Fig. 7: Effect of the acute manipulation of membrane region specific affinity of different lipid-anchored and integral membrane proteins.
From: A dynamic partitioning mechanism polarizes membrane protein distribution
a Schematic for increasing the back-state region specific affinity of uniformly distributed transmembrane protein cAR1. Upon 488 nm irradiation, the cytosolic CRY2PHR, which is fused to positively charged peptide (R+) or blank (ϕ, CTRL), gets globally recruited to CIBN-fused cAR1. b Live-cell images of Dictyostelium co-expressing cAR1-CIBN, CRY2PHR-mCherry-R+, and Lifeact-HaloTag(Janelia Fluor 646), before and after global 488 nm illumination (in all cases, laser was turned on at time t = 0 s). White arrowheads: F-actin rich protrusions before or right after recruitment; Green arrowheads: F-actin rich protrusions from where cAR1-CIBN/recruited CRY2PHR-mcherry-R+ was excluded. c, d Live-cell images of ventral wave propagation in Dictyostelium cell co-expressing cAR1-CIBN, CRY2PHR-mCherry-R+, along with PHCrac-YFP (c) or Lifeact-HaloTag(Janelia Fluor 646) (d), before and after global 488 nm irradiation. First two time point images in (c) and the inset image of second time point in (d) are showing confocal slices around the middle z-section (proving successful recruitment); other images are focusing on the substrate-attached surface of cell to visualize wave propagation. e Live-cell images of differentiated HL-60 cells, before and after recruitment of cytosolic CRY2PHR-mCherry-R+ to membrane bound Lyn11-CIBN-GFP. Blue arrowheads: The uropods or back-state regions of neutrophils where CRY2PHR-mCherry-R+ was localized upon recruitment, which in turn polarized the membrane distribution of Lyn11 there. f Box and whisker plots and aligned dot plots of front-state regions to back-state region intensity ratio of F-actin biosensor LimE (tan) and cAR1-CIBN (purple), after the recruitment of CRY2PHR-mCherry-R+ or CRY2PHR-mCherry(CTRL). For each of the nc = 11 (for CTRL) or nc = 12 (for R+) cells, intensity ratio values for nf = 5 frames were plotted (nc: number of cells; nf : number of frames); p values (two-sided, by Mann–Whitney-Wilcoxon test): n.s.: 0.1391, ****: ≤0.0001. Boxes extend from the 25th to 75th percentiles, median is at the center, and whiskers and outliers are graphed as per Tukey’s convention (as computed by Graphpad Prism). Source data are provided as a Source Data file.
