Sequestration of GPI-Anchored Proteins in Caveolae Triggered by Cross-Linking

Abstract

Glycosyl-phosphatidylinositol (GPI)-anchored proteins have been reported to reside in clusters collected over small membrane invaginations called caveolae. The detection of different GPI-anchored proteins with fluorescently labeled monoclonal antibodies showed that these proteins are not constitutively concentrated in caveolae; they enter these structures independently after cross-linking with polyclonal secondary antibodies. Analysis of the cell surface distribution of the GPI-anchored folate receptor by electron microscopy confirms these observations. Thus, multimerization of GPI-anchored proteins regulates their sequestration in caveolae, but in the absence of agents that promote clustering they are diffusely distributed over the plasma membrane.

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

“…The distribution of both GPIanchored proteins and CTXB also appears to be independent of caveolin/caveolae. This is consistent with the finding that GPI-anchored proteins are not enriched in caveolae unless they are cross-linked with antibodies (Mayor et al, 1994;Fujimoto, 1996) and that CTXB labeling is not confined exclusively to caveolae (Montesano et al, 1982;Parton, 1994). In fact, the highest E we observed was for CTXB in Fao cells ( Figures 3C and 5B), in which no caveolin labeling could be detected by immunofluorescence microscopy (our unpublished results).…”

“…If cells were instead labeled with monoclonal antibodies followed by anti-mouse Ig and briefly incubated at 37°C before fixation, discrete spots of label were seen (our unpublished results). These puncta were similar to those reported in previous studies (Rothberg et al, 1990;Mayor et al, 1994;Fujimoto, 1996;Harder et al, 1998;Kenworthy and Edidin, 1998). The plasma membrane distribution of CTXB and GPI-anchored proteins was more diffuse and extensive than that of caveolin-1 or -2, which could be detected by indirect immunofluorescence microscopy in HeLa and NRK cells but not in Fao cells (our unpublished results).…”

“…The plasma membrane distribution of CTXB and GPI-anchored proteins was more diffuse and extensive than that of caveolin-1 or -2, which could be detected by indirect immunofluorescence microscopy in HeLa and NRK cells but not in Fao cells (our unpublished results). That neither the GPI-anchored proteins nor CTXB appear to be exclusively associated with caveolin/caveolae is in agreement with previous reports (Montesano et al, 1982;Mayor et al, 1994;Parton, 1994;Fujimoto, 1996). Because clustering of CTXB in caveolae in living cells is enhanced with increasing the temperature of incubation (Parton, 1994), in our experiments cells were labeled on ice, a condition in which minimal clustering occurs, and then immediately fixed.…”

“…Such domains containing clustered GPI-anchored proteins can sometimes be detected by conventional light or electron microscopy (Matsuura et al 1984;Latker et al 1987;Kobayashi and Robinson, 1991;van den Berg et al, 1995) (reviewed in Anderson [1998]). In other cases clusters of GPI-anchored proteins or other lipid raft components are only apparent when they have been crosslinked with secondary antibodies (Howell et al 1987;Rothberg et al, 1990;Fra et al, 1994;Mayor et al, 1994;Parton et al, 1994;Fujimoto, 1996;Harder et al, 1998). This suggests that in vivo, lipid rafts may be small or dynamic structures that are aggregated and stabilized by detergent extraction or by antibody-induced cross-linking (Edidin, 1997;Harder and Simons, 1997;Simons and Ikonen, 1997;Weimbs et al, 1997;Brown and London, 1998;Jacobson and Dietrich, 1999).…”

Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy

“…Because folate gold nanoparticles and anti-folate receptor antibodies are multivalent, and because multimerization of folate receptors by antibodies has been shown to affect the internalization itinerary (34), data from these studies may not reflect the intracellular pathway taken by monomeric folate conjugates after internalization (2, 3). Although studies with [ 3 H]folate uptake into Fig.…”