Figure 1. Binding of HypF-N oligomers to cells with differing GM1 contents and the ensuing Ca²⁺ influx.

(A) Representative confocal scanning microscopy images of GM1-depleted (25 μM PDMP), basal and GM1-enriched (100 μg/ml GM1) cells treated for 1 h with type A or type B HypF-N oligomers (12 μM, monomer equivalents). Red and green fluorescence indicates the cell membranes and the HypF-N oligomers (small dots), respectively. Large aggregates (big dots) result from oligomer clustering on the cell membrane. (B) Plot showing the extent of oligomer binding to the membrane against GM1 content, for type A (red) or type B (blue) HypF-N oligomers (12 μM, monomer equivalents). Red and blue lines represent the best fits to hyperbolic and linear functions, respectively. Variable numbers of cells (12–22) in three different experiments were analysed for each condition. Error bars refer to standard deviation (S.D.) (C) Representative confocal scanning microscopy images of GM1-depleted (25 μM PDMP), basal and GM1-enriched (100 μg/ml GM1) cells showing levels of intracellular free Ca²⁺ following treatment for 1 h with type A or type B oligomers of HypF-N (12 μM, monomer equivalents); the green fluorescence arises from Ca²⁺ binding to the intracellular Fluo3-AM probe. (D) Intracellular Ca²⁺ fluorescence against GM1 content for type A (red) or type B (blue) HypF-N oligomers. Other details are as for panel B.