IFITM Proteins Restrict Viral Membrane Hemifusion
Figure 8
Expression of IFITM proteins increases the lipid order of cell membranes.
293/LH2SN cells stably expressing IFITM1, 2 or 3, or mock controls were incubated with 1.8 µM Laurdan for 40 min at 37°C, and were imaged using two-photon fluorescence microscope. Parental 293/LH2SN cells were treated with 10 mM MβCD for 1 h to serve as controls. (A) Fluorescence intensity images. The fluorescence signal was acquired from 416 nm to 474 nm for the blue channel and from 474 nm to 532 nm for the green channel. (B) Generalized polarization (GP) images. The GP image was generated according to the GP function which is a normalized ratio between the blue and the green channels (GP scale from −1 to +1). According to the calculated GP values, we restricted the GP scale from −0.3 to 0.5. (C) GP histograms. The GP histogram was fitted using two Gaussian distributions. The lower GP values are associated with internal membranes, and the higher GP values are associated with plasma membranes. (D) Pseudo-colored GP images. The lower and higher GP distributions were pseudo-colored in green and red, respectively. (E) Averaged GP values. The GP values of individual cell lines were averaged and plotted; for each cell lines, a total of 12–18 images were used for statistical analysis. Significant differences were observed between mock control and IFITM1 (p = 0.00672), IFITM3 (p = 0.00107) in the plasma membrane. See text for details.