Fig. 3.

Presence of M does not affect the arrangement of the envelope glycoproteins’ clusters on the membrane. PK13 cells were co-transfected with NiV-M, -F, and -G and fixed at 24 h post transfection. Without permeabilization, F was immunostained using a mouse anti-FLAG primary antibody and an anti-mouse Cy3B secondary antibody, and G an anti-HA primary antibody and an anti-rabbit Alexa Fluor 647 secondary antibody. a, b x–y cross section (100 nm thick in z) of a representative cell shows the superimposition of a wide field image for M (blue) and the corresponding SMLM images of F (red) and G (green) on the cell body (a) and membrane protrusions (b). Scale bar: 1 μm. c x–y cross section (100 nm thick in z) of the M-positive sites (c1, 2) boxed in b. Scale bar: 0.1 μm. d 3D surface reconstruction of the dome-like structure in c1, with F (red) and G (green) localization densities projected on the surface. A higher brightness indicates a higher localization density. e Comparison of the localization densities of F (red) or G (green) on the M-positive and M-negative regions at the dorsal surface of the cell. f Comparison of the Hopkins’ indices of F (red) and G (green) in the M-positive and M-negative regions at the plasma membrane. All data in e and f were normalized to the mean of the M-negative regions of the same cell. Lines represent the mean value and SD. The sample size is indicated in the parentheses. The p values were determined by two-tailed, unpaired t-test with Welch correction. g The distribution of the DoC values between F and G molecules in the M-negative and M-positive regions. h VLPs produced in PK13 cells expressing M, F, and G were adhered to fibronectin-coated coverslips, fixed, and stained for NiV-F and G via tags described above. The z-stacks of the x–y cross section of a VLP show the super-imposition of a wide field image of M (blue) and the corresponding SMLM images of F (red) and G (green). Scale bar: 0.1 μm