Figure 6. IRSp53 I-BAR domain enhances Gag recruitment to GUV-membranes and at the tip of I-BAR domain-induced tubes.

(a) (Left) AX488 Gag fluorescence intensity on membranes in the absence of I-BAR domain (named ‘Gag only’), in the presence of I-BAR domain where GUVs were first incubated with I-BAR domain and then Gag (named ‘I-BAR + Gag’) and GUVs were first incubated with Gag and then I-BAR domain (named ‘Gag + I-BAR’). Each circle presents one GUV analysis. N = 82 GUVs, n = 4 sample preparations for ‘Gag only,’, N = 67 GUVs, n = 4 sample preparations for ‘I-BAR + Gag,’ and N = 104 GUVs, n = 4 sample preparations for ‘Gag + I-BAR’. To pool all data points from the four sample preparations, in each preparation for all three conditions, Gag intensities were normalized by the mean Gag intensity in the ‘Gag only’ condition. Protein bulk concentrations: 0.3 µM for AX488 Gag and 0.5 µM for I-BAR domain (not fluorescently labeled). (Right) Representative confocal images of AX488 Gag on GUV membranes in ‘I-BAR + Gag’ condition. To visualize GUV membranes, 0.5 mol% of BODIPY-TR-C5-ceramide was incorporated in the membranes. (b) Representative confocal images of AX594 Gag in I-BAR domain-induced tubules. Inverted grayscale images are shown for I-BAR domain and Gag. Protein bulk concentrations: 0.3 μM for AX594 Gag and 0.05 µM for I-BAR domain (70% unlabeled and 30% AX488 labeled I-BAR domain). Cyan arrowheads point out I-BAR domain-induced tubules and white arrows indicate the colocalization of Gag and I-BAR domain at the tips of the tubules. (c and d) Representative confocal images of AX488 Gag (green) in I-BAR domain-induced tubules. Protein bulk concentrations: in (c) 0.1 µM for AX488 Gag and 0.05 µM for I-BAR domain (not fluorescently labeled); in (d) 0.3 µM for AX488 Gag and 0.05 µM for I-BAR domain (not fluorescently labeled). To visualize GUV membranes, 0.5 mol% of BODIPY-TR-C5-ceramide (magenta) was incorporated in the membranes. In (c), inverted grayscale images were shown for membranes and Gag. The cyan arrowhead points out an I-BAR domain-induced tubule and white arrow indicates Gag signals at the tip of the tubule. Sorting map was obtained by calculating the fluorescence intensity ratio of Gag and membranes (see Material and Methods for more details). In (d), dashed white lines indicate I-BAR domain-induced tubules. Scale bars, (a–c) 5 µm and (d) 2 μm. GUV, giant unilamellar vesicle.

Figure 6—figure supplement 1. In vitro interplay between IRSp53 I-BAR and HIV-1 Gag binding to GUV.

(a, b) In vitro GUV experiments showing an increase of IRSp53 I-BAR binding on GUV upon HIV-1 Gag addition. (a) Representative confocal images of AX488 labeled IRSp53 I-BAR and AX546 labeled Gag on GUV membranes in ‘I-BAR + Gag’ condition. (b) Measurement of AX488 IRSp53 I-BAR fluorescence intensity on membranes in the absence of Gag (named ‘I-BAR only’), in the presence of Gag where GUVs were first incubated with IRSp53 I-BAR domain and then with HIV-1 Gag (named ‘I-BAR + Gag’). Each circle presents one GUV analysis. N = 27 GUVs, n = 2 sample preparations for ‘I-BAR only,’ N = 32 GUVs, n = 2 sample preparations for ‘I-BAR + Gag.’ To pool all data points from the two sample preparations, I-BAR intensities were normalized by the mean I-BAR intensity in the ‘I-BAR only’ condition. Protein bulk concentrations: 0.05 µM for I-BAR domain and 0.3 µM for Gag. **p=0.01133, Student’s t-test. (c, d) In vitro GUV experiments. (c) Representative confocal images of a GUV (the membrane contains 0.5 mol% of BODIPY-TR-C5-ceramide, red) incubating with a low concentration of IRSp53-I-BAR domain (0.05 μM, composed of 70% of unlabeled I-BAR domain and 30% of AX488 labeled I-BAR domain, green). The I-BAR domain induces membrane tubulation toward the interior of the GUV. (d) Representative confocal images of a GUV first incubated with IRSp53 I-BAR domain (0.05 μM, unlabeled), followed by addition of HIV-1 Gag (0.3 μM, AX488 labeled, green). GUV membranes contained 0.5 mol% of BODIPY-TR-C5-ceramide (red). The white arrow points to the accumulation of AX488 Gag singles at the tip of an I-BAR induced tube. Scale bars, = 5 µm. GUV, giant unilamellar vesicle.

Figure 6—figure supplement 2. Dotty signals of Gag in GUVs with I-BAR induced tubules.

Representative confocal images of GUVs first incubated with IRSp53 I-BAR domain (0.05 μM, unlabeled), followed by addition of HIV-1 Gag (0.3 μM, AX488 labeled, green). GUV membranes contained 0.5 mol% of BODIPY-TR-C5-ceramide (magenta). White arrows point to the accumulation of AX488 Gag singles at the tip of I-BAR induced tubes. Scale bars = 5 µm. GUV, giant unilamellar vesicle.