Figure 1.
Outer- and inner-leaflet lipid-anchored molecules employed in this study and their cross-linking schemes.(A) The outer-leaflet molecules employed in this work were a prototypical GPI-AR, CD59; a prototypical ganglioside, GM1; and a prototypical nonraft phospholipid, DNP-DOPE. The inner-leaflet molecules examined here were (G and GFP represent EGFP) the following: Lyn-FG, Lyn conjugated at its C-terminus to two molecules of FKBP in series and then to GFP; Myrpal-N20Lyn-GFP, myristoyl, palmitoyl-anchored Lyn peptide conjugated to GFP, where the peptide was the 20-aa N-terminal sequence of Lyn, which contains the conjugation sites for both myristoyl and palmitoyl chains; TM-Lyn-GFP, the TM mutant of Lyn-GFP, in which the TM domain of a prototypical nonraft molecule LDLR was conjugated to the N-terminus of the full-length Lyn-GFP (which cannot be fatty acylated); Palpal-N16GAP43-GFP, palmitoyl, palmitoyl-anchored GAP43 peptide conjugated to GFP, in which the peptide was the 16-aa N-terminal sequence of GAP43 containing two palmitoylation sites (likely to be raft associated); GFP-C5Rho-geranylgeranyl, GFP anchored by a geranylgeranyl chain, in which GFP was conjugated at its C-terminus to the five-aa C-terminal sequence of Rho, which contains a site for attaching an unsaturated geranylgeranyl chain (likely to be non–raft associated); FGH-Ras, H-Ras chimera molecule in which two tandem FKBP molecules linked to GFP were then conjugated to H-Ras; and GFP-tH, GFP linked to the 10-aa C-terminal sequence of H-Ras containing two sites for palmitoylation and a site for farnesylation. These molecules were expressed and observed in live HeLa cells. (B–D) The schemes for clustering (cross-linking) CD59 (B), GM1 (C), and FGH-Ras (D). CD59 was clustered by the sequential additions of anti-CD59 mAb IgG labeled with the fluorescent dye A633 and secondary Abs (+2°-antibodies; B). GM1 was clustered by the sequential additions of CTXB conjugated with A633 and anti-CTXB Abs (C). FGH-Ras (as well as Lyn-FG) was clustered by the addition of AP20187 (cross-linker for FKBP; D). After the induction of clustering of these molecules, the possible recruitment of lipid-anchored molecules in the other leaflet of the PM at these clusters was examined.

Outer- and inner-leaflet lipid-anchored molecules employed in this study and their cross-linking schemes. (A) The outer-leaflet molecules employed in this work were a prototypical GPI-AR, CD59; a prototypical ganglioside, GM1; and a prototypical nonraft phospholipid, DNP-DOPE. The inner-leaflet molecules examined here were (G and GFP represent EGFP) the following: Lyn-FG, Lyn conjugated at its C-terminus to two molecules of FKBP in series and then to GFP; Myrpal-N20Lyn-GFP, myristoyl, palmitoyl-anchored Lyn peptide conjugated to GFP, where the peptide was the 20-aa N-terminal sequence of Lyn, which contains the conjugation sites for both myristoyl and palmitoyl chains; TM-Lyn-GFP, the TM mutant of Lyn-GFP, in which the TM domain of a prototypical nonraft molecule LDLR was conjugated to the N-terminus of the full-length Lyn-GFP (which cannot be fatty acylated); Palpal-N16GAP43-GFP, palmitoyl, palmitoyl-anchored GAP43 peptide conjugated to GFP, in which the peptide was the 16-aa N-terminal sequence of GAP43 containing two palmitoylation sites (likely to be raft associated); GFP-C5Rho-geranylgeranyl, GFP anchored by a geranylgeranyl chain, in which GFP was conjugated at its C-terminus to the five-aa C-terminal sequence of Rho, which contains a site for attaching an unsaturated geranylgeranyl chain (likely to be non–raft associated); FGH-Ras, H-Ras chimera molecule in which two tandem FKBP molecules linked to GFP were then conjugated to H-Ras; and GFP-tH, GFP linked to the 10-aa C-terminal sequence of H-Ras containing two sites for palmitoylation and a site for farnesylation. These molecules were expressed and observed in live HeLa cells. (B–D) The schemes for clustering (cross-linking) CD59 (B), GM1 (C), and FGH-Ras (D). CD59 was clustered by the sequential additions of anti-CD59 mAb IgG labeled with the fluorescent dye A633 and secondary Abs (+2°-antibodies; B). GM1 was clustered by the sequential additions of CTXB conjugated with A633 and anti-CTXB Abs (C). FGH-Ras (as well as Lyn-FG) was clustered by the addition of AP20187 (cross-linker for FKBP; D). After the induction of clustering of these molecules, the possible recruitment of lipid-anchored molecules in the other leaflet of the PM at these clusters was examined.

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