Correctly sorted molecules of a GPI-anchored protein are clustered and immobile when they arrive at the apical surface of MDCK cells.

Abstract

Abstract. Glycosyl-phosphatidylinositol (GPI)-anchored proteins are sorted to the apical surface of many epithelial cell types. To better understand the mechanism for apical segregation of these proteins, we analyzed the lateral mobility and molecular associations of a model GPI-anchored protein, herpes simplex virus gD1 fused to human decay accelerating factor (gD1-DAF) (Lisanti, M. P., I. W. Caras, M. A. Davitz, and E. . J. Cell Biol. 109:2145-2156 shortly after arrival and after longterm residence at the surface of confluent, polarized MDCK cells. FRAP measurements of lateral diffusion showed that the mobile fraction of newly arrived gD1-DAF molecules was much less than the mobile fraction of long-term resident molecules (40 vs. 80-90%). Fluorescence resonance energy transfer measurements showed that the newly arrived molecules were clustered, while resident molecules were not. Newly delivered gD1-DAF molecules were clustered but not immobilized in mutant, Concanavalin A-resistant MDCK cells that failed to sort gD1-DAE Our results indicate that GPI-anchored proteins in MDCK cells are clustered before delivery to the surface. However, clustering alone does not target molecules for apical delivery. The immobilization observed when gD1-DAF is correctly sorted suggests that the clusters must associate some component of the cell's cytoplasm.

GPI-anchored protein organization and dynamics at the cell surface

“…It was speculated that the size, glycosylation, and interactions of the ecto-domain of the GPIanchored proteins with the extracellular matrix, other TM receptors, or cortical cytoskeletal corrals could be a potential explanation ( 37,39 ). FRAP-based studies also showed that apically sorted GPIs are immobile just after reaching the cell surface, and their mobility increases as they become long-term residents at the cell surface, suggesting that GPI-anchored proteins are delivered to the membrane in a clustered form ( 40 ). Studying the dynamics of GPI-anchored proteins ( 41 ) provided a scale dependence to GPI-anchored protein diffusion, which suggested that they diffuse freely as individual molecules over a larger length scale, but may dynamically partition into raft domains.…”

The epithelial polarity program: machineries involved and their hijacking by cancer

“…Today, the concept of lipid rafts has evolved and association with lipid rafts is considered not sufficient for apical targeting. Fifteen years old observations (Hannan et al, 1993) and recent work (Paladino et al, 2004) suggest that the ability of glycosylphosphatidyl inositol anchors to act as apical sorting signals depends on clustering through their carbohydrate or proteinaceous moieties (RodriguezBoulan et al, 2005). It has been proposed that glycosylphosphatidyl inositol-protein clustering may be mediated by lectins of the galectin class (reviewed by Fullekrug and Simons, 2004).…”

Apical trafficking in epithelial cells: signals, clusters and motors

“…Early experiments with a clonal MDCK cell line resistant to killing by the lectin concanavalin A (ConA) (Meiss et al, 1982) suggested an explanation. ConA-resistant MDCK cells were unable to correctly sort GPI-APs to the apical surface, and fluorescence recovery after photobleaching (FRAP) experiments showed that, unlike wild-type MDCK cells, they were unable to 'cluster' GPI-APs into immobile aggregates, suggesting that "correctly sorted GPI-APs are clustered before arrival at the apical surface" (Hannan et al, 1993). More recent experiments by Zurzolo and co-workers (Paladino et al, 2004) provided additional evidence for a key clustering event in apical targeting of GPI-APs.…”

“…Journal of Cell Science 122 (23) (Lisanti et al, 1989a;Brown et al, 1989;Brown and Rose, 1992;de Marco et al, 2006;Delacour et al, 2005;Hannan et al, 1993;Paladino et al, 2008;Paladino et al, 2004;Vieira et al, 2005) (Gundersen et al, 1991;Deora et al, 2005;Diaz et al, 2009) (Hoekstra et al, 2004;Slimane et al, 2003;Wakabayashi et al, 2006) ATP7B (Menkes) copper transporter ? ?…”