NiV-F forms regular-sized clusters that are not affected by the surface expression level.

(A and B) Cross-section (Δz = 600 nm) of SMLM images of NiV-F in high-expression (A) and low-expression (B) PK13 cells. Scale bar: 1 μm. The yellow boxed region is enlarged to show the detailed distribution pattern. Scale bar: 0.2 μm. (C) Hopkin’s index of the F localizations in low- and high-expression cells. n = 35 and 40. (D and E) Cluster maps (left) and localization density maps (right) of the enlarged regions in A and B. Cluster contours are highlighted with gray lines. Normalized relative density is pseudocolored according to the scale on the right. (F) The percentage distribution of the NiV-F cluster diameter from 13 cells. n = 133. (G) The cluster diameters in low- and high-expression cells. n = 58 and 56. (H) The localization density (# of localizations per μm2) within clusters in low- and high-expression cells. n = 58 and 50. (I) The number of clusters per μm2 in low- and high-expression cells. n = 59 and 74. The cut-off fluorescence intensity for low- and high-expression cells is 8000 (Arb. Unit). Sample size n is the number of total regions from 6-8 cells. Bars represent mean ± SD. P value was obtained using the Mann-Whitney test. ns: p > 0.05; * p<0.01; ** p<0.001; *** p<0.0001.

Endosomal cleavage does not affect the nanoscale distribution of NiV-F.

(A) First column: Cross-section (Δz = 600 nm) of SMLM images of NiV-F in HeLa cells untreated (NC) and treated with 20 μM E64d (E64d). HeLa cells were co-transfected by expression plasmids coding for NiV-G and NiV-F. Twenty μM E64d or the same volume of solvent methanol was added to cells at 2 hrs post-transfection. Scale bar: 1 μm. Second column: The yellow boxed region in the first column is enlarged to show individual clusters. Scale bar: 0.2 μm. Third column: Cluster maps from the enlarged regions. Fourth column: Localization density maps show the normalized relative density of the enlarged regions. (B-F) Quantitative analyses of NiV-F clusters formed in HeLa cells without (NC) and with (E64d) the E64d treatment: (B) Hopkin’s index, n = 40 and 40; (C) percentage of localizations in clusters, n = 101 and 101; (D) relative density, n = 100 and 103; (E) average cluster diameters, n = 100 and 101; (F) total density of the region, n = 101 and 102. Bars represent mean ± SD. p value was obtained using Mann-Whitney test. ns: p > 0.05; * p<0.01; ** p<0.001; *** p<0.0001. Sample size n is the number of total regions from 4-10 cells.

Mutations at the NiV-F hexameric interface affect its nano-organization.

(A) First column: Cross-section (Δz = 600 nm) of SMLM images of the FLAG-tagged NiV-F-WT (WT), L53D, V108D, and Q393L on PK13 cell membrane. Scale bar: 1 μm. Second column: The yellow boxed regions in the first column are enlarged to show individual clusters. Scale bar: 0.2 μm. Third column: Cluster maps from enlarged regions. Fourth column: Localization density maps show normalized relative density of the enlarged regions. (B-F) Quantitative analyses of the distribution of the FLAG-tagged NiV-F constructs: (B) Hopkin’s index, n = 57-70; (C) Percentage of localizations in clusters, n = 106-198; (D) Relative density, n = 90-187; (E) Average cluster diameters, n = 106-196; (F) total density of the region (a ratio of total localizations in a region to the size of the region), n = 105-242. Bars represent mean ± SD. Sample size n is the number of total regions from 11-20 cells. p value was obtained using Mann-Whitney test. ns: p > 0.05; * p<0.01; ** p<0.001; *** p<0.0001.

The distribution and organization of NiV-F constructs in VLPs.

(A). The incorporation of F-WT and mutants in VLPs. NiV-M-GFP, G-HA, and FLAG-tagged F-WT or mutants were transfected to 293T cells. The supernatants were collected at 48 hrs post-transfection and analyzed on SDS-PAGE followed by western blotting. NiV-M was probed by polyclonal goat anti-GFP, NiV-G polyclonal rabbit anti-HA, F0 and F2 M2 monoclonal mouse anti-FLAG antibody. (B) Cross-section (Δz = 100 nm) of SMLM images of the FLAG-tagged NiV-F-WT (WT), L53D, V108D, and Q393L on individual VLPs. Scale bar: 0.2 μm. (C) The classification of the ordered sequence of reachability distances of the NiV-F constructs localizations. Orange: F-WT (n = 306) and Q393L (n = 323); Blue: L53D (n = 310) and V108D (n = 329). n is the number of VLPs used for classification analysis.

Mutations in the LI zipper of the NiV-F transmembrane domain disturbs the NiV-F distribution.

A) First column: Cross-section (Δz = 600 nm) of SMLM images of the FLAG-tagged NiV-F-WT (WT) and NiV-F-LI4A (LI4A) mutant on PK13 cell membrane. Scale bar: 1 μm. Second column: The yellow boxed region in the first column is enlarged to show individual clusters. Scale bar: 0.2 μm. Third column: Cluster maps from enlarged regions. Fourth column: Localization density maps show normalized relative density of the enlarged regions in the second column. (B-E) Quantitative analyses of the WT and LI4A clusters: (B) Hopkin’s index, n=40 and 40 (C) Percentage of localizations in clusters, n = 171 and 211; (D) Average cluster diameters, n = 171 and 211; (E) Relative density, n = 166 and 211. Bars represent mean ± SD. p value was obtained using Mann-Whitney test. ns: p > 0.05; * p<0.01; ** p<0.001; *** p<0.0001. Sample size n is the number of total regions from 13-16 cells.

The NiV-F nanoclusters are stabilized by endocytosis components.

(A) First column: Cross-section (Δz = 600 nm) of SMLM images of the FLAG-tagged NiV-F-WT (WT) and NiV-F-YA (YA) mutant on PK13 cell membrane. Scale bar: 1 μm. Second column: The yellow boxed region in the first column is enlarged to show individual clusters. Scale bar: 0.2 μm. Third column: Cluster maps from enlarged regions. Fourth column: Localization density maps show normalized relative density of the enlarged regions in the second column. (B-F) Quantitative analyses of the WT and YA clusters: (B) Hopkins index of WT and YA, n = 40 and n = 40; (C) Percentage of localizations in clusters, n = 72 and 77; (D) Average cluster diameters, n = 72 and 77; (E) Relative density, n = 71 and 76; (F) total density of the region, n = 72 and 77. (G) First column: Cross-section (Δz = 600 nm) of SMLM images of the FLAG-tagged NiV-F-WT treated without (NC) and with pitstop2 (pitstop2) on HeLa cell membrane. Scale bar: 1 μm. Second column: The yellow boxed region in the first column is enlarged to show individual clusters. Scale bar: 0.2 μm. Third column: Cluster maps from enlarged regions. Fourth column: Localization density maps show normalized relative density of the enlarged regions in the second column. (H-L) Quantitative analyses of NiV-F without (NC) and with pitstop2 (pitstop2): (H) Hopkins index, n = 40 and n = 40; (I) Percentage of localizations in clusters, n = 82 and 85; (J) Average cluster diameters, n = 82 and 81; (K) Relative density, n = 82 and 81; (L) total density of the region, n = 82 and 81. Bars represent mean ± SD. p value was obtained using Mann-Whitney test. ns: p > 0.05; * p<0.01; ** p<0.001; *** p<0.0001. Sample size n is the number of total regions from 4-9 cells.