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. 2012 Apr 3;10(4):e1001299. doi: 10.1371/journal.pbio.1001299

Figure 4. The ROP2-RIC4 pathway inhibits PIN1 endocytosis through the promotion of the accumulation of cortical actin filaments.

Figure 4

(A) A time-course analysis of PIN1-dendra2 internalization after photo-conversion in Arabidopsis PCs. PIN1-dendra2 was transiently expressed in WT (Ws-2) or ric4-1 PCs for 24 h. Compared to WT cells, formation of vesicles (arrowheads) and decrease of PM signal were accelerated in ric4-1 cells as in rop4 R2i cells (Figure 1D). Stabilizing F-actin by TIBA treatment for 2 h suppressed both vesicle formation and decrease of PM signal in ric4-1 cells. (B) Quantitative analysis of PM signal shown in (A). Relative intensity was measured by absolute value of intensity divided by value of intensity in the first image. Error bars represent SD (n = 5). (C) An analysis of FM1-43 uptake in Col-0 WT and RIC4 overexpressing lines. Images were taken 1 h after leaves were incubated in liquid MS medium containing FM1-43. Each image is a stack image of 40 images taken for around 4 min to visualize internalized signal. Vesicles containing FM1-43 (arrowheads) were accumulated in Col-0 but not in RIC4 overexpressing cells (RIC4 OX). Application of Latrunculin B (100 nM, LatB) induced uptake of FM1-43 into small vesicles in RIC4 OX cells. (D) Quantitative analysis of vesicle numbers shown in (C). Images were taken from ten different leaves in at least three independent experiments, and the mean number of vesicles per area of cells was determined. Error bars represent SD (n = 30). p-Values were determined by two-tailed Student's t test assuming equal variances (***, p<0.001). (E) A model for a ROP-signaling-based feed-forward mechanism for PIN1 polarization to the lobe region of PCs. Extracellular auxin activates ROP2 in the lobing region, and the activated ROP2-RIC4 pathway inhibits PIN1 internalization through RIC4-dependent cortical F-actin, leading to PIN1 polarization to the lobing region. PIN1-based export of auxin induces further ROP2 activation to complete the feed-forward cycle. In addition to its inhibition of endocytosis, the ROP2-RIC4 pathway is also required for the endosomal trafficking of PIN1 from Ara7-marked endosomes to recycling endosomes.