Fig 1.
(A) A simplified schematic of the interaction scheme between HIV-1 assembly sites and the ESCRT recruitment pathway. The constructs analyzed in this study are highlighted in purple. (B) An overview of the proposed models of membrane scission where ESCRT factors interact either via external constriction from the cytosol (cytosolic model, Model 1), via internal constrict of the budding virus in the neck region (neck model, Model 2) or from within the bud (bud model, Model 3).
Fig 2.
Super-resolution images of Tsg101 at HIV-1 assembly sites.
HeLa cells were transfected with HIV:HIVmCherry and endogenous Tsg101 was immunostained at 14 – 15 h post transfection. (A) Cells were imaged using TIRFM. A time projection of the TIRFM images are shown (HIVmCherry, left panel; Tsg101, middle panel; Overlay, magenta: HIVmCherry, green: Tsg101, right panel), scale bars: 10 μm. (B) Left panel. A zoomed image of the region highlighted in panel A. Scale bar: 2 μm. Middle panel. A zoomed image of the individual colocalizing Tsg101 cluster highlighted in the left panel. Scale bar: 500 nm. Right panel. A drift-corrected STORM image of the Tsg101 complex shown in the middle panel. Scale bar: 500 nm. (C) The size distribution of all Tsg101 structures colocalizing with HIVmCherry, revealing an average cluster size of 58 ± 7 nm. (D) Size distribution of all non-colocalizing Tsg101 clusters in cells expressing HIVmCherry revealing an average cluster size of 60 ± 15 nm or (E) or in cell expressing HIVmCherry late- revealing average cluster size of 58 ± 16 nm. Number N represents the number events contributing to the respective histogram.
Fig 3.
Super-resolution imaging of endogenous ALIX at HIV-1 assembly sites.
HeLa cells were transfected with HIV:HIVmCherry and endogenous ALIX was immunostained at 14 – 15 h post transfection. Cells were imaged by TIRFM. (A) A time projected image of ALIX (green) overlaid with TIRF image of HIVmCherry assembly sites (magenta) is shown. Circles indicate non-colocalizing ALIX clusters and the rectangles highlight different colocalized ALIX classes. The crops show drift-corrected super-resolution STORM images of the three identified object classes: condensed ALIX structures without a surrounding cloud (1), ALIX structures with a central, condensed spot surrounded by a cloud-like structure (2) and diffuse ALIX membrane assemblies without a central spot (3). Scale bars: 10 μm (large image), 100 nm (crops). (B) Size distribution of all central spots of colocalizing ALIX protein clusters with an average cluster size (FWHM) of 64 ± 18 nm. (C) Size distribution of all central spots of non-colocalizing ALIX protein clusters with an average cluster size (FWHM) of 74 ± 26 nm. (D) Cloud cluster size characterization for all cloud structures of ALIX colocalizing with HIVmCherry obtained by Ripley’s cluster analysis with an average diameter of 164 ± 31 nm. (E) The distribution of the minimum number of ALIX proteins detected per cloud estimated from the super-resolution data.
Fig 4.
Super-resolution imaging of CHMP4B-HA clusters.
HeLa cells were transfected with both HIV:HIVmCherry and CHMP4B-HA and CHMP4B-HA was detected by immunostaining 14–15 h post transfection. Cells were imaged by TIRFM. (A) Left panel. A time projected overlay of HeLa cells transfected with HIVmCherry (magenta) and CHMP4B-HA (green). The circles indicate partially diffuse CHMP4B clusters at the membrane, which are ignored in further analyses. Scale bar: 10 μm. Middle panel. A zoomed-in image of a selected CHMP4B-HA cluster (highlighted in grey in the left panel) colocalizing with HIVmCherry. Right panel. The corresponding drift-corrected STORM image. Scale bars: 500 nm (B) Size distribution of all CHMP4B-HA structures colocalizing with HIVmCherry with an average cluster size (FWHM) of 56 ± 12 nm. (C) Size distribution of all non-colocalizing CHMP4B-HA clusters in cells co-expressing HIVmCherry wildtype with an average cluster size (FWHM) of 69 ± 30 nm. (D) Size distribution of all non-colocalizing CHMP4B clusters in cells co-expressing HIVmCherry late- with an average cluster size (FWHM) of 53 ± 16 nm.
Fig 5.
Super-resolution imaging of CHMP2A at HIV-1 assembly sites.
HeLa cells were transfected with HIV:HIVmCherry and endogenous CHMP2A was detected by immunostaining 14–15 h post transfection. HeLa cells were imaged using TIRFM. (A) Left panel. A merged time-projected TIRF image of HIVmCherry (magenta) and CHMP2A (green) prior to STORM analysis. Middle panel. A zoomed-in image of the selected CHMP2A cluster highlighted in grey in the left panel colocalizing with an HIV assembly site. Right panel. The corresponding drift-corrected STORM image of the CHMP2A cluster. Scale bars: 500 nm. (B) The size distribution of all CHMP2A structures colocalizing with HIVmCherry with an average cluster size (FWHM) of 56 ± 12 nm. (C) Size distribution of all non-colocalizing CHMP2A clusters in cells co-expressing HIVmCherry with an average cluster size (FWHM) of 59 ± 20 nm.