Fig. 6

Imaging the intracellular plasma membrane-connected compartment (IPMC) by serial block-face scanning electron microscopy (EM) and 3D reconstruction. Monocyte-derived macrophages (MDMs) were infected with HIV-1 R3A PTAP⁻YP⁻ as in Fig. 5, stained with CellMask orange to visualise the plasma membrane and IPMCs, fixed and processed for serial block-face scanning EM. a, b Fluorescence and phase contrast images of a selected cell with prominent intracellular staining. The circle in a marks the position of the nucleus (based on the phase image) and I and II identify two portions of the IPMC. c–e Serial block-face EM images from this cell. c shows the first of a set of 300 sections. N, nucleus. d, e Higher magnification details of the two marked areas in (c) corresponding to portions I and II of the IPMC, respectively, reveal the presence of virus buds. Membrane boundaries of the compartment were segmented (green). The arrows mark the connection between the two portions of the IPMC. f 3D reconstruction of the IPMC shown with xy and xz ortho-slices to indicate the context of the cell volume. The portions of the IPMC are labelled I and II. Arrowheads mark a short channel connecting portion I to the cell surface, the double-headed arrows show branches associated with region II, and P indicates a pocket from region II. g Detail of area I with xy and yz ortho-slices. Internal virus buds are shown in magenta. h View of IPMC portion II showing the mesh-like interconnected IPMC membranes. i–n Analysis of virus buds at the cell surface. i Detail from section 220. The boxed area is enlarged in (j), with the IPMC and cell surface segmented in green and yellow, respectively. Arrows mark clusters of virus buds at the cell surface between membrane protrusions. k Side view of the IPMC (xz) with virus buds in the IPMC in magenta and at the cell surface in cyan. l–n Views looking down onto the cell surface (xy orientation) showing cell surface virus buds only (l), cell surface virus buds with the IPMC (m), or cell surface virus buds and the cell surface in yellow (n). For all reconstructions, the IPMC is shown in green, cell surface yellow, IPMC virus buds magenta, cell surface virus buds cyan and the nucleus in blue. White arrowheads mark the channel from IPMC portion I towards the cell surface (in f, g and k) or the cell surface virus buds above the channel opening in l–n. Scale bars b, 20 μm, c, i and n, 5 μm and d, e and j, 1 μm. See also Additional file 6: Figure S6, Additional file 7: Movie S1, Additional file 8: Figure S7 and Additional file 9: Movie S2