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. 2000 Jul;11(7):2359–2371. doi: 10.1091/mbc.11.7.2359

Figure 7.

Figure 7

Formation of transient RH intermediates requires the cooperative action of HA trimers activated in the presence of the target membrane. (A) RH formation requires HA structures that do not develop in the absence of cell contacts. Bars 1 and 2, fusion of X31 HA-cells with bound RBC was triggered by a 2-min pulse of pH 5.3 at 22°C followed immediately (bar 1) or after 30 min of incubation at 4°C (bar 2) by a 1-min application of 0.25 mM CPZ applied at 22°C. Bars 3 and 4, X31 HA-cells were treated with the same pulse of low pH in the absence of RBC. Immediately after the end of the low pH pulse, the temperature was decreased to 4°C and RBC were added immediately (bar 3) or 10 min later (bar 4). After a 30-min incubation at 4°C, unbound RBC were washed out, the temperature was returned to 22°C, and 0.25 mM CPZ was applied for 1 min. Bars represent the mean extents of lipid mixing ± SE, n > 3. (B) RH formation requires the concerted action of multiple HA trimers. The same number of HA molecules formed more RH intermediates if these HA molecules were activated at the same time rather than in portions. Fusion of X31 HA-cells with bound RBC was triggered at room temperature by a 1-min (bar 1) or 6-min (bar 3) pulse of pH 5.3 followed by a 1-min application of 0.5 mM CPZ. Bar 2, after a 1-min application of pH 5.3, cells were incubated at neutral pH for 10 min and then treated with a 5-min pulse of pH 5.3 followed immediately by CPZ (0.5 mM, 1 min). Bar 4 represents the sum of bars 1 and 2. No measurable lipid mixing was observed after each of these low pH pulses with no CPZ applied or after a 1-min pulse of pH 5.3 followed by a 15-min incubation and then by CPZ pulse. Bars 1–3 represent the mean extents of lipid mixing ± SE, n > 3.

HHS Vulnerability Disclosure