Limits...
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.

Show MeSH

Related in: MedlinePlus

Immunogold localization of gD1–DAF on the surface  of caveolin-expressing FRT cells. Cav1-FRT cells were grown to  subconfluence on coverslips, incubated with an anti-gD1–DAF  monoclonal antibody on ice for 1 h and, after washing in PBS,  with a secondary antibody conjugated with 10-nm colloidal gold  on ice for 1 h. After fixation, cells were dehydrated through  graded ethanol and were scraped from the coverslips in 70% ethanol. The pellets were then processed as described in Materials  and Methods. A–D show different sections of apical and basolateral plasma membranes of cav1-FRT cells that contain gD1–DAF  clustered in newly formed (A and B) or classical flask-shaped caveolae (C and D; small arrows). A racemose cluster of caveolae  containing gD1–DAF is shown on the apical surface (D; large arrow). Arrowheads indicate the gold particles bound to the anti-gD1–DAF antibody. Bar, 100 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2140173&req=5

Figure 9: Immunogold localization of gD1–DAF on the surface of caveolin-expressing FRT cells. Cav1-FRT cells were grown to subconfluence on coverslips, incubated with an anti-gD1–DAF monoclonal antibody on ice for 1 h and, after washing in PBS, with a secondary antibody conjugated with 10-nm colloidal gold on ice for 1 h. After fixation, cells were dehydrated through graded ethanol and were scraped from the coverslips in 70% ethanol. The pellets were then processed as described in Materials and Methods. A–D show different sections of apical and basolateral plasma membranes of cav1-FRT cells that contain gD1–DAF clustered in newly formed (A and B) or classical flask-shaped caveolae (C and D; small arrows). A racemose cluster of caveolae containing gD1–DAF is shown on the apical surface (D; large arrow). Arrowheads indicate the gold particles bound to the anti-gD1–DAF antibody. Bar, 100 nm.

Mentions: To determine whether gD1–DAF was able to localize in these caveolae at the surface of the transfected FRT cells, we performed an immuno-EM localization of gD1–DAF after cross-linking with antibodies. We found that gD1– DAF was distributed upon the entire surface of transfected FRT cells, and that it was able to cluster in caveolae upon antibody cross-linking (Fig. 9, A–D) as was previously shown in other cell lines (Mayor et al., 1994; Parton et al., 1994; Mayor and Maxfield 1995). These experiments indicated that in FRT cells, the newly formed caveolae are able to interact with GPI-anchored proteins and, together with our other data, suggest that cav1 and caveolae are not involved in GPI-anchored protein trafficking.


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)

Immunogold localization of gD1–DAF on the surface  of caveolin-expressing FRT cells. Cav1-FRT cells were grown to  subconfluence on coverslips, incubated with an anti-gD1–DAF  monoclonal antibody on ice for 1 h and, after washing in PBS,  with a secondary antibody conjugated with 10-nm colloidal gold  on ice for 1 h. After fixation, cells were dehydrated through  graded ethanol and were scraped from the coverslips in 70% ethanol. The pellets were then processed as described in Materials  and Methods. A–D show different sections of apical and basolateral plasma membranes of cav1-FRT cells that contain gD1–DAF  clustered in newly formed (A and B) or classical flask-shaped caveolae (C and D; small arrows). A racemose cluster of caveolae  containing gD1–DAF is shown on the apical surface (D; large arrow). Arrowheads indicate the gold particles bound to the anti-gD1–DAF antibody. Bar, 100 nm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Immunogold localization of gD1–DAF on the surface of caveolin-expressing FRT cells. Cav1-FRT cells were grown to subconfluence on coverslips, incubated with an anti-gD1–DAF monoclonal antibody on ice for 1 h and, after washing in PBS, with a secondary antibody conjugated with 10-nm colloidal gold on ice for 1 h. After fixation, cells were dehydrated through graded ethanol and were scraped from the coverslips in 70% ethanol. The pellets were then processed as described in Materials and Methods. A–D show different sections of apical and basolateral plasma membranes of cav1-FRT cells that contain gD1–DAF clustered in newly formed (A and B) or classical flask-shaped caveolae (C and D; small arrows). A racemose cluster of caveolae containing gD1–DAF is shown on the apical surface (D; large arrow). Arrowheads indicate the gold particles bound to the anti-gD1–DAF antibody. Bar, 100 nm.
Mentions: To determine whether gD1–DAF was able to localize in these caveolae at the surface of the transfected FRT cells, we performed an immuno-EM localization of gD1–DAF after cross-linking with antibodies. We found that gD1– DAF was distributed upon the entire surface of transfected FRT cells, and that it was able to cluster in caveolae upon antibody cross-linking (Fig. 9, A–D) as was previously shown in other cell lines (Mayor et al., 1994; Parton et al., 1994; Mayor and Maxfield 1995). These experiments indicated that in FRT cells, the newly formed caveolae are able to interact with GPI-anchored proteins and, together with our other data, suggest that cav1 and caveolae are not involved in GPI-anchored protein trafficking.

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