REM1.3's phospho-status defines its plasma membrane nanodomain organization and activity in restricting PVX cell-to-cell movement
Fig 4
REM1.3's dynamic localization in PD and PM nanodomains is regulated by its phospho-status.
(A) Representative confocal mages showing aniline blue staining of callose deposition at the PD pitfields in N. benthamiana leaf epidermal cells transiently expressing YFP-REM1.3 or phosphomutants. Color-coding indicates fluorescence intensity. (B) Graphs show aniline blue fluorescence intensities in cells transiently expressing YFP-REM1.3 and phosphomutants relative to control cells expressing YFP alone. Three independent biological experiments were performed and at least 15 cells per condition and per experiment were analyzed. Letter indicate significant differences revealed by Dunn’s multiple comparisons test p<0.001. (C) PD index of YFP-REM1.3 phosphomutants was calculated as described in S1 Fig. Graphs present quantifications from 3 independent biological experiments. Letter indicate significant differences revealed by Dunn’s multiple comparisons test p<0.002. (D) Super-resolved trajectories (illustrated by different colours) of transiently expressed EOS-REM1.3, and phosphomutants, transiently expressed in N. benthamiana cells, observed by spt-PALM. Scale bars, 2 μm. (E) Distribution of diffusion coefficients (D) represented as log(D) of the different fusion proteins. Mean Square Displacement (MSD) over time for the global trajectories of each EOS-REM1.3 construct followed during at least 600ms. 27 cells for EOS-REM1.3, 15 cells for EOS-REM1.3AAA and 17 cells for EOS-REM1.3DDD were analyzed in 3 independent experiments. Statistical analysis was performed by Mann-Whitney test * p<0.05 ** p<0.01. (F) Live PALM analysis of EOS-REM1.3 phosphomutants by tessellation-based automatic segmentation of super-resolution images. (G) Computation of EOS-REM1.3 and phosphomutants single molecule organization features based on tessellation-based automatic segmentation images. For REM1.3 and phosphomutants nanodomain size distribution and the Gaussian fits are indicated. Total nanodomain area is expressed as percentage of the total PM surface. Percentage of EOS-REM1.3 molecules localizing into nanodomains, relative to all molecules observed. Localization density refers to the number of molecules observed per μm2 per second. Statistics were performed on at least 13 data sets per condition extracted from 3 independent experiments. Statistical differences determined by Mann-Whitney test * p<0.05, ** p<0.01.