Figure 5.
Stability of transient RH (A) and UH (B, C) upon proteolysis of low pH form of HA (A, B) and in the presence of LPC (C). (A) Although mild treatment of transient RH intermediates with thermolysin facilitated lipid mixing, harsher treatment caused complete dissociation of RH. Fusion of X31 HA-cells with bound RBC was triggered by a 90-s pulse of pH 5.2 at room temperature. Bar 1, lipid mixing extent observed in the control experiment with neither CPZ nor thermolysin applied. Bar 2, low pH application was followed by a 1-min pulse of 0.25 mM CPZ. Bar 3, immediately after the end of the low pH pulse, cells were treated with thermolysin (2 μg/ml, 10 s) and no CPZ was applied. Bars 4 and 5, harsher treatment with thermolysin (40 μg/ml, 1 min) followed or not followed (bar 5 and 4, respectively) by the CPZ pulse. Bars represent the mean extents of lipid mixing ± SE, n > 3. (B) The development of HA-independent binding between PKH26- and carboxyfluorescein-labeled RBC and GPI-HA cells at later stages of UH. Cells were incubated at pH 4.9 for 5 min at 37°C. Immediately after the pulse (bar 1), or after 5 or 10 min of incubation at neutral pH (bars 2 and 3, respectively), the cells were treated for 20 min with proteinase K (0.1 mg/ml) and neuraminidase (1 U/ml). Then, we screened at least 200 cells to find the average number of carboxyfluorescein-labeled RBC bound to each PKH26-labeled GPI-HA cell. In the control experiment, neither proteinase K nor neuraminidase was applied (bar 4). The number of hemifused (i.e., PKH26-labeled) GPI-HA cells achieved the maximal level (66 ± 6%) within 5 min of low pH application and remained constant throughout the experiment. (C) Hemifusionconnections can be reversibly suppressed by LPC only at an early stage of the UH. Fusion of X31 HA-cells with bound PKH26- and carboxyfluorescein-labeled RBC was triggered at 37°C by a 2-min pulse of pH 5.2. After the pulse, the cells were incubated in PBS (pH 7.4) for 5 min, still at 37°C. During this time, the percentage of PKH26-labeled (closed bars) and carboxyfluorescein-labeled (open bars) HA-cells reached the maximum (“final”) levels (bars 1). After this 5-min incubation (bars 2–4), or after longer, 35-min incubation intervals (bars 5–7), the cells were treated with a 1-min pulse of 0.25 mM CPZ at 22°C (bars 2 and 5) or with CPZ in the presence of 150 μM LPC (bars 3 and 6). In the experiments indicated by bars 4 and 7, the cells were incubated with LPC for 2 min, LPC was washed out, and a CPZ pulse was applied. Bars represent the mean extents of lipid mixing ± SE, n > 3.