Accepted Manuscript Spatiotemporal Dynamics of 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 PII:
S1674-2052(15)00202-6
DOI:
10.1016/j.molp.2015.04.005
Reference:
MOLP 123
To appear in:
MOLECULAR PLANT
Received Date: 10 March 2015 Revised Date:
10 April 2015
Accepted Date: 12 April 2015
Please cite this article as:
Wang L., Li H., Lv X., Chen T., Li R., Xue Y., Jiang J., Jin B., Baluška F., Šamaj J., Wang X., and Lin J. (2015). Spatiotemporal Dynamics of BRI1 Receptor and Its Regulation by
Membrane Microdomains in Living Arabidopsis Cells. Mol. Plant. doi: 10.1016/j.molp.2015.04.005.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Spatiotemporal Dynamics of BRI1 Receptor and Its
2
Regulation by Membrane Microdomains in Living
3
Arabidopsis Cells
4
Li Wang1,2, #, Hong Li1,2, #, Xueqin Lv1,3, Tong Chen1, Ruili Li3, Yiqun Xue1, Jianjun
5
Jiang4, Biao Jin2, František Baluška5, Jozef Šamaj6, Xuelu Wang4,7, Jinxing Lin1,3*
RI PT
1
6 7
1
8
China;
9
2
College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, China;
10
3
College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China;
11
4
State Key Laboratory of Genetic Engineering and Institute of Plant Biology, School of Life Sciences,
12
Fudan University, Shanghai 200433, China;
13
5
Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany;
14
6
Department of Cell Biology, Palacky University Olomouc, Olomouc 78371, Czech Republic
15
7
College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
TE D
M AN U
SC
Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing 100093,
16
*
17
#
18
The authors declare no conflict of interest.
19
Running title: Membrane microdomains regulate endocytosis of BRI1
EP
L.W. and H.L. contributed equally to this work.
AC C
20
Correspondence: [email protected]
21
Short Summary: How membrane microdomains are involved in regulating
22
BRI1 endocytosis and BR signaling is still unknown. In this study, using
23
VA-TIRFM in combination with FCS and FCCS, we identified membrane
24
microdomains mediated a novel endocytic pathway involving in BRI1
25
endocytosis. Significant light has been shed on the molecular mechanisms
26
through which BRI1 can be recruited into functional membrane microdomains
27
and internalized in response to BR signal.
28
1
ACCEPTED MANUSCRIPT 29
ABSTRACT
30 31
The
32
INSENSITIVE1 (BRI1) plays fundamental roles in BR signaling, but the
33
molecular
34
internalization and assembly state remain unclear. Here, we applied
35
variable
36
(VA-TIRFM) and fluorescence cross-correlation spectroscopy (FCCS) to
37
analyze the dynamics of GFP-tagged BRI1. We found that, in response to
38
BR stimulation, the degree of colocalization of BRI1-GFP with
39
AtFlot1-mcherry increased; in particular, BR stimulated the membrane
40
microdomain-associated pathway of BRI1 internalization. We also verified
41
this in endocytosis-defective chc2-1 mutants and the AtFlot1 amiRNA
42
15-5 lines. Furthermore, examination of the phosphorylation status of
43
bri1-EMS-suppressor 1 (BES1) and measurement of BR-responsive gene
44
expression revealed that membrane microdomains affect BR signaling.
45
These results suggested that BR promotes the partitioning of BRI1 into
46
functional membrane microdomains to activate BR signaling.
mechanisms
total
underlying
internal
receptor
the
reflection
BRASSINOSTEROID
effects
of
BR
on
BRI1
fluorescence
microscopy
TE D
M AN U
SC
angle
(BR)
RI PT
brassinosteroid
EP
47
major
Keywords: BRI1; BR signaling; endocytosis; membrane microdomains;
49
spatiotemporal dynamics
50 51 52 53
AC C
48
54 55 56 57 58 2
ACCEPTED MANUSCRIPT INTRODUCTION
60
Brassinosteroids (BRs) are essential plant steroid hormones that regulate a
61
wide range of developmental and physiological processes, including cell
62
elongation and root growth (Clouse and Sasse, 1998; Wu et al., 2011). The major
63
BR receptor BRASSINOSTEROID INSENSITIVE1 (BRI1) plays fundamental
64
roles in BR signaling and plant development (Wang et al., 2001; Wang and He,
65
2004). For example, knock-out mutants of BRI1 are extremely dwarfed, and
66
completely BR-insensitive (Clouse et al., 1996; Li and Chory, 1997; Kinoshita et
67
al., 2005). BRI1 belongs to the leucine-rich repeat receptor-like kinase
68
(LRR-RLK) class and has an extracellular domain containing 24 leucine-rich
69
repeats (LRRs), a transmembrane domain, and a cytoplasmic domain
70
containing a serine/threonine (Ser/Thr) kinase domain (Li and Chory, 1997; Vert
71
et al., 2005). Upon perceiving BRs, plant cells activate BRI1 kinase to trigger
72
the dissociation of the inhibitory BRI1 KINASE INHIBITOR 1 (BKI1), thus
73
enabling sequential transphosphorylation of BRI1 and its co-receptor BRI1
74
associated kinase 1 (BAK1) to form an active receptor complex, thereby
75
initiating BR signaling (Wang and Chory, 2006; Bücherl et al., 2013).
TE D
M AN U
SC
RI PT
59
In animals and yeast, endocytosis regulates protein abundance at the
77
plasma membrane during signaling and acts to retarget or degrade membrane
78
proteins (Royle and Murrell-Lagnado, 2003; Grant and Donaldson, 2009). Plant
79
cells use endocytosis to internalize exogenous material, ligands, and plasma
80
membrane proteins or regulate signaling at the plasma membrane (Murphy et al.,
81
2005; Šamaj et al., 2005). Constitutively internalized and ligand-inducible
82
receptors are subject to endocytosis (Royle and Murrell-Lagnado, 2003;
83
Scarselli and Donaldson, 2009). The constitutive endocytosis of plant
84
transmembrane proteins such as receptors, channels and transporters has
85
been extensively reported; these proteins include PINs, PIP2, and BRI1
86
(Paciorek et al., 2005; Geldner et al., 2007; Li et al., 2011). The constitutive
87
internalization of PIN1, PIN2 and PIP2;1 requires clathrin-dependent
AC C
EP
76
3
ACCEPTED MANUSCRIPT endocytosis (Baisa et al., 2013; Dhonukshe et al., 2007; Kitakura et al., 2011).
89
Furthermore, plant structural sterols occur within endocytic networks, and play
90
a role in a constitutive endocytic cycling of some plasma membrane transport
91
proteins (Šamaj et al., 2005). BRI1 mainly localizes to the plasma membrane
92
and early endosomal compartments in Arabidopsis root cells (Russinova et al.,
93
2004; Geldner et al., 2007; Hink et al., 2008). Recent work reported clathrin and
94
ARF GTPase (ARF-GEF) dependent endocytic regulation of BR signaling from
95
the plasma membrane (Irani et al., 2012; Rubbo et al., 2013), and ubiquitination
96
can promote BRI1 internalization (Martins et al., 2015). However, the molecular
97
mechanisms underlying internalization and assembly state changes of BRI1 in
98
response to exogenous BR stimulation remain poorly understood.
M AN U
SC
RI PT
88
BR regulates the activity and dynamic behaviors of BRI1 particles at the
100
plasma membrane. Therefore, monitoring and tracking the behavior of
101
individual BRI1 particles is important for understanding BRI1 function,
102
especially in relation to BR stimuli and regulation of downstream signaling.
103
Single particle analysis (SPT) provides a powerful technique to visualize the
104
dynamic behavior of individual particles inside living cells; these behaviors
105
would otherwise be lost in ensemble averages (Groves et al., 2008; Vale, 2008;
106
Vizcay-Barrena et al., 2011; Gowrishankar et al., 2012). Measurements of
107
fluorophore
108
simultaneous monitoring of heterogeneous molecular populations and their
109
complex kinetics at or near physiological conditions (Bacia et al., 2006; Jaiswal
110
and Simon, 2007; Vale, 2008; Lord et al., 2010). Here, we used a BRI1-GFP
111
fusion construct driven by the native BRI1 promoter to monitor the assembly
112
state and dynamics of the BR receptor. To mark other membrane components,
113
we co-expressed BRI1-GFP with clathrin light chain (CLC)-mCherry or AtFlot1
114
(a membrane microdomain marker)-mCherry in Arabidopsis. We showed the
115
dynamic behavior and partitioning of BRI1-GFP at the plasma membrane, by
116
using single-particle tracking and variable angle total internal reflection
EP
TE D
99
and
temporal
fluorescence
fluctuations
allow
AC C
distributions
4
ACCEPTED MANUSCRIPT 117
fluorescence microscopy (VA-TIRFM). Fluorescence correlation spectroscopy
118
(FCS) analyses revealed membrane microdomain associated pathways
119
function
120
microdomains in endocytic pathways are critically involved in the activation of
121
BRI1 signaling, an essential step in the response to BR.
122
RESULTS
123
Single-Particle Imaging of BRI1 at the Plasma Membrane
124
We followed the subcellular localization of BRI1 in Arabidopsis seedlings
125
overexpressing a BRI1-GFP fusion protein driven by the native BRI1 promoter,
126
a system suitable for cell biology studies. BRI1-GFP clearly targeted to the
127
plasma membrane and intracellular compartments, particularly in root
128
meristem cells (Figure 1A and B). To visualize the state of single BRI1-GFP
129
particles at the plasma membrane by VA-TIRFM, we used single particle
130
analysis (SPT) method (Jaqaman et al., 2008), which can not only eliminate
131
the fluctuation particles, but also exclude the BRI1-GFP particles from the
132
cytosol. By VA-TIRM, we found that the moving BRI1-GFP particles appeared
133
as well-dispersed diffraction-limited fluorescent spots at the plasma membrane
134
(Figure 1C and D; Supplemental Movie 1). The sequential images showed that
135
individual BRI1-GFP particles appeared at the membrane as isolated
136
fluorescent spots and demonstrated dramatically different dynamics. Generally,
137
some BRI1-GFP particles stayed at certain positions for a few frames and then
138
rapidly disappeared, possibly due to endocytosis or photobleaching. By
139
contrast, some BRI1-GFP particles appeared gradually in the plane of focus or
140
diffused laterally (Supplemental Figure 1). Two representative fluorescence
141
intensity tracks of BRI1-GFP particles are shown in Figure 1E and F.
endocytosis.
We
further
showed
that
membrane
RI PT
BRI1
AC C
EP
TE D
M AN U
SC
in
142
To examine the diffusion of BRI1-GFP particles at the plasma membrane,
143
we quantified the diffusion modes of individual BRI1-GFP particles by
144
analyzing the mean square displacement curves obtained from single-particle 5
ACCEPTED MANUSCRIPT tracking in consecutive images. BRI1-GFP particles exhibited multiple motion
146
types, which can be classified as: simple Brownian diffusion, restricted diffusion
147
limited by corrals or impaired by obstacles, directed diffusion displaying a high
148
velocity, or mixed diffusion combining Brownian and restricted modes (Figure
149
1G).
150
Motion and Diffusion of BRI1 Particles at the Plasma Membrane
151
We were also curious as to whether BRs affect the dynamic behavior of BRI1
152
proteins at the plasma membrane; therefore we monitored the motion of BRI1
153
particles in plant cells with different BR levels (Figure 2A and B). In Col-0 (wild
154
type, WT) seedlings expressing BRI1-GFP, the motion ranges of BRI1-GFP
155
distributed into two subpopulations, long distance motion (39.4%, 1.05 ± 0.27
156
µm, the position of the peak) and short distance motion (60.6%, 0.55 ± 0.13
157
µm, the position of the peak) (Figure 2C and G). After BRZ (BR biosynthesis
158
inhibitor) treatment, the motion ranges of BRI1-GFP particles were different
159
from those in Col-0 wild type; only 32.78% of the particles showed long distance
160
motion and 67.22% showed short distance motion (Figure 2D and G). We
161
further confirmed in the brassinosteroid-deficient det2 mutants (with low
162
endogenous BR levels) expressing BRI1-GFP, and found that long distance
163
motion and short distance motion were 27.2% and 72.8%, respectively (Figure
164
2E and G). This indicates that the majority of BRI1-GFP particles in the resting
165
state were immobile and their diffusion was restricted within a limited domain at
166
the plasma membrane. When the Col-0/BRI1-GFP seedlings were treated with
167
0.1 µM of the BR analog 24-epibrassinolide (eBL), BRI1-GFP diffusion
168
changed significantly, with 56.8% showing long distance motion and 43.2%
169
showing short distance motion, indicating that activated BRI1 proteins can
170
diffuse from limited zones to relative wide zones (Figure 2F and G).
AC C
EP
TE D
M AN U
SC
RI PT
145
171
We then determined the dynamic properties of BRI1 proteins in more
172
detail by calculating diffusion coefficients at different BR levels. For this
6
ACCEPTED MANUSCRIPT purpose, the distribution of diffusion coefficients was plotted on histograms and
174
fitted using Gaussian functions, in which the Gaussian peaks (noted as Ĝ)
175
were considered as the characteristic diffusion coefficients. We found that Ĝ
176
was 8.8 × 10-3 µm2/s (SE 8.17 to 9.44 × 10-3 µm2/s) in control Col-0/BRI1-GFP
177
seedlings (WT) (Figure 2H and L). In det2/BRI1-GFP seedlings, or after BRZ
178
treatment, Ĝ was 6.11× 10-3 µm2/s (SE 5.51 to 6.77 × 10-3 µm2/s) / 6.58× 10-3
179
µm2/s (SE 6.04 to 7.06 × 10-3 µm2/s), representing a significant decrease in
180
comparison to Col-0/BRI1-GFP seedlings (Figure 2I,J and L). Only slight
181
changes occurred in the mock control treated with 0.85‰ EtOH (Supplemental
182
Figure 2A-C), but, in contrast, eBL treatment caused a significant increase in Ĝ
183
(1.39 × 10-2 µm2/s, SE 1.28 to 1.51 × 10-2 µm2/s) (P
S1674-2052(15)00202-6
DOI:
10.1016/j.molp.2015.04.005
Reference:
MOLP 123
To appear in:
MOLECULAR PLANT
Received Date: 10 March 2015 Revised Date:
10 April 2015
Accepted Date: 12 April 2015
Please cite this article as:
Wang L., Li H., Lv X., Chen T., Li R., Xue Y., Jiang J., Jin B., Baluška F., Šamaj J., Wang X., and Lin J. (2015). Spatiotemporal Dynamics of BRI1 Receptor and Its Regulation by
Membrane Microdomains in Living Arabidopsis Cells. Mol. Plant. doi: 10.1016/j.molp.2015.04.005.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Spatiotemporal Dynamics of BRI1 Receptor and Its
2
Regulation by Membrane Microdomains in Living
3
Arabidopsis Cells
4
Li Wang1,2, #, Hong Li1,2, #, Xueqin Lv1,3, Tong Chen1, Ruili Li3, Yiqun Xue1, Jianjun
5
Jiang4, Biao Jin2, František Baluška5, Jozef Šamaj6, Xuelu Wang4,7, Jinxing Lin1,3*
RI PT
1
6 7
1
8
China;
9
2
College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, China;
10
3
College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China;
11
4
State Key Laboratory of Genetic Engineering and Institute of Plant Biology, School of Life Sciences,
12
Fudan University, Shanghai 200433, China;
13
5
Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany;
14
6
Department of Cell Biology, Palacky University Olomouc, Olomouc 78371, Czech Republic
15
7
College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
TE D
M AN U
SC
Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing 100093,
16
*
17
#
18
The authors declare no conflict of interest.
19
Running title: Membrane microdomains regulate endocytosis of BRI1
EP
L.W. and H.L. contributed equally to this work.
AC C
20
Correspondence: [email protected]
21
Short Summary: How membrane microdomains are involved in regulating
22
BRI1 endocytosis and BR signaling is still unknown. In this study, using
23
VA-TIRFM in combination with FCS and FCCS, we identified membrane
24
microdomains mediated a novel endocytic pathway involving in BRI1
25
endocytosis. Significant light has been shed on the molecular mechanisms
26
through which BRI1 can be recruited into functional membrane microdomains
27
and internalized in response to BR signal.
28
1
ACCEPTED MANUSCRIPT 29
ABSTRACT
30 31
The
32
INSENSITIVE1 (BRI1) plays fundamental roles in BR signaling, but the
33
molecular
34
internalization and assembly state remain unclear. Here, we applied
35
variable
36
(VA-TIRFM) and fluorescence cross-correlation spectroscopy (FCCS) to
37
analyze the dynamics of GFP-tagged BRI1. We found that, in response to
38
BR stimulation, the degree of colocalization of BRI1-GFP with
39
AtFlot1-mcherry increased; in particular, BR stimulated the membrane
40
microdomain-associated pathway of BRI1 internalization. We also verified
41
this in endocytosis-defective chc2-1 mutants and the AtFlot1 amiRNA
42
15-5 lines. Furthermore, examination of the phosphorylation status of
43
bri1-EMS-suppressor 1 (BES1) and measurement of BR-responsive gene
44
expression revealed that membrane microdomains affect BR signaling.
45
These results suggested that BR promotes the partitioning of BRI1 into
46
functional membrane microdomains to activate BR signaling.
mechanisms
total
underlying
internal
receptor
the
reflection
BRASSINOSTEROID
effects
of
BR
on
BRI1
fluorescence
microscopy
TE D
M AN U
SC
angle
(BR)
RI PT
brassinosteroid
EP
47
major
Keywords: BRI1; BR signaling; endocytosis; membrane microdomains;
49
spatiotemporal dynamics
50 51 52 53
AC C
48
54 55 56 57 58 2
ACCEPTED MANUSCRIPT INTRODUCTION
60
Brassinosteroids (BRs) are essential plant steroid hormones that regulate a
61
wide range of developmental and physiological processes, including cell
62
elongation and root growth (Clouse and Sasse, 1998; Wu et al., 2011). The major
63
BR receptor BRASSINOSTEROID INSENSITIVE1 (BRI1) plays fundamental
64
roles in BR signaling and plant development (Wang et al., 2001; Wang and He,
65
2004). For example, knock-out mutants of BRI1 are extremely dwarfed, and
66
completely BR-insensitive (Clouse et al., 1996; Li and Chory, 1997; Kinoshita et
67
al., 2005). BRI1 belongs to the leucine-rich repeat receptor-like kinase
68
(LRR-RLK) class and has an extracellular domain containing 24 leucine-rich
69
repeats (LRRs), a transmembrane domain, and a cytoplasmic domain
70
containing a serine/threonine (Ser/Thr) kinase domain (Li and Chory, 1997; Vert
71
et al., 2005). Upon perceiving BRs, plant cells activate BRI1 kinase to trigger
72
the dissociation of the inhibitory BRI1 KINASE INHIBITOR 1 (BKI1), thus
73
enabling sequential transphosphorylation of BRI1 and its co-receptor BRI1
74
associated kinase 1 (BAK1) to form an active receptor complex, thereby
75
initiating BR signaling (Wang and Chory, 2006; Bücherl et al., 2013).
TE D
M AN U
SC
RI PT
59
In animals and yeast, endocytosis regulates protein abundance at the
77
plasma membrane during signaling and acts to retarget or degrade membrane
78
proteins (Royle and Murrell-Lagnado, 2003; Grant and Donaldson, 2009). Plant
79
cells use endocytosis to internalize exogenous material, ligands, and plasma
80
membrane proteins or regulate signaling at the plasma membrane (Murphy et al.,
81
2005; Šamaj et al., 2005). Constitutively internalized and ligand-inducible
82
receptors are subject to endocytosis (Royle and Murrell-Lagnado, 2003;
83
Scarselli and Donaldson, 2009). The constitutive endocytosis of plant
84
transmembrane proteins such as receptors, channels and transporters has
85
been extensively reported; these proteins include PINs, PIP2, and BRI1
86
(Paciorek et al., 2005; Geldner et al., 2007; Li et al., 2011). The constitutive
87
internalization of PIN1, PIN2 and PIP2;1 requires clathrin-dependent
AC C
EP
76
3
ACCEPTED MANUSCRIPT endocytosis (Baisa et al., 2013; Dhonukshe et al., 2007; Kitakura et al., 2011).
89
Furthermore, plant structural sterols occur within endocytic networks, and play
90
a role in a constitutive endocytic cycling of some plasma membrane transport
91
proteins (Šamaj et al., 2005). BRI1 mainly localizes to the plasma membrane
92
and early endosomal compartments in Arabidopsis root cells (Russinova et al.,
93
2004; Geldner et al., 2007; Hink et al., 2008). Recent work reported clathrin and
94
ARF GTPase (ARF-GEF) dependent endocytic regulation of BR signaling from
95
the plasma membrane (Irani et al., 2012; Rubbo et al., 2013), and ubiquitination
96
can promote BRI1 internalization (Martins et al., 2015). However, the molecular
97
mechanisms underlying internalization and assembly state changes of BRI1 in
98
response to exogenous BR stimulation remain poorly understood.
M AN U
SC
RI PT
88
BR regulates the activity and dynamic behaviors of BRI1 particles at the
100
plasma membrane. Therefore, monitoring and tracking the behavior of
101
individual BRI1 particles is important for understanding BRI1 function,
102
especially in relation to BR stimuli and regulation of downstream signaling.
103
Single particle analysis (SPT) provides a powerful technique to visualize the
104
dynamic behavior of individual particles inside living cells; these behaviors
105
would otherwise be lost in ensemble averages (Groves et al., 2008; Vale, 2008;
106
Vizcay-Barrena et al., 2011; Gowrishankar et al., 2012). Measurements of
107
fluorophore
108
simultaneous monitoring of heterogeneous molecular populations and their
109
complex kinetics at or near physiological conditions (Bacia et al., 2006; Jaiswal
110
and Simon, 2007; Vale, 2008; Lord et al., 2010). Here, we used a BRI1-GFP
111
fusion construct driven by the native BRI1 promoter to monitor the assembly
112
state and dynamics of the BR receptor. To mark other membrane components,
113
we co-expressed BRI1-GFP with clathrin light chain (CLC)-mCherry or AtFlot1
114
(a membrane microdomain marker)-mCherry in Arabidopsis. We showed the
115
dynamic behavior and partitioning of BRI1-GFP at the plasma membrane, by
116
using single-particle tracking and variable angle total internal reflection
EP
TE D
99
and
temporal
fluorescence
fluctuations
allow
AC C
distributions
4
ACCEPTED MANUSCRIPT 117
fluorescence microscopy (VA-TIRFM). Fluorescence correlation spectroscopy
118
(FCS) analyses revealed membrane microdomain associated pathways
119
function
120
microdomains in endocytic pathways are critically involved in the activation of
121
BRI1 signaling, an essential step in the response to BR.
122
RESULTS
123
Single-Particle Imaging of BRI1 at the Plasma Membrane
124
We followed the subcellular localization of BRI1 in Arabidopsis seedlings
125
overexpressing a BRI1-GFP fusion protein driven by the native BRI1 promoter,
126
a system suitable for cell biology studies. BRI1-GFP clearly targeted to the
127
plasma membrane and intracellular compartments, particularly in root
128
meristem cells (Figure 1A and B). To visualize the state of single BRI1-GFP
129
particles at the plasma membrane by VA-TIRFM, we used single particle
130
analysis (SPT) method (Jaqaman et al., 2008), which can not only eliminate
131
the fluctuation particles, but also exclude the BRI1-GFP particles from the
132
cytosol. By VA-TIRM, we found that the moving BRI1-GFP particles appeared
133
as well-dispersed diffraction-limited fluorescent spots at the plasma membrane
134
(Figure 1C and D; Supplemental Movie 1). The sequential images showed that
135
individual BRI1-GFP particles appeared at the membrane as isolated
136
fluorescent spots and demonstrated dramatically different dynamics. Generally,
137
some BRI1-GFP particles stayed at certain positions for a few frames and then
138
rapidly disappeared, possibly due to endocytosis or photobleaching. By
139
contrast, some BRI1-GFP particles appeared gradually in the plane of focus or
140
diffused laterally (Supplemental Figure 1). Two representative fluorescence
141
intensity tracks of BRI1-GFP particles are shown in Figure 1E and F.
endocytosis.
We
further
showed
that
membrane
RI PT
BRI1
AC C
EP
TE D
M AN U
SC
in
142
To examine the diffusion of BRI1-GFP particles at the plasma membrane,
143
we quantified the diffusion modes of individual BRI1-GFP particles by
144
analyzing the mean square displacement curves obtained from single-particle 5
ACCEPTED MANUSCRIPT tracking in consecutive images. BRI1-GFP particles exhibited multiple motion
146
types, which can be classified as: simple Brownian diffusion, restricted diffusion
147
limited by corrals or impaired by obstacles, directed diffusion displaying a high
148
velocity, or mixed diffusion combining Brownian and restricted modes (Figure
149
1G).
150
Motion and Diffusion of BRI1 Particles at the Plasma Membrane
151
We were also curious as to whether BRs affect the dynamic behavior of BRI1
152
proteins at the plasma membrane; therefore we monitored the motion of BRI1
153
particles in plant cells with different BR levels (Figure 2A and B). In Col-0 (wild
154
type, WT) seedlings expressing BRI1-GFP, the motion ranges of BRI1-GFP
155
distributed into two subpopulations, long distance motion (39.4%, 1.05 ± 0.27
156
µm, the position of the peak) and short distance motion (60.6%, 0.55 ± 0.13
157
µm, the position of the peak) (Figure 2C and G). After BRZ (BR biosynthesis
158
inhibitor) treatment, the motion ranges of BRI1-GFP particles were different
159
from those in Col-0 wild type; only 32.78% of the particles showed long distance
160
motion and 67.22% showed short distance motion (Figure 2D and G). We
161
further confirmed in the brassinosteroid-deficient det2 mutants (with low
162
endogenous BR levels) expressing BRI1-GFP, and found that long distance
163
motion and short distance motion were 27.2% and 72.8%, respectively (Figure
164
2E and G). This indicates that the majority of BRI1-GFP particles in the resting
165
state were immobile and their diffusion was restricted within a limited domain at
166
the plasma membrane. When the Col-0/BRI1-GFP seedlings were treated with
167
0.1 µM of the BR analog 24-epibrassinolide (eBL), BRI1-GFP diffusion
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changed significantly, with 56.8% showing long distance motion and 43.2%
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showing short distance motion, indicating that activated BRI1 proteins can
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diffuse from limited zones to relative wide zones (Figure 2F and G).
AC C
EP
TE D
M AN U
SC
RI PT
145
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We then determined the dynamic properties of BRI1 proteins in more
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detail by calculating diffusion coefficients at different BR levels. For this
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ACCEPTED MANUSCRIPT purpose, the distribution of diffusion coefficients was plotted on histograms and
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fitted using Gaussian functions, in which the Gaussian peaks (noted as Ĝ)
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were considered as the characteristic diffusion coefficients. We found that Ĝ
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was 8.8 × 10-3 µm2/s (SE 8.17 to 9.44 × 10-3 µm2/s) in control Col-0/BRI1-GFP
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seedlings (WT) (Figure 2H and L). In det2/BRI1-GFP seedlings, or after BRZ
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treatment, Ĝ was 6.11× 10-3 µm2/s (SE 5.51 to 6.77 × 10-3 µm2/s) / 6.58× 10-3
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µm2/s (SE 6.04 to 7.06 × 10-3 µm2/s), representing a significant decrease in
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comparison to Col-0/BRI1-GFP seedlings (Figure 2I,J and L). Only slight
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changes occurred in the mock control treated with 0.85‰ EtOH (Supplemental
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Figure 2A-C), but, in contrast, eBL treatment caused a significant increase in Ĝ
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(1.39 × 10-2 µm2/s, SE 1.28 to 1.51 × 10-2 µm2/s) (P
