Fig. 5
BRAF regulates FREE1 recruitment to MVB/PVCs. a The number of GFP-FREE1 punctae in braf mutant was increased compared to WT. Confocal images were collected from the root epidermal cells of the basal meristem region. Analysis of the number of GFP-FREE1 punctae per root section by Z-stack projection is quantified on the right. Ten slices were collected in a total thickness of 5 μm for generating the 3D projection image. FM4-64 was used to label and visualize the cell plasma membranes (PM). The results were obtained from 10 individual seedlings. Error bars represent the S.D. of puncta per field. **P < 0.01 in Student’s t-test. Scale bars, 10 μm. b The GFP-FREE1 punctae in braf mutant localizes to MVB/PVCs. Immunofluorescent labeling with the MVB/PVC marker anti-VSR antibody in WT or braf-2 mutant expressing GFP-FREE1. Colocalization was quantified from five individual labeling roots. The percentage of anti-VSR-labeled MVB/PVCs with GFP-FREE1 vesicle colocalization is included in the bottom. Total numbers of vesicles counted were n = 1245 in WT, and n = 1265 in braf-2 mutant. Scale bars, 10 μm. c The sof524 mutation enhanced FREE1 cell membrane distribution. Immunoblot and quantification analysis of the FREE1 and BRAF membrane association in WT, DEX::FREE1-RNAi, and sof524 plants. The FREE1 or BRAF intensity was normalized by the loading control of anti-VSR as cell membrane (CM) fraction and anti-cFBPase as cell soluble (CS) fraction, and the CM/CS ratio of FREE1 or BRAF in indicated genotype seedlings was quantified on the right. Note the increased FREE1 distribution in CM fraction of sof524 seedlings as indicated by the arrow. Error bars are the S.D. from three independent experiments. d The BRAF(A330V) mutation disturbs its membrane association with the endosome, but not the PM. Confocal images were taken from root epidermal cells of 7-day-old seedlings of indicated genotypes after DEX induction. The numbers of intracellular punctae per root section of BRAF-YFP (column 1) or BRAF(A330V)-YFP (column 2) are quantified on the right. The results were obtained from 10 individual seedlings. FM4-64 was used as a plasma membrane (PM) marker. **P < 0.01 in Student’s t-test. Scale bars, 10 μm
