968), we observed that wild-type FRT cells have no plasmalemmal caveolae (Fig. 8, A and B). In contrast, FRT cells expressing caveolin displayed large numbers of normal flask-shaped caveolae (Palade and Bruns, 1968), frequently organized into characteristic racemose clusters (Fig. 8, C–E). These data showed that cav1 was necessary and sufficient to promote efficient caveolae formation in FRT cells.


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)

Electron micrographs of wild-type and  caveolin-expressing FRT cells. Cav1-transfected  and nontransfected FRT cells were grown on filters for 5 d, and then fixed and treated for EM as  described in Materials and Methods. Apical side  (A) and basal side (B) of nontransfected FRT  cells. Note the absence of plasmalemmal caveolae on both surfaces. FRT cells transfected with  cav1 contain caveolae both on the apical (C) and  basolateral membranes (D and E) characterized  as coat-free flask-shaped plasmalemmal invaginations with the diaphragm at the neck (small arrows). Note on the basal side (E) the characteristic cluster of caveolae into racemose structures  (small arrow). cp indicates coated pit; n indicates  nucleus; arrowheads show the edge of the filter.
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Related In: Results  -  Collection

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Figure 8: Electron micrographs of wild-type and caveolin-expressing FRT cells. Cav1-transfected and nontransfected FRT cells were grown on filters for 5 d, and then fixed and treated for EM as described in Materials and Methods. Apical side (A) and basal side (B) of nontransfected FRT cells. Note the absence of plasmalemmal caveolae on both surfaces. FRT cells transfected with cav1 contain caveolae both on the apical (C) and basolateral membranes (D and E) characterized as coat-free flask-shaped plasmalemmal invaginations with the diaphragm at the neck (small arrows). Note on the basal side (E) the characteristic cluster of caveolae into racemose structures (small arrow). cp indicates coated pit; n indicates nucleus; arrowheads show the edge of the filter.
Mentions: Previous experiments showed that transient expression of VIP21/cav1 using a Semliki Forest virus vector in lymphocytes lacking caveolin promoted the formation of plasmalemmal caveolae (Fra et al., 1995). Furthermore, it was suggested that the formation of large caveolin oligomers might be required for caveolar assembly (Parton and Simons, 1995; Sargiacomo et al., 1995). To definitively address the role of caveolin in caveolar formation, we assayed for the presence of caveolae in the stably transfected and nontransfected FRT cells. Using 0.1% tannic acid to enhance visualization of caveolar invaginations by EM (Palade and Bruns, 1968), we observed that wild-type FRT cells have no plasmalemmal caveolae (Fig. 8, A and B). In contrast, FRT cells expressing caveolin displayed large numbers of normal flask-shaped caveolae (Palade and Bruns, 1968), frequently organized into characteristic racemose clusters (Fig. 8, C–E). These data showed that cav1 was necessary and sufficient to promote efficient caveolae formation in FRT cells.

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