Expanding Insights: Harnessing Expansion Microscopy for Super-Resolution Analysis of HIV-1-Cell Interactions

Abstract

Expansion microscopy has recently emerged as an alternative technique for achieving high-resolution imaging of biological structures. Improvements in resolution are achieved by physically expanding samples through embedding in a swellable hydrogel before microscopy. However, expansion microscopy has been rarely used in the field of virology. Here, we evaluate and characterize the ultrastructure expansion microscopy (U-ExM) protocol, which facilitates approximately four-fold sample expansion, enabling the visualization of different post-entry stages of the HIV-1 life cycle, focusing on nuclear events. Our findings demonstrate that U-ExM provides robust sample expansion and preservation across different cell types, including cell-culture-adapted and primary CD4+ T-cells as well as monocyte-derived macrophages, which are known HIV-1 reservoirs. Notably, cellular targets such as nuclear bodies and the chromatin landscape remain well preserved after expansion, allowing for detailed investigation of HIV-1-cell interactions at high resolution. Our data indicate that morphologically distinct HIV-1 capsid assemblies can be differentiated within the nuclei of infected cells and that U-ExM enables detection of targets that are masked in commonly used immunofluorescence protocols. In conclusion, we advocate for U-ExM as a valuable new tool for studying virus-host interactions with enhanced spatial resolution.

Keywords: HIV-1; HIV-1 capsid; HIV-1 nuclear import; HIV-1 post-entry; expansion microscopy; super-resolution microscopy; ultrastructure expansion microscopy; virus–host interaction.

Figure 1

U-ExM enables robust 4.3-fold expansion with minor macroscopic distortions. (A) A schematic overview illustrating the different steps involved in the U-ExM procedure. (B) TZM-bl cells, fixed with PFA/GA, stained for chromatin (Hoechst, cyan) and membranes (BODIPY, right panel, gray) and imaged at different steps of the U-ExM process. Scale bar: 100 µm. (C) TZM-bl cells seeded onto an 18 mm GelMap slide patterned with R2-myc-his-ATTO 647N (magenta), fixed with PFA/GA, and stained for chromatin (Hoechst, cyan). Scale bar overview, 100 µm; scale bar enlargement, 50 µm. Arrowhead marks the position of the same cluster of cells before and after expansion in (C,D). (D) TZM-bl cells seeded onto an 18 mm GelMap slide, fixed with PFA/GA, expanded by U-ExM, and stained for chromatin (Hoechst, cyan), membranes (BODIPY, gray), and R2-myc (magenta). Scale bar, 200 µm. (E) Measurement of the Feret diameter of the nuclei of the same set of TZM-bl cells seeded onto an 18 mm GelMap slide, fixed with PFA/GA and stained for chromatin, before and after expansion; n = 168; median_before = 17.03 µm, median_after = 72.63 µm; data plotted according to Tukey box plot. (F,G) TZM-bl cells stained for chromatin (Hoechst, cyan) and membranes (BODIPY, gray) exhibiting expansion artifacts. Scale bar, 100 µm. All scale bars in expanded samples reflect post-expansion sizes.

Figure 2

Sample preparation and imaging optimization are critical for expanded samples. (A) TZM-bl cells fixed with PFA/GA in either PBS or PHEM buffer, expanded and stained for chromatin (Hoechst, cyan), membranes (BODIPY, yellow), and α-tubulin (magenta). Scale bar overview, 20 µm; scale bar enlargements, 10 µm. (B) 3D structure of an expanded TZM-bl cell, stained for chromatin (Hoechst, cyan) and membranes (BODIPY, green). First panel, zy-slice; second panel, xy-slice. The magenta dashed line denotes the position of the xy slice in z. Scale bar, 10 µm. (C) Single slices of the same expanded TZM-bl cell as in (B), stained for chromatin (Hoechst). Scale bar, 10 µm. All scale bars in expanded samples reflect post-expansion sizes.

Figure 3

U-ExM allows expansion of various cell types and cellular structures. (A) TZM-bl cells, resting and activated CD4+ T-cells, and MDM fixed with PFA/GA, expanded and stained for chromatin (Hoechst, cyan), membranes (BODIPY, green), and Nup153 (magenta). Scale bar, 10 µm. (B) Cropped region of the nucleus of an MDM fixed with PFA/GA, expanded and stained for chromatin (Hoechst, cyan), membranes (BODIPY, green), and Nup153 (magenta) showing double membrane layers of the nuclear envelope in membrane staining. Scale bar, 2 µm. (C) Cropped region of the nucleus of an MDM fixed with PFA/GA, expanded and stained for chromatin (Hoechst, cyan), membranes (BODIPY, not shown in merge), and Nup153 (magenta), showing heterochromatin-free channels beneath the NPCs in the nucleus. Scale bar, 2 µm; arrowheads mark the position of NPC. (D) A TZM-bl cell fixed with PFA/GA, expanded and stained for chromatin (Hoechst, cyan), protein density (NHS-ester, green), SRRM2 (yellow), and SON (magenta), showcasing different intranuclear compartments. Scale bar, 10 µm. (E) Enlargements from (D) highlighting nuclear speckles and nucleolus. Scale bar, 5 µm. All scale bars in expanded samples reflect post-expansion sizes.

Figure 4

NPC diameter is well conserved after U-ExM. (A) Different MDM fixed with PFA/GA and either stained for Nup153 and imaged with Airyscan microscopy or STED microscopy or expanded, stained for Nup153 and imaged with Airyscan microscopy. Scale bar of Airyscan and STED panel, 1 µm; scale bar U-ExM panel, 4 µm. (B) Maximum intensity projection (MIP) of the bottom of an expanded U2OS cell nucleus, stained for chromatin (Hoechst, gray) and Nup153 (magenta). Scale bar, 10 µm; scale bar enlargement, 1 µm. Arrowheads mark the position of select NPC. (C) Analysis of NUP153 signal to determine NPC dimension, measured as maximum and minimum Feret diameters. Sample size (n = 222); median_{max diameter} = 109 nm, median_{min diameter} = 91 nm; data blotted according to Tukey box plot with outliers shown as single data points. All scale bars in expanded samples reflect post-expansion sizes.

Figure 5

U-ExM allows visualization of nanoscopic structures. (A) MIP of a TZM-bl cell fixed with PFA/GA, expanded and stained for ELYS (magenta), showing ring-like structures (enlargements). Scale bar overview, 10 µm; scale bar enlargement, 1 µm. (B) Single z-slice of a TZM-bl cell fixed with PFA/GA, expanded and stained for chromatin (Hoechst, cyan), membranes (BODIPY, green), Nup88 (yellow), and ELYS (magenta). Scale bar overview, 10 µm; scale bar enlargement, 1 µm. (C) U2OS cells stably expressing Nup96-SNAP fixed with PFA/GA, permeabilized with saponine, and stained with BG-Biotin before expansion and staining for chromatin (Hoechst, cyan), membranes (BODIPY, green), Nup153 (yellow), and biotin-labeled Nup96 via fluorescently conjugated Streptavidin (magenta). Scale bar, 20 µm. (D) Enlargement from (C) showing a portion of the nuclear envelope. Scale bar, 5 µm. (E) Composite image of the region shown in (D). Scale bar: 5 µm; line denotes position of line profile for (F) normalized signal intensity of the Nup153 (yellow) and Nup96-SNAP (magenta, dotted line) signal over the line shown in (E). Line profiles were aligned to the maximum peak. All scale bars in expanded samples reflect post-expansion sizes.

Figure 6

U-ExM can visualize HIV-1 CA-containing structures upon nuclear entry and within the nucleus. (A) A TZM-bl cell infected with NNHIV, fixed with PFA/GA at 16 h.p.i., expanded and stained for CA with two different antisera (rabbit α-CA, cyan; sheep α-CA, magenta) as well as Nup153 (yellow) and membranes (BODIPY, green). The sheep α-CA channel was corrected for bleedthrough from the Nup153 channel. Scale bar overview, 20 µm. Enlargements show HIV-1 CAs (cyan/magenta) interacting with NPC complexes (yellow), marked by arrowheads. Scale bar enlargements, 2 µm. (B) Enlargements from (A) showing intranuclear CA signals (rabbit α-CA, cyan; sheep α-CA, magenta). Scale bar, 1 µm. (C,D) TZM-bl cells, infected with NNHIV, fixed with PFA/GA at 16 h.p.i., expanded and stained for the nuclear speckle marker SRRM2 (cyan), membranes (BODIPY, green), CPSF6 (yellow), and CA (sheep α-CA, magenta). Scale bar overview, 20 µm. (C) Enlargements show intranuclear CA signals (magenta) showing colocalization with SRRM2 (cyan) and CPSF6 (yellow). The CA channel and the SRRM2 channel were corrected for bleedthrough from the BODIPY channel. Scale bar enlargements, 2 µm. (D) Enlargement shows intranuclear CA signals (magenta) with CPSF6 (yellow) close to nuclear envelope marked with BODIPY (green). Scale bar enlargements, 1 µm. All scale bars in expanded samples reflect post-expansion sizes.