Visualization of BRI1 and SERK3/BAK1 Nanoclusters in Arabidopsis Roots
Visualization of BRI1 and SERK3/BAK1 Nanoclusters in Arabidopsis Roots
Stefan J. Hutten 0 1 2
Danny S. Hamers 0 1 2
Marije Aan den Toorn 0 1 2
Wilma van Esse 0 1 2
Antsje Nolles 0 1 2
Christoph A. BuÈ cherl 0 1 2
Sacco C. de Vries 0 1 2
Johannes Hohlbein 0 2
Jan Willem Borst 0 1 2
0 a Current address: Department Plant Breeding and Genetics, Max Planck Institute for Plant Breeding Research , Carl-von-LinneÂ -Weg 10, KoÈln, Germany. ¤b Current address: The Sainsbury Laboratory, Norwich Research Park, Norwich , United Kingdom
1 Laboratory of Biochemistry, Wageningen University & Research , Stippeneng 4, WE Wageningen , The Netherlands , 2 Laboratory of Biophysics, Wageningen University & Research , Stippeneng 4, WE Wageningen , The Netherlands , 3 Microspectroscopy Centre, Wageningen University & Research , Stippeneng 4, WE Wageningen , The Netherlands
2 Editor: Marisa Otegui, University of Wisconsin Madison , UNITED STATES
Brassinosteroids (BRs) are plant hormones that are perceived at the plasma membrane (PM) by the ligand binding receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) and the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE 3/BRI1 ASSOCIATED KINASE 1 (SERK3/BAK1). To visualize BRI1-GFP and SERK3/BAK1-mCherry in the plane of the PM, variable-angle epifluorescence microscopy (VAEM) was employed, which allows selective illumination of a thin surface layer. VAEM revealed an inhomogeneous distribution of BRI1-GFP and SERK3/BAK1-mCherry at the PM, which we attribute to the presence of distinct nanoclusters. Neither the BRI1 nor the SERK3/BAK1 nanocluster density is affected by depletion of endogenous ligands or application of exogenous ligands. To reveal interacting populations of receptor complexes, we utilized selective-surface observationÐ fluorescence lifetime imaging microscopy (SSO-FLIM) for the detection of FoÈ rster resonance energy transfer (FRET). Using this approach, we observed hetero-oligomerisation of BRI1 and SERK3 in the nanoclusters, which did not change upon depletion of endogenous ligand or signal activation. Upon ligand application, however, the number of BRI1-SERK3 /BAK1 hetero-oligomers was reduced, possibly due to endocytosis of active signalling units of BRI1-SERK3/BAK1 residing in the PM. We propose that formation of nanoclusters in the plant PM is subjected to biophysical restraints, while the stoichiometry of receptors inside these nanoclusters is variable and important for signal transduction.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: Financial support from the Graduate
School VLAG (Wageningen, The Netherlands) is
gratefully acknowledged. J. H. acknowledges
support from FP7-PEOPLE-2013-CIG - Marie-Curie
Action: "Career Integration Grants" (grant ID
630992). The FRET-FLIM experiments were
performed on a multimode confocal microscope
supported by a NWO Middelgroot Investment
Grant (721.011.004; J.W.B.). D.S.H. was supported
Brassinosteroids (BRs) are plant steroid hormones that regulate cellular expansion,
differentiation and proliferation [
]. The brassinosteroid signalling pathway starts at the plasma
membrane (PM), where BRs bind to the island domain in the extracellular part of the
leucine-richby Netherlands Organization for Scientific Research
(NWO; ALW 821.02.003).
repeat receptor like kinase (LRR-RLK) BRASSINOSTEROID-INSENSITIVE1 (BRI1). Ligand
binding induces phosphorylation and subsequent disassociation of the inhibitor protein BRI1
KINASE INHIBITOR 1 (BKI1) from the cytoplasmic kinase domain of BRI1 [
prevents BRI1 from interacting with its co-receptor SERK3/BAK1 (SOMATIC
EMBRYOGENESIS RECEPTOR KINASE 3/ BRI1 ASSOCIATED KINASE 1) .
Hetero-oligomerization of BRI1 and SERK3/BAK1 results in sequential trans phosphorylation events on their
cytoplasmic kinase domains , which is a prerequisite for successful BR signal transduction
]. Trans phosphorylation between SERK3/BAK1 and BRI1 leads to full activation of the
signalling pathway resulting in phosphorylation of downstream signalling components [
The signal is further relayed to the transcription factors BZR1 and BES1, resulting in regulated
expression of BR- responsive genes. Recent investigations suggest that hetero-oligomers of
BRI1 and SERK3/BAK1 are, at least partially, preformed in absence of ligand and form a
functional unit able to perceive BRs and initiate downstream signalling . Extracellular domain
interactions between SERK1, a highly homologous family member of SERK3/BAK1, and BRI1
are ligand dependent [
], suggesting that domains such as the transmembrane domain or other
cytoplasmic domains are essential for the observed ligand independent hetero-oligomerisation.
BRI1 and SERK3/BAK1 have a fundamental role in BR signalling and regulation of
BRrelated developmental processes in root and shoot [
]. Although there are indications of
endosomal BR signalling , the initial recognition of BRs and activation of the receptor
complex via ligand binding occurs at the PM [
]. The PM is a highly organized lipid bilayer
interspersed with proteins, of which most of them show restricted diffusion through the lipid
bilayer and even clear inhomogeneous patterning across the PM [14 and references therein].
Diffusion of proteins in the PM can be restricted via the cortical cytoskeleton, protein
`crowding', interaction between membrane components or heterogeneity in membrane composition
and state [
]. In Arabidopsis, the lateral movement of PM localized proteins is further
restricted by the presence of the cell wall, although not necessarily due to direct interactions
]. As a result of these restrictions, protein distribution across the membrane is likely to lead
to cluster formation. In animal cells, the presence of so-called nanoclusters of receptor proteins
in the PM has been established [
] and is thought to be essential for signal transduction. In
addition, internalization of receptors upon ligand-binding- through endocytosis has been
reported to take place via specific endosomal locations  and by relocalization within the
PM itself .
Recently, the plant receptor kinase BRI1 was found to localize in nanoclusters or membrane
microdomains in the PM . Upon BR stimulation, an increased colocalization of BRI1-GFP
with a membrane microdomain marker protein (AtFlot1-mCherry) was observed. This
partitioning of BRI1 into microdomains has been suggested to be essential for BR signalling.
Here, we investigate the PM distribution of BRI1 and SERK3/BAK1 in live Arabidopsis
thaliana epidermal root cells using variable-angle epifluoresence microscopy (VAEM) 
and fluorescence lifetime imaging microscopy (FLIM) for the detection of FoÈrster resonance
energy transfer (FRET). Different lines of BRI1-GFP were used in this study of which
BRI1GFP line 1 has endogenous protein expression levels whereas BRI1-GFP line 2 showed a
threefold higher expression . Our results show an inhomogeneous distribution of BRI1 and
SERK3/BAK1 depicted by cluster formation across the membrane. The cluster density is not
altered by activating the signalling complex; neither by over-expression of the receptor nor by
changing endocytosis rate of the receptor. To characterize BRI1-SERK3/BAK1
hetero-oligomers, we performed FRET in combination with FLIM in the plane of the PM of root epidermal
cells adopted as Selective Surface ObservationÐFLIM (SSO-FRET-FLIM). Using this approach,
we revealed that BRI1-SERK3/BAK1 hetero-oligomers are present as the active ligand
perception units within nanoclusters in epidermal cells of Arabidopsis roots.
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BRI1 and SERK3 are present in plasma membrane nanoclusters
To investigate the distribution of BRI1-GFP and SERK3/BAK1-mCherry at the PM we imaged
these receptors using VAEM. Both receptors form hetero-oligomeric complexes required for
signal transduction and are part of the same signalling pathway [
VAEM showed that neither BRI1 nor SERK3/BAK1 is homogenously distributed.
Furthermore, a clear punctuated pattern was seen for both receptors (Fig 1), in contrast to the
homogeneous distribution of PM-marker LT16-B (S1 Fig), an integral membrane protein [
Similar receptor distributions in animal cells are referred to as nanoclusters [
], a term that
we will employ here as well. Nanoclusters of clearly variable fluorescence intensities were
observed with an average size of approximately 5 pixels per cluster, which corresponds to a
diameter of 300±500 nm. These nanoclusters were observed for both the main receptor BRI1
and the coreceptor SERK3/BAK1 (S2±S6 Movies; a step by step guide for image analysis is
provided in S2 Fig). The receptors can be visualized by VAEM in the epidermal root cells of the
Fig 1. VAEM reveals a heterogeneous distribution of BRI1-GFP and SERK3-mCherry in the PM.
Typical VAEM images of live root epidermal cells of 6 day old A. thaliana seedlings showing PM distribution of
(A) BRI-GFP line1, (B) BRI1-GFP line 2 and (C) SERK3-mCherry, (D) BRI1-GFP in a serk1serk3 mutant
plant. Images taken are of epidermal cells in the early elongation zone. Scale bars represent 10 μm.
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elongation zone, an area where BR signalling is reported to be active [
]. The root meristem
zone itself cannot be visualized due to curvature of the root, which causes the epidermal cell
layer to be outside the critical range for VAEM. Our VAEM setup was capable of visualizing
endosomes and other internal membrane compartments demonstrated by imaging of the
ARA6/Rab F1-mRFP marker  (S1 Fig). Also, the ER marker WAVE6-mCherry [
visualized using VAEM. In plant cells, the ER forms a net-like basket occupying the cortical
space just below the plasma membrane. Time-lapse imaging revealed that the ER is a mobile and
dynamic organelle (S1 Movie). We used additional markers of Golgi stacks (WAVE18-mRFP,
], Trans Golgi Network (VHAa1-mRFP, [
]) and retrograde trafficking (ARA7/Rab F2b,
] to track intracellular membrane compartments in conjunction with the PM of
Arabidopsis root epidermal cells (S1 Fig).
Endosomal vesicles on the cytoplasmic side of the PM were clearly visible for both BRI1 and
SERK3/BAK1, with a higher number of BRI1-containing vesicles compared to SERK3/BAK1.
Especially BRI1-GFP line 2, which has approximately 3x higher expression of BRI1-GFP
compared to wild type, shows a large number of fluorescent endosomal compartments, which are
reminiscent of LE/EE compartments (S2 and S3 Movies). Interestingly, only a small number of
nanoclusters containing BRI1-GFP were seen to disappear from the PM (S4 Movie) even in the
presence of brassinazole (BRZ), a potent brassinosteroid synthesis inhibitor [
application of BL, we did not observe a difference in the rate of internalization of nanoclusters from
the PM by endocytosis. Recently, the existence of ligand-independent BRI1 endocytosis was
discussed in reference [
Brassinosteroid signalling is dependent on both the presence of the main ligand binding
receptor BRI1 and the SERK co-receptors [
]. In the root, the active signalling complex
consists of BRI1 with SERK1 and/or SERK3/BAK1 [
]. We recently showed that BRI1 and
SERK3/BAK1 co-localize in the PM and that a minor amount of BRI1 and SERK3/BAK1
receptors is already present in preformed complexes . To test whether the co-receptors
influence the distribution of the main receptor in the PM, the BRI1-GFP line 2 was crossed
into a serk1serk3 mutant background. VAEM showed that in the absence of both co-receptors
the distribution of the main ligand binding receptor and the overall fluorescence intensity (Fig
1D) are barely affected compared to BRI1-GFP line 2 in wild-type roots. Roots of serk1serk3
double mutants are almost completely insensitive to BL [
], indicating that no active
signalling complexes are formed. Our results thus imply that SERK co-receptors do not participate
in maintaining the PM distribution of BRI1 in epidermal cells of Arabidopsis roots.
By combining the BRI1 and SERK3/BAK1 PM receptor density data from  with the
number of nanoclusters per μm2 PM determined in this work, we estimated the number of
receptors present in each nanocluster (Table 1). In this analysis, we assumed perfect
maturation efficiency of fluorescent proteins leading to a possible underestimation of receptor
The average number of receptors (second column taken from reference ) and the number of nanoclusters per μm2 (third column) allows calculating the
average number of receptors per nanocluster (fourth column). Numbers are given with their respective standard error of the mean (SEM). For each
experiment, three different roots were recorded using confocal imaging (n = 9). n = number of individual cells. Values are given ±SEM.
* data from 
4 / 19
quantities, but still provide information between expression levels between different lines. For
example, at near-endogenous receptor levels we estimated that at least 6 fluorescent BRI1
receptors are present in each nanocluster. Intriguingly, while overexpression of BRI1-GFP
resulted in about a three-fold increase in PM receptor density , there is no concomitant
increase in nanocluster density. In fact, the density of BRI1 nanoclusters appeared to be even
slightly lower in cells of the overexpression line (Table 1). Two explanations are conceivable:
first, either the nanoclusters harbouring PM located receptors can accommodate variable
numbers or, second, a larger number of receptors is distributed more uniformly outside of the
PM nanocluster domains upon overexpression. In the SERK3-GFP line about two-fold more
nanoclusters were present compared to BRI1-GFP line 1, of which each contained at least two
receptors. A similar calculation for the SERK3/BAK1-mCherry line resulted in about 1 ± 1
receptors per nanocluster. The number of receptors per nanocluster is in line with estimates
based on single-molecule photo bleaching analysis (S3 Fig).
A third plant receptor was investigated to rule out that the observed nanocluster
distribution pattern was an inherent property of only these two receptors. BIR3 (BAK1-interacting
receptor kinase 3) is an abundant PM receptor-like kinase for which the related members
] and BIR2 [
] have been implicated as stabilizing components of PM receptor
complexes involved in pathogen triggered immunity. Similar to BRI1 and SERK3, BIR3 is
distributed heterogeneously in the PM including punctuated pattern, suggesting that a distribution
into nanoclusters is a common configuration for plant membrane receptors (S4 Fig). Other
PM proteins such as remorins and flottilins also showed nanocluster arrangements harbouring
about 0.1±1.3 domains per μm 2 [
BRI1 PM-distribution is not altered upon signal activation or absence of endogenous ligands
In order to determine whether BRI1 and SERK3/BAK1 receptor distribution across the PM is
affected by activation of the BR signalling pathway, seedlings were first depleted of endogenous
ligands by incubation with BRZ. Prior to VAEM, the BL depleted seedlings were incubated
with 1 μM BL, which is the biologically most active brassinosteroid, for 1 h. This treatment is
routinely used to fully activate the BR signalling pathway and optimized to achieve a near
maximal receptor-ligand occupancy [
]. Interestingly, under these conditions of full activation,
only a minor amount of BRI1-GFP and SERK3/BAK1-mCherry was previously found to
]. It was therefore of great interest to test whether changes in the distribution or number
of nanoclusters would be visible upon full activation of the signalling pathway. Surprisingly,
we could not detect any significant change in the number of clusters per μm2 at the PM or
cluster size for both BRI1-GFP and SERK3/BAK1-mCherry between the BRZ treated roots and the
BRZ+BL treated roots (Table 2). Roots expressing SERK3/BAK1-mCherry showed a
non-significant increase in the number of clusters upon BRZ treatment. BRI1-GFP distribution was
also not affected in the brassinosteroid synthesis mutant det2 (S5 Fig), which contains less than
10% of the normal WT levels of brassinosteroids [
BRI1 and SERK3/BAK1 mobility in the PM investigated using FRAP
Using VAEM, we observed different mobility states, ranging from static for BRI1 and SERK 3/
BAK1 to rapid directional movement ER marker (S1±S6 Movies). In order to characterize the
mobility of BRI1 and SERK3/BAK1 receptors in the PM, we performed fluorescence recovery
after photobleaching (FRAP) on fluorescently labelled receptors expressed in Arabidopsis
roots. In the elongation zone of Arabidopsis root cells, BRI1-GFP and SERK3/BAK1-GFP
showed a slow diffusion (below 0.1 μm2 s-1) and a mobile fraction of 35 and 60% for BRI1 and
5 / 19
ligand application. Ligand application was accomplished by incubation of seedlings with 1 μM BL for 1h. All experiments were performed twice; three images
were collected for three seedlings per experiment (n = 9). Numbers are given with their respective pooled standard error of the mean (SEM).
SERK3/BAK1, respectively. These values were at the lower end of 0.1±1.0 μm2 s-1, reported for
free lateral diffusion of a PM receptor [
] and of control measurement (Fig 2). In meristem
cells, the diffusion coefficients were even lower, rendering both receptors virtually immobile
(Table 3 and S6 Fig). Further analysis employing a one-dimensional Gaussian fit of the
fluorescence intensity over the cross section of the PM suggests that most of the observed mobility in
the elongation zone is due to replenishment from internal receptor pools (S7 Fig). In a similar
study by Wang and coworkers , BRI1-GFP was expressed under its native promoter in Col
Fig 2. FRAP analysis of BRI1-GFP and KNOLLE-GFP in epidermal cells of Arabidopsis roots. (A)
Typical images of FRAP experiment prior and post bleach pulse of KNOLLE-GFP and BRI1-GFP. (B)
Recovery-curves of KNOLLE-GFP (blue line) and BRI1-GFP (black line) in epidermal cells in the root
meristem. Around 200 s after bleaching, fluorescence intensity of KNOLLE-GFP at the PM is fully restored.
No such recovery is observed for BRI1-GFP. For KNOLLE-GFP n = 7, for BRI1-GFP n = 15, measured in
independent replicas, error bars indicate standard error of means (SEM).
6 / 19
n = number of individual measurements. Mf = mobile fraction. D = diffusion coef®cient. Values are given ± SEM
0 background and a diffusion coefficient of 8.8x10-3 μm2 s-1 was found, which is in similar
range to our observations in meristem cells. Taken together, our VAEM and FRAP data suggest
that both BRI1 and SERK3/BAK1 receptors are distributed in PM nanoclusters, which are
largely immobile and contain only a limited number of BRI1 and SERK3/BAK1 receptors.
BRI1 and SERK3/BAK1 distribution is not influenced by various inhibitors
All three integral membrane receptors investigated in this study, BRI1, SERK3/BAK1 and
BIR3, showed a similar distribution of nanoclusters. In addition, BRI1 and SERK3/BAK1
nanocluster distribution was not affected upon ligand induced activation. To investigate the
underlying mechanism regulating membrane distribution, we treated Arabidopsis roots with
various inhibitors of the endocytic, cytoskeleton or biosynthetic pathways. Treatment of
BRI1-GFP and SERK3/BAK1-mCherry seedlings with cycloheximide, latrunculin B or
brefeldin A either alone or in combination with BRZ or with BRZ and BL did not result in any
consistent change in distribution of either receptor (data not shown).
Receptor interactions visualized by selective-surface observation
A limitation of VAEM is the inability to report on the interaction between BRI1-SERK3/BAK1
hetero-oligomers. We therefore performed SSO FRET-FLIM by focussing the confocal spot at
the PM of root epidermal cells. As expected, fluorescence intensity images obtained with this
approach (Fig 3B) revealed a similar distribution of BRI1-GFP nanoclusters as found using
VAEM (Fig 3A). Notably, VAEM can acquire single images within a few hundred
milliseconds, whereas a SSO confocal image requires an acquisition time between one and two
Fig 3. BRI1-GFP nanoclusters imaged using VAEM and SSO-confocal imaging. BRI1-GFP line 2
expressed in root epidermal cells imaged using VAEM (A) or SSO-confocal imaging (B). Both imaging
modalities show similar nanocluster distributions. Scale bars represent 10 μm.
7 / 19
a The mean difference is signi®cant at the (p<0.001) when compared to the donor only sample (BRI1-GFP).
All seedlings were depleted of endogenous brassinosteroids with BRZ and treated with 1 μM BL. τ represents the average ¯uorescence lifetime of the GFP
in picoseconds ± SEM. IPS represents the percentage of interacting pixels, and n the number of ¯uorescence lifetime images analysed.
The spatial distribution of fluorescence lifetimes of BRI1-GFP nanoclusters is not
homogeneous even though the standard deviation of all samples analysed is small (τ = 2402 ± 33 ps,
Table 4). At present, it is not clear whether this is due to small local variations in the immediate
environment of the receptors inside nanoclusters or whether it is due to technical limitations,
such as the precision by which the confocal volume can be positioned with respect to the plane
of the PM. In root cells expressing both BRI1-GFP and SERK3-mCherry, a small number of
nanoclusters showed reduced fluorescence lifetimes (τ = 2227 ± 134 ps, Table 4). This
observation indicated that a few nanoclusters located in the root epidermal cell PM contain both
receptors in close proximity, leading to a detectable FRET signal (Fig 4). BRZ treatment
followed by application of BL showed only a minor reduction in overall fluorescence lifetimes of
BRI1-GFP or BRI1-GFP/SERK3/BAK1-mCherry expressing root cells as determined by SSO
FRET-FLIM (τ = 2241 ±103 ps, Table 4). This finding is in full accordance with recently
published data using conventional FRET-FLIM, showing only a small increase in interaction
between BRI1 and SERK3/BAK1 upon full activation of the signalling pathway [
significant change of donor fluorescence lifetimes was observed in roots depleted of endogenous BL
or stimulated with BL (Table 4) independent of analysis conditions (see materials and
methods) suggesting that that no significant changes in the size or composition of nanoclusters
occurred upon ligand application.
We recently introduced the concept of interaction pixels (IPS) to obtain a more quantitative
description of the occurrence of FRET detected by FLIM [
]. Briefly, this method determines
the number of pixels in an image that have sufficiently high photon counts as well as a strongly
reduced fluorescence lifetime and reports these as a percentage of the total number of pixels.
Using IPS in conventional FRET-FLIM, we previously showed that upon BL stimulation in
Fig 4. SSO-FRET-FLIM of BRI1-GFP and BRI1-GFP/SERK3/BAK1-mCherry in absence and presence
of BL. (A) SSO fluorescence intensity image of BRI1-GFP expressed in root epidermal cells pretreated with
50 μM BRZ (B) SSO fluorescence lifetime image of BRI1-GFP expressed in root epidermal cells pretreated
with 50 μM BRZ (C) SSO fluorescence lifetime image of BRI1-GFP/SERK3/BAK1-mCherry expressed in root
epidermal cells pretreated with 50 μM BRZ. (D) SSO fluorescence lifetime image of BRI1-GFP/SERK3/
BAK1-mCherry expressed in root epidermal cells pretreated with 50 μM BRZ and subsequent incubation with
1 μM BL for 1 hour. The color bar represents the false color code for fluorescence lifetime (τ) distribution.
Scale bar represents 10 μm.
8 / 19
Fig 5. Fluorescence lifetime distribution of high intensity nanoclusters of BRI1-GFP and
BRI1-GFPSERK3/BAK1-mCherry in the absence and presence of BL. Fluorescence lifetime distribution plots of
nanoclusters with fluorescence intensities above 2000 photons per pixel. (A) Fluorescence lifetime
distribution of BRI1-GFP nanoclusters pretreated with 50 μM BRZ (n = 91). (B) Fluorescence lifetime
distribution of BRI1-GFP/SERK3-mCherry nanoclusters after treatment with 50 μM BRZ (n = 126). (C)
Fluorescence lifetime distribution of BRI1-GFP-SERK3/BAK1-mCherry nanoclusters pretreated with 50 μM
BRZ followed by application of 1 μM BL for 1 hour (n = 95). The data were obtained from 3 independent series
BRZ pre-treated BRI1-GFP/SERK3/BAK1-mCherry expressing roots the IPS increased from
about 8% to 13% [
Using the same analysis method for our SSO FRET-FLIM results, we determined an IPS of
15% in ligand-depleted roots (Table 4). This percentage is about two-fold higher compared to
the 8% found previously using conventional FRET-FLIM [
]. The increase of IPS in SSO
FRET-FLIM is possibly due to imaging a larger area of PM located receptors oriented
perpendicular to the focal plane. Surprisingly, ligand application resulted in a reduction in the
percentage of IPS to about 10% (Table 4) instead of the increase noted earlier [
]. A plausible
explanation for this discrepancy can be found in movies of root cells recorded either in VAEM
or in SSO mode. During active signalling, occasionally nanoclusters were seen to be
endocytosed and thus rapidly disappearing from the PM (S2 Movie). Given the fact that there is no
overall change in the number of BRI1 or SERK3/BAK1 containing nanoclusters in the PM
after BL ligand application, we conclude that the reduction in IPS is mainly due to endocytosis
of nanoclusters containing interacting BRI1 and SERK3/BAK1 receptors.
We evaluated a series of individual nanoclusters to determine whether the fluorescence
lifetimes of individual bright nanoclusters changed during active BR signalling, possibly reflecting
a change in the composition of interacting and non-interacting BRI1 and SERK3/BAK1 pairs
within a cluster (Fig 5). No significant changes were observed upon ligand application,
suggesting that the composition per nanocluster remains unchanged. There also does not seem to
be any correlation between the intensity of the nanoclusters and the reduction of donor
We visualised the distribution of BRI1, SERK3/BAK1 and BIR3 in the PM of Arabidopsis root
cells using variable-angle epifluorescence microscopy (VAEM) and showed that these
receptors are forming nanoclusters. In addition, these nanoclusters contain hetero-oligomers of
BRI1-GFP and SERK3/BAK1-mCherry receptors as demonstrated using SSO FRET-FLIM.
9 / 19
Cluster formation of receptors in general was reported earlier. In plants, protein clusters are
found for RbohH and PIN proteins [
]. In mammalian cells, the Epidermal Growth
Factor Receptor (EGFR) is present in oligomeric clusters in the membrane . These clusters
consist of approximately two receptors in an unstimulated situation, which is in the same
range we determined for BRI1 and its coreceptor SERK3/BAK1. For EGFR, the nanocluster
distribution is thought to be coupled to the biological activity of the receptor [
doubling in the number of receptors per cluster is observed upon ligand binding (from ~2 to ~4
receptors per nanocluster; [
]). In contrast, we observed no change in nanocluster
distribution for BRI1 or SERK3/BAK1 upon ligand application or depletion of endogenous ligand.
Our observation of nanoclusters in plant cells are supported by the work of Jarsch and
], who identified membrane structures in the form of microdomains for 20 different
plasma membrane localised proteins predominantly belonging to the Remorin protein family.
We therefore suggest that the formation of microdomains or nanoclusters is a general feature
of plant transmembrane receptors.
Receptors that are arranged in nanoclusters are considered to be part of larger
arrangements of signalling proteins [
], where the stoichiometry between different components
can be altered without affecting the arrangement [
]. We made similar observations after
we compared the PM of two BRI1-GFP lines that differed about 3-fold in receptor density
while retaining a similar nanocluster density. Changing the receptor stoichiometry within
confinements of nanoclusters could be a mechanism of plant cells to regulate signalling
output, especially in the situation of SERK3/BAK1, which is part of different signalling
complexes in the same cell [
Our FRAP analysis revealed that BRI1-GFP is largely immobile showing a mobile fraction
of only 28 ± 2% with a diffusion coefficient of approximately (3.0 ± 1.0) 10−3 μm2 s-1 in
meristematic Arabidopsis root cells. This is conjunction with the work of Wang and coworkers in
which a diffusion coefficient of BRI1-GFP of (8.8 ± 0.6) 10−3 μm2 s-1 was found . As in the
case of EGFR, animal receptor nanoclusters are thought to be confined by the cortical actin
filament network and cholesterol rich domains [
]. However, for BRI1 and SERK3/BAK1,
treatment with Latrunculin B, which is an actin depolymerising agent, did not lead to
significant changes in nanocluster distribution. Lateral diffusion of plant receptors is restricted by
the presence of the plant cell wall [
]. Restricted diffusion of receptor proteins due to physical
barriers such as the underlying cytoskeleton or the cell wall of plant cells could induce
clustering of membrane proteins [
]. The classical model for PM receptor activation assumes
ligand-induced endocytosis which could imply removal of the entire nanocluster from the PM
or a change in their stoichiometry.
Given the observed restrictions in lateral movement shown by our FRAP analysis, it is
unlikely that BRI1 nanoclusters are formed after arrival of the proteins at the PM. At present,
it is unknown where plant PM receptor nanocluster assembly takes place. One way in which
this could be accomplished is via the preformation of higher order signalling complexes,
inserted in their respective position in the membrane as a fully assembled unit. Preformation
of complexes has been observed for BRI1 and SERK3/BAK1 [
], corroborating this idea.
Future research is needed to address the question whether these proteins are indeed inserted
in the membrane together, or whether minor mobility within the confinements of the clusters
is sufficient to form receptor complexes after insertion in the membrane.
VAEM allows for selective illumination of a thin surface layer and can be used to visualise
the plant PM and intracellular membrane compartments in close proximity to the PM, but
does not provide spatial information of BRI1-GFP and SERK3/BAK1-mCherry complexes
within the nanoclusters. We therefore developed SSO-FRET FLIM as a novel method to show
that BRI1-GFP and SERK3/BAK1-mCherry are present in nanoclusters as hetero-oligomers.
10 / 19
We observed a reduction of fluorescence lifetime when comparing BL treated BRI1-GFP/
SERK3/BAK1-mCherry Arabidopsis roots with donor only samples. Furthermore, SSO-FRET
FLIM showed no change of the interaction distribution of BRI1-GFP and
SERK3/BAK1-mCherry in nanoclusters in the absence or presence of ligand (see Fig 5). The numerical
evaluation of IPS significantly reduced fluorescence lifetimes revealing a reduction from 15% to 10%
in the number of hetero-oligomers of BRI1-GFP and SERK3/BAK1-mCherry upon signal
activation. This reduction in the percentage of IPS is in contradiction with the earlier observed
increase of IPS using conventional FRET-FLIM [
]. During active signalling, occasionally
nanoclusters were seen to be endocytosed and thus rapidly disappearing from the PM (S2
Movie), similar to what has been observed by Wang et al. 2015 . Using conventional
FRET-FLIM, the endocytosed complexes of BRI1-SERK3/BAK1 nanoclusters remain in the
focal imaging plane and are taken into account in the IPS analysis. We further conclude that
the observed nanoclusters containing interacting oligomers of BRI1 and SERK3/BAK1
represent preformed receptor complexes that upon ligand application do not change in composition
and are subsequently endocytosed after signal activation.
We observed nanoclusters with varying fluorescence lifetimes indicating either changes in
microenvironment of BRI1-GFP or interaction between BRI1 and SERK3/BAK1. We propose
that the varying fluorescence lifetimes in the observed nanoclusters are a result of different
receptor stoichiometries. When considering the scale on which proteins interact and the
observed cluster size, it has to be noted that the observed nanoclusters most likely contain
multiple receptor pairs and other proteins. It remains unknown if the decrease in IPS is due to a
conformational change of the receptor complex or due to endocytosis of active signalling
Further investigation of underlying organelles could provide interesting answers into the
dynamics of BR signal activation within the complex confinements of the plant PM and cell
wall. Plant receptor distribution at the PM could be a result of intrinsic properties of the PM
mediated by actin [
] or microtubule structures. Such clustering might be influenced by
endocytosis or incorporation rates of proteins in the PM through contact with the underlying
cortical ER. The existence of nanoclusters can play a gate way function in regulating signalling
responses, limiting effects of minor small increases in ligand availability and establishing a
threshold concentration for signal activation [
]. This way of signalling might be less
susceptible to variations in internal and external influences thereby increasing signalling fidelity.
In accordance to this trend exogenous application of BRs results in a hyperbolic root growth
response curve .
The receptor clusters observed with VAEM showed a diameter of 300±500 nm, which
represents an upper limit of the actual receptor cluster size, as the expected cluster size of the
receptors is probably below the diffraction limit. To further investigate the clustering of BRI1
and SERK3/BAK1, optical super-resolution techniques, namely photo-activated localization
microscopy (PALM) [
], should be employed in combination with VAEM.
It is known that SERK3/BAK1 plays an important role in multiple pathways such as BR
signalling and innate immunity (Flagellin) [
]. We hypothesise that each individual
nanocluster represents a signalling entity composed of preassembled receptor pairs. Similar
clustering has been proposed for the CLAVATA receptor family . The main advantage of
preassembly is that the signalling fidelity and response time is improved in comparison to a
scenario in which the receptors are homogeneously distributed across the PM, which would
require the assembly of signalling units post receptor activation. The nanoclusters would
provide spatial separation of receptor pairs and could explain how one protein can play an
essential role in multiple signalling pathways.
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Materials & Methods
Growth conditions and plant lines
Arabidopsis thaliana plants of ecotype Columbia (Col-0) were used as wild type. Seeds were
surface sterilized and germinated on ½ Murashige and Skoog medium (Duchefa)
supplemented with 1% sucrose (Sigma) and 1% Daishin agar (Duchefa). Plants were grown at 22ÊC
under fluorescent light, with 16 h light/8h dark photoperiods. Col-0 plants expressing BRI1
(AT4G39400) fused to GFP under its native promoter, here referred to as BRI1-GFP line 1,
were provided by N. Geldner [
]. BRI1-GFP line 2 is a BRI1-GFP line overexpressing the
transgene roughly three-fold, and was provided by J. Chory [
]. Col-0 plants expressing
SERK3/BAK1-mCherry or SERK3/BAK1-GFP under control of its native promoter were
generated as previously described [
]. BRI1-GFP line 2 was crossed with the SERK3-mCherry line
to create a plant harbouring both transgenes. Serk1 serk3 mutant plants harbouring BRI1-GFP
was produced by crossing BRI1-GFP line 2 with the double mutant serk1-3 (GABI-KAT line
448E10) serk3-2 (SALK_116202) resulting in the serk1serk3 BRI1-GFP line. The det2 seeds
were obtained from the Arabidopsis seed stock centre and crossed with BRI1-GFP line 2. Col-0
plants containing the transgenes Wave6-mCherry and Wave18-RFP were provided by N.
], LT16B-GFP was provided by C. ten Hove [
], VHAa1-mRFP [
F2B-mRFP and ARA6/ Rab F1-mRFP were provided by K. Schumacher, Heidelberg.
KNOLLE-GFP was used as a positive control for the FRAP experiments based on the data of [
The pBIR3:BIR3-GFP line was constructed by Walter van Dongen (Biochemistry, WU).
Hormone and inhibitor treatments
For hormone treatment, six-day-old seedlings were incubated in 1 mL ½ Murashige and
Skoog medium, supplemented with 1% sucrose and 1 μM 24-epi-brassinolide (BL, Sigma). For
brassinazole treatment, seeds were first germinated and grown for four days on ½ Murashige
and Skoog medium, supplemented with 1% sucrose and 1% Daishin agar. After four days the
seedlings were transferred to plates complemented with 5 μM brassinazole (BRZ, TCI Europe)
and grown on these plates for an additional two days.
For FRET-FLIM experiments, 5 day old seedlings were used. The seedlings were transferred
to 1 ml ½ MS medium containing 5 μM BRZ 3 days post germination for an additional two
days. BR signalling was induced by incubation of seedlings with 1 μM BL for 1 hour.
Variable-angle epifluorescence microscopy (VAEM)
In total-internal reflection fluorescence microscopy (TIRFM), the laser light is focussed into
the rim of the backfocal plane of a microscope objective with high numerical aperture. As a
result, the strong inclination of the passing laser light leads to the phenomena of total internal
reflection at the interface between the cover slip and the sample medium due to the lower
refractive index of the sample medium. Even though the light does not pass the interface, an
evanescent wave is generated which decays exponentially within a few hundred nanometers
]. In animal cells, TIRFM has been used to visualize proteins located in the PM [53±55]. In
plant cells, however, utilisation of TIRFM is hampered due to presence of the plant cell wall
] whose thickness is comparable to the effective excitation depth of the evanescent wave.
An alternative to TIRFM is VAEM in which the laser light is focussed closer to the centre of
the backfocal plane such that not all light is reflected at the interface between the cover glass
and water (buffer); instead, a thin band of illuminating light penetrates the sample allowing for
greater penetration depth and yielding a high signal to noise ratio for visualizing biological
processes at or near the PM of living cells. By varying the position of the focus in the backfocal
12 / 19
plane, we can adjust the depth at which the sample is illuminated. Due to the curvature of the
plant root, only a narrow region of the outer PM of the epidermal root cells in close proximity
to the cover slip was visualized.
Live root imaging was performed on a home-build microscopy setup described in [
setup is equipped with a 100x/1.49NA TIRF objective (Nikon) and an Ixon Ultra 897 emCCD
camera with 512 x 512 pixel (Andor) for imaging. The total magnification of the microscope is
125 x corresponding to a pixel size of 130 nm. Data was recorded using micromanager [
GFP was excited with a 473 nm laser (laser power in front of the polychroic mirror 0.98 mW)
and fluorescence emission was detected from 480±550 nm. mCherry was excited with a 561
nm laser (laser power set at 0.35 mW) and fluorescence emission was detected from 570±625
nm. Movies containing 250 or 500 frames were recorded every 100 msec, with an exposure
time of 100 msec. ImageJ and FIJI were used for data processing (FIJI software, IMAGEJA,
51.45j, Max Planck Society; [
]. For all images of PM localized proteins, a background
subtraction (rolling ball radius = 50.0 pixels) was performed. For the analysis of nanoclusters, a
Gaussian blur filter of 2 μm (σ) was applied to the fluorescence intensity image. The resulting
binary image was thresholded at 80 a.u. and subsequently analysed using the plugin for particle
analysis of Image J. For quantification of the number of receptors per nanocluster, we defined
areas of 5x5 pixels as region of interest and analysed bleaching decay curves by plotting z-axis
Confocal microscopy and FRAP experiments
Roots of Arabidopsis seedlings expressing BRI1-GFP line 1, SERK3/BAK1-GFP or
KNOLLE-GFP were imaged with a Zeiss CONFOCOR2/LSM510 confocal microscope equipped
with a 40x/1.2NA water objective, and an argon laser (output of 6.1 A). For FRAP analysis, the
PM was scanned at 488 nm excitation with a laser power of 5% and 9% for BRI1-GFP and
SERK3/BAK1-GFP respectively. The fluorescence intensity of GFP was detected with a
bandpass filter at 505±550 nm. The image size was set to 512x512 pixels and four scans were
averaged for each picture. After 3 scans, a high intensity bleach pulse (50 iterations at 50% laser
power) at 488 nm was applied over the selected area. Subsequently, the fluorescence recovery
was followed for 499 s and 436 s for BRI1-GFP and SERK3/BAK1-GFP respectively.
FRAP data analysis was performed by subtracting the background signal from the raw data
followed by a normalisation of the fluorescence intensity between zero and one (Fig 2 and S8
Fig). The normalised data were plotted using MS Excel, which was also used for curve fitting.
Selective-surface observation FRET-FLIM
FoÈrster resonance energy transfer (FRET) is a process in which excitation energy is transferred
from a donor fluorophore to an acceptor chromophore through nonradiative dipole±dipole
]. This process can only occur if fluorescent donor and acceptor molecules are at
very close proximity. The energy transfer rate is proportional to the inverse 6th power of the
distance R between donor and acceptor, which makes this method extremely sensitive for
distances at protein level dimensions (<10 nm). FRET determined using fluorescence lifetime
imaging microscopy (FLIM) is independent of protein concentration, but very sensitive for
the local microenvironment of the fluorophores. In FRET-FLIM, the fluorescence lifetime of
the donor molecule is reduced in presence of an acceptor molecule nearby since energy
transfer to the acceptor will introduce an additional relaxation path from the excited to the ground
state of the donor. The FRET efficiency (E) is given by E = (1 - τDA/ τD) where τDA is the
fluorescence lifetime of the donor in the presence of acceptor and τD is the fluorescence lifetime of
the donor alone.
13 / 19
Selective-surface observation (SSO)-FRET-FLIM was performed on a Leica TCS SP5 X
equipped with a 63X/1.2NA water immersion objective. In SSO-FRET-FLIM, the confocal
spot is positioned perpendicular to the PM of the root epidermal cells. In this configuration, it
was possible to observe signals from the PM whilst largely omitting signals from underlying
organelles such as the cortical ER. A 40 MHz tunable supercontinuum laser was used to excite
GFP and mCherry at 488 nm and 587 nm, respectively. Fluorescence emission was detected
using an internal Hybrid (HyD) detector with 100 ps time resolution and collected in a spectral
window of 495±550 nm for GFP and 500±540 nm for mCherry provided by an
Acousto-Optical Beam Splitter. The signal output from the HyD was coupled to an external time-correlated
single photon counting module (Becker&Hickl) for acquiring FLIM data. Typical images had
128 x 128 pixels (pixel size ± 300 nm), and 256 time channels per pixel with an acquisition
time of 90±120 seconds per image.
From the time resolved fluorescence intensity images, the fluorescence decay curves were
calculated for each pixel and fitted with either a mono- or double-exponential decay model
using the SPCImage v5.0 software (Becker & Hickl). Fitting was performed without fixing any
parameters. FRET-FLIM analysis provided fluorescence intensity as well as false-colored
fluorescence lifetime images. The raw data was subjected to the following criteria to analyze and
omit false positive negatives in the fluorescence lifetime scoring: minimum photon count per
pixel of 1200 photons, goodness of fit (χ2<2) and fluorescence lifetime range of 1500±2500 ps.
For data analysis, we set pixel binning at 1 to have sufficient number of photons per pixel
required for accurate fluorescence lifetime analysis.
In addition, a numerical evaluation of the observed fluorescence lifetime values and
interaction pixels (IPS) was determined by exporting the fitted data from SPCImage into a Phyton
written script, which calculates the number of pixels that adheres to the above-mentioned
fitting criteria. The fraction of IPS was established by applying a fluorescence lifetime threshold
as a percentage of the total number of pixels [
]. This threshold, corresponding to a FRET
efficiency of about 13%, was used and only pixels with fluorescence lifetimes below this
interaction threshold were collected as IPS.
Sequence data from this article can be found in the Arabidopsis Genome Initiative or EMBL/
GenBank data libraries under accession numbers: BRI1 (AT4G39400), SERK3/BAK1
(AT4G22430), WAVE6/NIP1;1 (AT4G19030), WAVE18/Got1p homolog (AT3G03180),
ARA6/RABF1 (AT3G54840), ARA7/RABF2B (AT4G19640), VHA-A1 (AT2G28520) LTI6B/
RCI2B (AT3G05890), DET2 (AT2G38050) KNOLLE (AT1G08560) and BIR3 (AT1G27190).
S1 Fig. Visualization of A. thaliana membrane compartments with VAEM. Live-cell VAEM
imaging performed on 6 day old Arabidopsis seedling roots expressing fluorescent markers for
different membrane compartments. (A) PM localized LT16B-GFP, (B) ER localized
WAVE6mCherry, (C) Golgi localized WAVE18-mRFP, (D) TGN localized VHAa1-mRFP, (E) EE/LE
localized ARA7-mRFP and (F) LE localized ARA6/Rab F1-mRFP. The exposure time for all
images was 100 msec except for B in which 40 sequential images of 100 msec each were
combined. Scale bars represent 10 μm.
S2 Fig. ImageJ processing for nanocluster analysis. VAEM images of cluster forming
BRI1-GFP (A) and the PM marker LT16B-GFP (B) were analysed. The respective original
14 / 19
image was processed by application of a Gaussian blur filter with of 2 μm (σ) followed by
subtraction of the blurred image from the original image. Subsequently, a threshold of 80 a.u. and
a black-white inversion was applied. (A) Nanocluster analysis was performed only in regions
within each cell (here exemplified by an ROI marked in green). For further details on the
cluster analysis please see the materials and methods section. (B) LT16B-GFP does not show the
formation of nanoclusters. In fact, only single, non-connected pixels appear in the final image.
S3 Fig. VAEM fluorescence decay curves. Shown are two representative decay curves of
SERK/BAK1-mCherry clusters, and of BRI1-GFP line 1 clusters. As can be seen, the decay
curves of SERK3-mCherry sometimes portrays almost a single molecule behaviour, but at
other times, more receptors are present in a cluster. For both receptors, discreet decrease in
fluorescence is observed, indicating that the number of receptors in the cluster must be
S4 Fig. PM Receptor distribution of PIN2-GFP, BIR3-GFP, BRI1-GFP line 1 and Col0
Arabidopsis thaliana in live epidermal root cells using VAEM. Live-cell VAEM imaging
performed on 6 day old Arabidopsis seedling roots expressing (A) PM localized PIN2-GFP, (B)
PM localized BIR3-GFP, (C) PM localized BRI1-GFP line 1, (D) Arabidopsis thaliana ecotype
S5 Fig. VAEM images of BRZ treated BRI1-GFP Line 2 epidermal root cells upon BL
stimulation. Live-cell VAEM imaging performed on 6 day old Arabidopsis seedling roots
expressing BRI1-GFP (A) PM localized BRI1-GFP, (B) PM localized BRI1-GFP treated with 5 μM
brassinazole for 3 days, (C) PM localized BRI1-GFP treated with 5 μM brassinazole for 3 days
and subsequently with 1 μM 24-epi-brassinolide for 1 h, (D) PM distribution of BRI1-GFP in
the det2 BR biosynthesis mutant.
S6 Fig. Example of typical FRAP experiment. In Fig S6 A and B, images of BRI1-GFP at
different scanning iterations are shown. (A) shows images that undergo only scan bleaching
whereas (B) contains features of FRAP region convoluted with scan bleaching. (C) Plots of the
fluorescence intensity versus number of scans (top orange line: ROI in (A), middle grey line:
ROI in (B), blue line: background intensity). (D) Normalised FRAP curve, corrected for scan
bleaching. As shown, BRI1-GFP receptors are largely immobile. Furthermore, scan bleaching
strongly interferes with the interpretation of the dynamics of the recovery.
S7 Fig. Gaussian fits of receptor molecules in PM surrounding the anticlinal cell wall
before and after photobleaching. The line represents the fit of a Gaussian distribution on the
fluorescence intensity data across the anticlinal cell wall. Distance 0 is the midpoint of two
adjacent plasma membranes in a confocal image. The cytoplasm is situated between 1±0,5 μm
on either side of the midpoint. (A) Fluorescence intensity of BRI1-GFP at the bleached area
(left) compared to the intensity at a non-bleached area of the PM (right). After 400 seconds,
the fluorescence intensity at the non-bleached area (right panel) was reduced significantly due
to scan bleaching. (B) Same as A, except now for SERK3/BAK1-GFP. n = 5 different roots; 20
fits per image (n 100 data points).
15 / 19
S8 Fig. FRAP data analysis.
S1 Movie. Dynamics of ER marker (VMA21-GFP).
S2 Movie. Movie of BRI1-GFP in presence of BL.
S3 Movie. Movie of BRI1-GFP in presence of BL.
S4 Movie. Movie of BRI1-GFP in absence of BL.
S5 Movie. Movie of SERK3-GFP.
S6 Movie. Movie of SERK3-GFP.
We thank Adrie Westphal for valuable discussions on data interpretation. Data and materials
availability: All reagents, chemicals and plant lines are available upon request.
Conceptualization: SCdV JH JWB.
Data curation: SJH DSH.
Formal analysis: SJH DSH JH JWB.
Funding acquisition: SCdV JH JWB.
Investigation: SJH DSH WvE AN.
Methodology: SJH DSH JH JWB.
Project administration: SCdV JH JWB.
Writing ± original draft: SJH DSH MT.
Writing ± review & editing: SCdV JH JWB.
16 / 19
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