1. Volume 8, Issue 9, Pages 1334-1349 (September 2015)
Spatiotemporal Dynamics of the BRI1 Receptor and its Regulation by Membrane Microdomains in Living Arabidopsis Cells 
Li Wang, Hong Li, Xueqin Lv, Tong Chen, Ruili Li, Yiqun Xue, Jianjun Jiang, Biao Jin, František Baluška, Jozef Šamaj, Xuelu Wang, Jinxing Lin 
Molecular Plant 
Volume 8, Issue 9, Pages (September 2015)
DOI: /j.molp
Copyright © 2015 The Author Terms and Conditions

2. Figure 1
Subcellular Localization of BRI1-GFP in the Root Meristem of a Transgenic Arabidopsis Col-0 Seedling.
(A) BRI1-GFP fluorescence was detected in plasma membrane and intracellular compartments in Arabidopsis root meristem cells. An enlarged image of the boxed area is presented in (B) (scale bar represents 20 μm).
(B) Pseudocolor images (blue-yellow-red palette) show the fluorescence intensity of BRI1-GFP at the plasma membrane (color code bar indicates the relative intensities). Arrows indicate BRI1-GFP signals that are associated with intracellular compartments (scale bar represents 10 μm).
(C) VA-TIRFM image of BRI1-GFP at the plasma membrane of living cells. Individual BRI1-GFP puncta are circled (scale bar represents 2 μm).
(D) Three-dimensional luminance plots of the BRI1-GFP spots in (C) showing varied fluorescence intensity among different spots.
(E and F) Fluorescence intensity tracks of single BRI1-GFP particles showing their disappearance (E) and appearance (F).
(G) Mean square displacement (MSD) analysis for various trajectories and classification into diffusion modes. The resulting curves were fitted with Brownian, directed, restricted, and multimodal diffusion models.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

3. Figure 2
Dynamic In Vivo Behavior of Individual BRI1-GFP Spots in the Cell Membrane at Different BR Levels.
(A) VA-TIRFM image of BRI1-GFP at the plasma membrane (scale bar represents 5 μm).
(B) Trajectories of BRI1-GFP in the area indicated by the white box in (A).
(C) Distribution of BRI1-GFP motion range in Col-0 seedlings (WT).
(D) Distribution of BRI1-GFP motion range in Col-0 seedlings after BRZ treatment.
(E) Distribution of BRI1-GFP motion range in det2 seedlings.
(F) Distribution of BRI1-GFP motion range in Col-0 seedlings after eBL treatment.
(G) Frequency of long distance and short distance motions for BRI1-GFP under different BR levels.
(H) Distribution of BRI1-GFP spot diffusion coefficients in Col-0 seedlings (WT).
(I) Distribution of BRI1-GFP spot diffusion coefficients in det2 seedlings.
(J) Distribution of BRI1-GFP spot diffusion coefficients in Col-0 seedlings after BRZ treatment.
(K) Distribution of BRI1-GFP spot diffusion coefficients in Col-0 seedlings after eBL treatment.
(L) Diffusion coefficients of BRI1-GFP spots under different BR levels.
**P < 0.01, *P < 0.05, t-test. Error bars represent mean ± SD.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

4. Figure 3
In Vivo Distribution of Individual BRI1-GFP Spot Fluorescence Intensities and Photobleaching Steps in the Cell Membrane at Different BR Levels.
(A) VA-TIRFM image of BRI1-GFP at the plasma membrane, individual BRI1-GFP puncta are circled (scale bar represents 5 μm).
(B) Distribution of purified GFP fluorescence intensity.
(C) Distribution of BRI1-GFP fluorescence intensity in Col-0 seedlings (WT).
(D and E) Time course of GFP emission after background correction showing one-step and two-step bleaching.
(F) Frequency of one-step and two-step bleaching events for BRI1-GFP under different BR levels.
(G) Distribution of BRI1-GFP fluorescence intensity in Col-0 seedlings after BRZ treatment.
(H) Distribution of BRI1-GFP fluorescence intensity in det2 seedlings.
(I) Distribution of BRI1-GFP fluorescence intensity in Col-0 seedlings after eBL treatment.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

5. Figure 4 BRI1 Endocytosis Is Clathrin-Dependent.
(A) Internalization of BRI1-GFP into FM4-64-labeled BFA compartments after BFA treatment.
(B) TyrA23 efficiently inhibited BFA-induced intracellular accumulation of BRI1-GFP.
(C) BFA-induced intracellular accumulation of BRI1-GFP decreased in chc2-1 mutants.
(D) TyrA51 did not inhibit BFA-induced intracellular accumulation of BRI1-GFP.
(E) VA-TIRFM image of BRI1-GFP clusters proximal to the plasma membrane.
(F) VA-TIRFM image of CLC-mCherry clusters proximal to the plasma membrane.
(G) Merged image of (E) and (F), the yellow dots (white arrows) indicate the co-localization of the two signals.
(H) An example of real-time dynamic observation of BRI1-GFP and CLC-mCherry co-diffusion (the circled puncta in G, see also Supplemental Movie 3).
Scale bars represent 10 μm (A–D); 2.5 μm (E–G); 0.5 μm (H).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

6. Figure 5
BRI1-GFP Internalization Is Perturbed by MβCD and BRI1-GFP Partially Co-Localizes with AtFlot1-mCherry.
(A) BRI1-GFP co-localized with FM4-64 both at the plasma membrane and endosomal compartments.
(B) MβCD reduced the abundance of intracellular BRI1-GFP.
(C) Intracellular BRI1-GFP was reduced in AtFlot1 amiRNA lines.
(D) Quantification of relative BRI1 uptake.
(E) BFA increased intracellular localization of BRI1-GFP co-localized with FM4-64 in BFA compartments.
(F) MβCD reduced the BFA-induced intracellular accumulation of BRI1-GFP.
(G) BFA-induced intracellular accumulation of BRI1-GFP decreased in AtFlot1 amiRNA15-5 lines.
(H) Quantification of relative BRI1 uptake after BFA treatment.
(I–K) VA-TIRFM image of BRI1-GFP (I) and AtFlot1-mCherry (J). Merged image of (I) and (J); the yellow dots (white arrows) indicate the co-localized particles (K).
(L) Real-time observation showed co-diffusion of BRI1-GFP and AtFlot1-mCherry fluorescence (the circled puncta in K, see also Supplemental Movie 5).
Scale bars represent 10 μm (A–C, E–G); 2.5 μm (I–K); 0.5 μm (L).
∗∗P < 0.01, t-test. Error bars represent mean ± SD.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

7. Figure 6
FCCS Analysis of BRI1-GFP/CLC-mCherry and BRI1-GFP/AtFlot1-mCherry at Different BR Levels.
(A) A confocal image detected by FCS; points 1 and 2 show the location where the laser beam was focused to monitor the fluorescence fluctuations. Scale bar represents 20 μm.
(B) The density of BRI1-GFP molecules in the presence of different inhibitors in chc2-1 mutants and AtFlot1 amiRNA lines as measured by FCS.
(C) Correlation percentage of BRI1-GFP with CLC-mCherry and AtFlot1-mCherry in WT and after treatments with BRZ and eBL.
(D and E) The fluorescence correlation curves of BRI1-GFP with CLC-mCherry (D) and with AtFlot1-mCherry (E) in WT plants.
(F and G) The fluorescence correlation curves of BRI-GFP with CLC-mCherry (F) and with AtFlot1-mCherry (G) in WT plants treated with BRZ.
(H and I) The fluorescence correlation curves of BRI-GFP with CLC-mCherry (H) and with AtFlot1-mCherry (I) in WT plants treated with eBL.
**P < 0.01; *P < 0.05, t-test. Error bars represent the mean ± SD.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

8. Figure 7
Association of BRI1 with Clathrin/AtFlot1 at Different BR Levels.
(A) VA-TIRFM image of cell co-expressing BRI1-GFP and CLC-mCherry.
(B) VA-TIRFM image of cell co-expressing BRI1-GFP and AtFlot1-mCherry.
(C) 3D plot of BRI1 and CLC cross-correlation versus pixel shift.
(D) 3D plot of BRI1 and AtFlot1 cross-correlation versus pixel shift.
(E) Mean protein proximity indexes showing BRI1-CLC degree of proximity under different BR levels.
(F) Mean protein proximity indexes showing BRI1-AtFlot1 degree of proximity under different BR levels.
Scale bars represent 5 μm (A, B).
**P < 0.01; *P < 0.05, t-test. Error bars represent the mean ± SD.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

9. Figure 8 MβCD Treatment Inhibits BR Signaling.
(A) MβCD-treatment promotes BES1-FLAG phosphorylation. Histogram shows the ratio of dephosphorylated to phosphorylated BES1-FLAG.
(B) MβCD antagonizes the eBL-induced BES1-FLAG dephosphorylation. Graph (right) shows the ratio of dephosphorylated to phosphorylated BES1-FLAG.
(C) MβCD treatment alters BR-regulated gene expression. CPD and DWF4 are BR down-regulated genes and SAUR-AC1 and BAS1 are BR up-regulated genes.
(D) MβCD enhanced BKI1-YFP association with the plasma membrane.
(E) eBL induced the dissociation of BKI1-YFP from the plasma membrane.
(F) MβCD treatment inhibited eBL-induced BKI1-YFP dissociation from the plasma membrane. Seedlings were pre-incubated with 10 mM MβCD for 30 min before incubation in 0.1 μM eBL.
(Scale bars represent 10 μm (D, E, F)).
**P < 0.01, t-test. Error bars represent the mean ± SD.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

10. Figure 9
A Diagram of the BRI1 Internalization Pathway and Assembly State in Response to BR in Arabidopsis.
(A) BRI1 assembly involves regulation of the monomer-predimer-dimer dynamic equilibrium.
(B) BR stimulus can promote the recruitment of BRI1 into functional membrane microdomains.
(C) The membrane microdomain-associated pathway was significantly enhanced and contributed to the stimulated BRI1 internalization in response to BR.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions