Volume 91, Issue 9, Pages 3349-3358 (November 2006) Heterogeneity of Early Intermediates in Cell-Liposome Fusion Mediated by Influenza Hemagglutinin  Mikhail A. Zhukovsky, Eugenia Leikina, Ingrid Markovic, Austin L. Bailey, Leonid V. Chernomordik  Biophysical Journal  Volume 91, Issue 9, Pages 3349-3358 (November 2006) DOI: 10.1529/biophysj.106.088005 Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 1 Lipid mixing at 4°C between HA-cells and DOPC-LS (curves 1 and 3), HA-cells and DSPC-LS (curves 2 and 4), recorded at pH 4.9 (curves 1 and 2; pH was lowered to 4.9 at the onset of measurements) and pH 7.4 (curves 3 and 4). Biophysical Journal 2006 91, 3349-3358DOI: (10.1529/biophysj.106.088005) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 2 (A) Lipid mixing at 37°C at neutral pH between HA-cells and DSPC-LS recorded without low-pH application (curve 1) and after the following durations of incubation at 4°C at pH 4.9: 1min (curve 2), 3min (curve 3), 5min (curve 4), 10min (curve 5), 60min (curve 6). (B) Lipid mixing at 37°C at neutral pH between HA-cells and SOPC-LS recorded without low-pH application (curve 1) and after the following durations of incubation at 4°C at pH 4.9: 1min (curve 2), 5min (curve 3), 10min (curve 4), 20min (curve 5). (C) Lipid mixing at 37°C at neutral pH between HA-cells and DSPC-LS, normalized to the level of fluorescence observed 10min after raising the temperature and recorded after the following durations of incubation at 4°C at pH 4.9: 1min, 2min, 3min, 5min, 10min, 30min, 60min, 90min (curves 1–8). Biophysical Journal 2006 91, 3349-3358DOI: (10.1529/biophysj.106.088005) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 3 (A) Lipid mixing at 37°C at neutral pH between HA-cells and DOPC-LS recorded without low-pH application (curve 1) and after the following durations of incubation at 4°C at pH 4.9: 1min (curve 2), 15min (curve 3), 30min (curve 4), and 60min (curve 5). (B) Lipid mixing at 37°C at neutral pH between HA-cells and DSPC/CL-LS recorded without low-pH application (curve 1) and after the following durations of incubation at 4°C at pH 4.9: 2min (curve 2), 5min (curve 3), 15min (curve 4), 60min (curve 5). Biophysical Journal 2006 91, 3349-3358DOI: (10.1529/biophysj.106.088005) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 4 Lipid mixing at 37°C at neutral pH between HA-cells and liposomes is dependent on the duration of incubation at 4°C at pH 4.9. The percentage of fluorescence dequenching of the lipid probe observed 4.5min after raising the temperature is plotted as function of the duration of incubation at 4°C at pH 4.9, normalized to the level of fluorescence after incubation at pH 4.9 for 1min. Liposomes are DOPC-LS (○), DSPC/CL-LS (●), SOPC-LS (□), and DSPC-LS (■). Biophysical Journal 2006 91, 3349-3358DOI: (10.1529/biophysj.106.088005) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 5 Lipid mixing between HA-cells and bound RBCs is dependent on the duration of incubation of the cell pairs at pH 5.2 at 4°C. After this incubation, pH of the medium was returned to neutral, and simultaneously the temperature was raised to 37°C. The extent of fusion was measured 10min after the low-pH application. Bars are mean±SE, n≥3. Biophysical Journal 2006 91, 3349-3358DOI: (10.1529/biophysj.106.088005) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 6 Lipid mixing at 37°C at neutral pH between HA-cells and liposomes is dependent on the duration of incubation at 4°C at pH 4.9. The percentage of fluorescence dequenching of the lipid probe observed 10min after raising the temperature is plotted as a function of the duration of incubation at 4°C at pH 4.9. Dequenching for ganglioside-containing DSPC-LS (●), dequenching for ganglioside-free DSPC-LS (▵), ratio of dequenching extents for ganglioside-containing and ganglioside-free DSPC-LS (□). Biophysical Journal 2006 91, 3349-3358DOI: (10.1529/biophysj.106.088005) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 7 Lipid mixing between virus and DOPC-LS (A) or DSPC-LS (B) was recorded at pH 4.9 at 37°C after R18-labeled viral particles with bound liposomes were preincubated at pH 4.9 at 4°C for 1min, 5min, and 10min (A) and 5min, 15min, and 45min (B). Recordings shown in blue and black represent experiments without pretreatment (blue) and the experiments where lipid mixing was detected at 4°C (black). Note that lipid mixing in the later experiments proceeded much more slowly and thus is presented with a different time scale. Biophysical Journal 2006 91, 3349-3358DOI: (10.1529/biophysj.106.088005) Copyright © 2006 The Biophysical Society Terms and Conditions