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Update on Plasma Membrane Compartmentalization in Signaling
The Nanoscale Organization of thePlasma Membrane and Its
Importance in Signaling:A Proteolipid Perspective1[OPEN]
Yvon Jaillais,a,2 and Thomas Ottb,3
aLaboratoire Reproduction et Développement des Plantes,
Université de Lyon, ENS de Lyon, UCB Lyon 1,CNRS, INRAE, F-69342
Lyon, FrancebCell Biology, Faculty of Biology, Centre for
Integrative Biological Signalling Studies (CIBSS), University
ofFreiburg, 79104 Freiburg, Germany
ORCID IDs: 0000-0003-4923-883X (Y.J.); 0000-0002-4494-9811
(T.O.).
Plasma membranes provide a highly selective environment for a
large number of transmembrane and membrane-associatedproteins.
Whereas lateral movement of proteins in this lipid bilayer is
possible, it is rather limited in turgid and cell
wall-shieldedplant cells. However, membrane-resident signaling
processes occur on subsecond scales that cannot be explained by
simplediffusion models. Accordingly, several receptors and other
membrane-associated proteins are organized and functional
inmembrane nanodomains. Although the general presence of membrane
nanodomains has become widely accepted as fact,fundamental
functional aspects, the roles of individual lipid species and their
interplay with proteins, and aspects ofnanodomain maintenance and
persistence remain poorly understood. Here, we review the current
knowledge ofnanodomain organization and function, with a particular
focus on signaling processes involving proteins, lipids, and
theirinteractions. Furthermore, we propose new and hypothetical
aspects of plant membrane biology that we consider importantfor
future research.
Together with the cell wall, the plasma membraneforms the
frontier of the cell. As such, it acts as aphysical barrier and
allows the generation and main-tenance of chemical gradients
between the outsideand inside of the cell. At the same time, the
plasmamembrane is a critical checkpoint for the perceptionand
integration of extracellular signals prior to signaltransduction in
the cytoplasm. The fluid mosaic modelinitially predicted that
biological membranes are fluids,with the underlying assumption that
their protein andlipid constituents can laterally diffuse in the
plane ofthe membrane without major restrictions (Singer
andNicolson, 1972). According to this view, membrane-embedded
receptors would distribute uniformlythroughout the cell surface and
irrespective of the plasma-membrane proteome. However, the very
opposite seems
to be the case. Unequivocal evidence shows that
theplasmamembrane itself is highly compartmentalized intosubdomains
and that lateral segregation of proteins and
1This work was funded by the German Research Foundation(Deutsche
Forschungsgemeinschaft) under Germany’s ExcellenceStrategy (Centre
for Integrative Biological Signalling Studies;EXC-2189 – Project ID
39093984 to T.O.) and by the FrenchNational Research Agency (Agence
Nationale de la Recherche) caL-IPSO (ANR-18-CE13-0025-02 to Y.J.)
and STAYING-TIGHT (ANR-18-CE13-0016-02 to Y.J.) on lipids and
ERA-NET Coordinating Actionin Plant Sciences SICOPID on receptor
kinase signaling (ANR-17-CAPS-0003-01 to Y.J.).
2Author for contact: [email protected]
author.Y.J. and T.O. wrote the article.[OPEN]Articles can be viewed
without a
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1682 Plant Physiology�, April 2020, Vol. 182, pp. 1682–1696,
www.plantphysiol.org � 2020 American Society of Plant Biologists.
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https://orcid.org/0000-0003-4923-883Xhttps://orcid.org/0000-0003-4923-883Xhttps://orcid.org/0000-0002-4494-9811https://orcid.org/0000-0002-4494-9811https://orcid.org/0000-0003-4923-883Xhttps://orcid.org/0000-0002-4494-9811http://crossmark.crossref.org/dialog/?doi=10.1104/pp.19.01349&domain=pdf&date_stamp=2020-03-30http://dx.doi.org/10.13039/501100001659http://dx.doi.org/10.13039/501100001659http://dx.doi.org/10.13039/501100001665http://dx.doi.org/10.13039/501100001665mailto:[email protected]://www.plantphysiol.org/cgi/doi/10.1104/pp.19.01349
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lipids is a critical facet of cell surface signaling,
modulat-ing signal perception, specificity, and integration.This
view of a compartmentalized plasmamembrane
first arose from biochemical fractionation, which couldseparate
biological membranes in a binary manner be-tween so-called
detergent-resistant (also referred to asdetergent-insoluble) and
detergent-sensitive mem-branes (Brown and Rose, 1992; Mongrand et
al., 2004;Borner et al., 2005; Morel et al., 2006; Laloi et al.,
2007;Lefebvre et al., 2007). However, fluorescent
microscopytechniques with ever increasing resolution power
havelargely replaced biochemical fractionation, as it rapidlybecame
clear that plasma membrane subdomains arenot binary but rather a
part of a large patchwork ofmany subdomains that coexist on various
spatial andtemporal scales. Such a view is supported by a wealthof
data from colocalization analyses using confocal,total internal
reflection fluorescence, and super-resolution microscopy
(Kleine-Vehn et al., 2011; Demiret al., 2013; Jarsch et al., 2014;
Hosy et al., 2015; Bücherlet al., 2017; Martinière et al., 2019;
Platre et al., 2019).Furthermore, the fact that different membrane
constit-uents display varying diffusion patterns within theplane of
the plasma membrane is also sustained bystudies on the dynamics of
protein/lipid lateral diffu-sion using fluorescence recovery after
photobleaching(FRAP), single-molecule imaging (e.g. single
particletracking photoactivated localization microscopy),
andfluctuation correlation spectroscopy (Li et al., 2011,2016b,
Martinière et al., 2012, 2019, Wang et al., 2013,2015; Jarsch et
al., 2014; Hosy et al., 2015; Gronnier et al.,2017; Cui et al.,
2018; McKenna et al., 2019; Platre et al.,2019). Technically,
receptor/scaffold complexes havealso often been studied using
distance-based imagingtechniques such as Förster resonance energy
transfer-fluorescence lifetime imaging (FRET-FLIM). However,a note
of caution has recently been put forward con-cerning the use of
bimolecular-fluorescence comple-mentation, which can artificially
stabilize membraneproteins in membrane contact sites of the
endoplasmicreticulum and the plasma membrane (Tao et al., 2019).In
this update, we review the current evidence for the
coexistence of a patchwork of membrane nanodomainsin the plant
plasma membrane and their functionalimportance and assess the roles
of lipids, the cell wall,and the cytoskeleton in shaping this
diverse plasmamembrane landscape. Finally, we discuss
plausiblescenarios for the functional importance of
proteinnanoclustering in signal transduction.
THE PLASMA MEMBRANE AS A PATCHWORK OFCOEXISTING
FUNCTIONALMEMBRANE NANODOMAINS
Receptor Scaffolding at the Nanoscale
Unequivocal evidence demonstrates that a signifi-cant number of
membrane-resident proteins cluster inhigher-order structures that
have been termed
“membrane nanodomains” or “membrane micro-domains,” for which a
nomenclature has been sug-gested recently (Ott, 2017). Briefly,
nanodomains aresubmicron protein and/or lipid assemblies (;20–300nm
and ,1 mm), whereas microdomains are signifi-cantly larger
assemblies (.1 mm, e.g. perimicrobialmembranes, the Casparian strip
domain, polar do-mains, plasmodesmata [PD], or membrane
contactsites; Ott, 2017). Herein, we will specifically focus
onplasma membrane nanodomains, which have oftenbeen termed “lipid
rafts.”Whereas the lipid raft model was mainly based on
biochemical evidence, recent cell biological approachesrevealed
that a number of proteins distribute hetero-geneously on plant cell
membranes mostly labelingpuncta-like structures (Kleine-Vehn et
al., 2011; Demiret al., 2013; Jarsch et al., 2014; Bücherl et al.,
2017;Martinière et al., 2019; Platre et al., 2019). Over
time,members of theflotillin (FLOT) andplant-specific
remorinprotein families have been described asmarker proteinsfor
thesemembrane nanodomains (Fig. 1; Raffaele et al.,2009; Jarsch and
Ott, 2011; Li et al., 2012; Marín et al.,2012; Perraki et al.,
2012; Hao et al., 2014; Jarsch et al.,2014; Wang et al., 2015;
Bücherl et al., 2017; Gronnieret al., 2017; Liang et al., 2018).
This allowed comparativestudies that revealed the coexistence of
multiple nano-domainswithin the same cell (Jarsch et al., 2014).
FLOTscomprise a small protein family with only two mem-bers in
Arabidopsis (Arabidopsis thaliana; Dan�ek et al.,2016), whereas at
least 16 remorins have been identi-fied with an additional two
legume-specific members(group 2; Fig. 1; Raffaele et al., 2007).
Whereas the precisemolecular function of most of these proteins
remainsunknown,most group 1 remorinswere repeatedly shownto
regulate viral spreading in leaves, possibly by modu-lating PD
conductance (Raffaele et al., 2009; Perrakiet al., 2012, 2018;
Ishikawa et al., 2017). A recent pre-print report described reduced
numbers of PDs in arem1.2 rem1.3 double knockout mutant, indicating
arole of remorins in PD biogenesis (Wei et al., 2019).Additionally,
remorins form higher-order oligomers
leading to filamentous structures in vitro (Bariolaet al., 2004;
Marín et al., 2012; Martinez et al., 2019)and in vivo (Wei et al.,
2019) and this may drive associ-ation of remorins with the plasma
membrane (Legrandet al., 2019). Remorin oligomer formation itself
is mainlymediated by the conserved C-terminal coiled-coil region,a
hallmark feature of these proteins (Raffaele et al., 2009;Marín et
al., 2012; Martinez et al., 2019), and furthersupported by the
intrinsically disordered N-terminal re-gion that harbors the
majority of phosphorylation sites(Marín and Ott, 2012; Marín et
al., 2012).The effect of remorin phosphorylation on their asso-
ciation patterns with membrane nanodomains remainselusive.
However, functionality of remorins in plas-modesmata is hampered in
phospho-mutants (Perrakiet al., 2018). Whereas remorin
phosphorylation wasmostly studied in vitro, several remorins are
ableto associate, or at least colocalize, with a number ofsoluble
or membrane-associated kinases such as
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CALCIUM-DEPENDENT PROTEIN KINASE 3 (CPK3;Perraki et al., 2018),
CPK21 (Demir et al., 2013),AVRPPHB SUSCEPTIBLE1 (PBS1; Albers et
al., 2019),SNF1 RELATEDKINASE (SnRK1; Son et al., 2014),
andreceptor-like kinases (RLKs) such as the
Arabidopsisbrassinosteroid receptor BRASSINOSTEROID INSEN-SITIVE 1
(BRI1) and innate immune receptor FLA-GELLIN SENSING2 (FLS2;
Bücherl et al., 2017), the rice(Oryza sativa) SOMATIC EMBRYOGENEIS
RECEP-TOR KINASE1 (SERK1) and OsBRI1 (Gui et al., 2016),and the
Medicago truncatula RLKs NOD FACTORPERCEPTION (NFP), LYSINMOTIF
KINASE3 (LYK3),and DOES NOTMAKE INFECTIONS2 (DMI2), as wellas their
corresponding homologs in Lotus japonicus(Lefebvre et al., 2010;
Tóth et al., 2012; Liang et al.,2018). Increasing evidence suggests
that remorins andFLOTs play versatile roles in nanodomain
stabilization(Huang et al., 2019a) and receptor recruitment
intothese structures (Haney et al., 2011; Bücherl et al.,
2017;Liang et al., 2018).
In rice, significant progress was made with respectto remorin
functionality and the functional relevanceof receptor recruitment
into nanodomains, whereligand-induced phosphorylation of the
remorinOsREM4.1 results in its dissociation from OsSERK1,which
consequently allows the assembly of thesignaling-competent
OsSERK1/OsBRI1 receptorcomplex (Gui et al., 2016). A different mode
of main-taining an active receptor complex has been proposedin the
legume M. truncatula, where receptor-mediatedligand perception
results in transcriptional activationof the group 2 remorin
SYMREM1, which in turnphysically associates with the entry receptor
LYK3.This interaction results in stabilization and physi-cal
recruitment of LYK3 in a laterally stable andFLOT4-positive
nanodomain. Genetic evidence fur-ther suggests that this process is
required for receptorstabilization at the plasma membrane (Haney et
al.,2011; Liang et al., 2018).
Nanodomain Targeting
Even though the evidence is still slightly scattered, anumber of
studies suggest that receptor nanoclusterscolocalize with one or
the other scaffold belonging tothe FLOT and/or REM protein
families. This impliesthat these proteins may act as organizing
centers, asrecently suggested for remorins (Gui et al., 2016;
Lianget al., 2018; Huang et al., 2019a) and at least for theM.
truncatula FLOT2/4 (Haney and Long, 2010; Haneyet al., 2011; Liang
et al., 2018).
Recruitment of remorins themselves to the plasmamembrane and, in
some cases, also to nanodomains ismediated by a C-terminal
hydrophobic stretch calledthe remorin C-terminal anchor (REM-CA;
Fig. 1). TheREM-CA peptide specifically binds in a
pH-dependentmanner to sterols and phosphoinositides and requiresthe
simultaneous presence of both b-sitosterol andphosphoinositides in
the same nanodomain (Legrandet al., 2019). This demonstrates the
tight link betweenspecific lipid species and nanodomain recruitment
ofproteins.
In addition, remorin association with membranes isfurther
supported by S-acylation of Cys residues, aswell as protein-protein
interactions (Raffaele et al.,2009; Perraki et al., 2012; Konrad et
al., 2014; Gronnieret al., 2017; Legrand et al., 2019). It remains,
however,still an open point of discussion whether
S-acylationrepresents a major and general posttranslational
mod-ification supporting nanodomain targeting of remorinsand other
proteins. Among the .600 acylated proteinsidentified in Arabidopsis
is also the immune receptorFLS2 (Hemsley et al., 2013).Whereas
S-acylation on twoSer residues (S830 and S831) within the
juxtamembranedomain of FLS2 have been mapped, these residues arenot
required for plasma membrane localization of thereceptor per se
(Hurst et al., 2019). However, a recentpreprint suggests that they
may contribute to the lo-calization of FLS2 in nanodomains (Chen et
al., 2019).
Figure 1. Remorin proteins and theirtargeting to plasma membrane
nano-domains. Remorins have anN-terminalintrinsically disordered
region, a cen-tral coiled-coil domain, and a REM-CA.The
intrinsically disordered region isregulated by posttranslational
modifi-cation, such as phosphorylation, andpossibly modulates
interaction withmany different proteins. The coiled-coil domain is
an oligomerization do-main and mainly contributes toremorin trimer
formation and interac-tions with other proteins. REM-CA isrequired
for both plasma membraneand nanodomain targeting via interac-tion
with inner leaflet lipids such assterols and PI4P.
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This study also claims that S-acylation might be ahallmark of
receptor targeting into nanodomains, as aposttranslational
modification-dependent recruitmentto FLOT1-labeled nanodomains was
also observed forthe receptors CERK1 and P2K1 (DORN1), which
me-diate perception of chitin and extracellular ATP, re-spectively
(Chen et al., 2019). However, these resultswere obtained using
transient expression in proto-plasts, in which the formation of
nanodomains isheavily impacted due to the absence of a cell wall
(seebelow). Furthermore, nanodomains were visualizedusing
deconvolution as an imaging postprocessingmethod, rather than
microscopy techniques that canactually resolve diffraction-limited
structures. There-fore, these results should be taken with caution
untilthey have been verified in situ. In addition, Hurst
andcoworkers found that FLS2mutants, in which C830 andC831 were
substituted by serines, were fully functional,arguing that
S-acylation at these sites is dispensable forFLS2 signaling
function. Receptor tagging and expres-sion levels are critical for
FLS2 function and thereforeneed to be considered in functional
studies (Hurst et al.,2018). Similar concerns have been raised for
the FLS2coreceptor BAK1 (Ntoukakis et al., 2011).
IMPORTANCE OF LIPIDS IN PLASMA MEMBRANELATERAL SEGREGATION
Asymmetric Localization of Lipids in the Two Leaflets ofthe
Plasma Membrane
Like proteins, lipids are not uniformly localized inthe plasma
membrane. In animal cells, they display a
strong asymmetry between the outer and inner mem-brane leaflets,
which face the cell wall and the cytosol,respectively (Fig. 2). The
outer membrane leaflet isrich in glycosphingolipids and sterols, as
well as thephospholipid phosphatidylcholine. In plants,
lipidasymmetry between the two membrane leaflets hasnot been
extensively addressed experimentally, butwas proposed to be largely
similar to animal cells(Gronnier et al., 2018), with the main plant
sphingo-lipids, glycosylinositol phosphorylceramides (GIPCs;Cacas
et al., 2016), being enriched in the outer mem-brane leaflet (Fig.
2). By contrast, the inner leaflet isenriched in phospholipids,
notably phosphatidyletha-nolamine and the minor anionic
phospholipids, whichinclude phosphatidic acid, phosphatidyl-Ser
(PS),phosphatidylinositol (PI) and its phosphorylated de-rivatives
phosphoinositides (Colin and Jaillais, 2019).These anionic lipids
confer a strong electronegativeproperty to the inner surface of the
plasma membrane,which drives the identity of the plasma membrane
andis crucial for the recruitment of many soluble or lipid-anchored
proteins to this compartment (Fig. 2; Simonet al., 2016; Noack and
Jaillais, 2017; Platre et al., 2018).
Lipid Order in Plasma Membrane Compartmentalization
In addition to lipid segregation between the outerand inner
plasma membrane leaflets, there is also lipidsegregation laterally
within each leaflet. The lipid rafthypothesis postulates that
lipids in biological mem-branes may be in a liquid-disordered or
liquid-orderedphase (Kusumi et al., 2012). The liquid-ordered phase
isenriched in sterols and glycosphingolipids, which
Figure 2. Schematic representation ofthe lipid distribution
within a plantplasma membrane. Note the asym-metric repartition of
lipids across thebilayer as well as lateral segregation oflipids in
both inner and outer mem-brane leaflets (Gronnier et al.,
2018).PIP, phosphoinositide; GluCer, gluco-sylceramide; VLCFA, very
long chainfatty acid; PA, phosphatidic acid.
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largely corresponds to the lipid composition of theouter
leaflets. Sphingolipids, such as GIPC, interactwith phytosterols to
increase lipid order (Fig. 2; Cacaset al., 2016).
Evidence for different ordered phases at the plantplasma
membrane recently emerged when using di-4-ANEPPDHQ (ANEPP), a probe
sensitive to the lipidorder (Roche et al., 2008; Frescatada-Rosa et
al., 2014;Zhao et al., 2015a, 2015b; Gerbeau-Pissot et al.,
2016;Gronnier et al., 2017; Grosjean et al., 2018; Huang et
al.,2019a; Laurent et al., 2019; Pan et al., 2019). Its
fluo-rescence emission spectrum is blue-shifted in liquid-ordered
compared with liquid-disordered phases (Jinet al., 2005, 2006).
Initially validated in vitro in modelmembranes, this dye is also
amenable to live imaging,as it is easy to apply and can detect both
the liquid-ordered and liquid-disordered lipid phases in
livingcells (Gerbeau-Pissot et al., 2016). ANEPP stainingin vivo
shows heterogeneous labeling of the plasmamembrane (Gronnier et
al., 2017; Pan et al., 2019). It issensitive to
methyl-b-cyclodextrin (mßcd), a sterol-depleting agent, which
decreases the ANEPP-stainedliquid-ordered phase at the expense of
the liquid-disordered phase (Huang et al., 2019a). Similarly, a
re-cent preprint suggested that the amount of staining ofthe
liquid-ordered phases is reduced in the sterol bio-synthesis mutant
fackel-J79 (fk-J79; Pan et al., 2019).
Together, these data support the notion that sterolsare required
for the formation of liquid-ordered lipidphases in planta. However,
these approaches also havelimitations, and conclusions obtained
with eitherANEPP or mßcd should be taken with caution. Indeed,ANEPP
is a membrane-intercalating dye and maytherefore impact membrane
properties. In addition,mßcd has amassive impact onmembrane
structure andintegrity, and protein-lipid/lipid-lipid
interactions.
Roles of Lipids in Protein Targeting in Nanodomains
Recent data imply that liquid-ordered phases arerelevant for
protein localization, since the ANEPP-stained liquid-ordered phases
colocalize with the Sola-num tuberosum group 1 remorin StREM1.3
(Gronnieret al., 2017). Interestingly, the localization of
StREM1.3in nanodomains is sensitive to fenpropimorph, an in-hibitor
that affects the plasma membrane sterol com-position but not the
total amount of sterols (Gronnieret al., 2017). Below, wewill use
StREM1.3 as an exampleof the role of protein/lipid interactions in
nanodomainlocalization, as this is to date the best studied case
inplants (Fig. 1). Although indirect, these data are con-sistent
with the notion that sterols participate in theformation of
liquid-orderedmembrane domains, whichthemselves are required for
localization of proteins,including remorins (Gronnier et al., 2017;
Legrand et al.,2019). However, as mentioned above, sterols
andsphingolipids mainly accumulate in the outer mem-brane leaflet,
whereas remorins are inserted into thecytosolic leaflet (Cacas et
al., 2016; Gronnier et al., 2017).
This raises two questions: (1) Are there any lipids di-rectly
involved in remorin nanodomain localization inthe cytosolic
leaflet? And (2) if yes, how do the liquid-ordered domains formed
in the outer leaflet influencethe nanoscale organization of the
plasma membrane atthe inner leaflet?
The first question is partially understood, at least
forStREM1.3. Indeed, biophysical and modeling approachesshowed that
StREM1.3 is recruited to plasma mem-brane nanodomains via its
C-terminal anchor and thatit directly binds to phosphoinositides
(Fig. 1; Gronnieret al., 2017). Accordingly, immuno-electron
microscopyon purified membranes showed that the phosphoino-sitide
phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) localizes to
nanodomains;40 nm in size (Furt et al.,2010). Furthermore,
inducible genetic perturbation in-dicates that
phosphatidylinositol-4-phosphate (PI4P)is required for StREM1.3
plasma membrane localiza-tion (Gronnier et al., 2017). Whether the
liquid-orderedphase induced by sterols and GIPCs at the outer
leafletmodulates the formation of nanodomains inside the
cellremains unknown. However, studies in animal cellssuggest
transbilayer coupling between outer- and inner-leaflet lipids via
very long-chain fatty acids (Raghupathyet al., 2015). Indeed, GIPCs
have very long-chain fattyacids, which would perfectly fit such a
role in trans-bilayer coupling (Fig. 2).
From the cytosolic side, the most prominent phos-pholipid for
transbilayer coupling is PS, which containsvery long-chain fatty
acids of 20–24 carbons in plants(Mamode Cassim et al., 2019).
Single-molecule super-resolution imaging of a PS biosensor suggests
that, in-deed, PS accumulates in nanodomains ;50–70 nm insize in
the inner plasma membrane leaflet (Fig. 2; Platreet al., 2019).
However, there are still many unansweredquestions concerning these
PS-containing domains, as itis unclear whether (1) they also
contain phosphoinosi-tides or sterols, (2) their formation depends
on sterolsand/or GIPCs, (3) there is a coupling between
liquid-ordered membrane domains in the outer leaflet
andPS-containing domains in the inner leaflet, and (4)remorins
themselves depend on PS for localization innanodomains. In vitro
analyses detected no interactionbetween PS and StREM1.3 (Legrand et
al., 2019), butthis does not exclude that PS could be indirectly
in-volved in StREM1.3 localization in vitro and in vivo.Indeed, PS
is required to stabilize RHO-OF-PLANTS6(ROP6) in nanodomains in
root epidermis (Platre et al.,2019). A recent preprint proposes
that ROP6 nano-clustering in the leaf epidermis also depends on
sterols(Pan et al., 2019). This suggests a combined action of PSand
sterols for Rho GTPase clustering, although to befully validated,
it needs to be investigated further in thesame developmental
context.
Protein Feedback on the Lateral Segregation of Lipids
Lipid nano-patterning at the plasma membrane ap-pears critical
for the localization of proteins. However,
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the experimental evidence for the presence of lipids indifferent
nanodomains is comparatively scarce and thefull extent of lipid
segregation in nanodomains is notfully appreciated. Although lipids
alone may clusterinto distinct domains in vitro depending on the
com-position, it is also important to note that proteins
highlyinfluence such lateral sorting of lipids. For
example,remorins themselves affect the local membrane order(Huang
et al., 2019a; Legrand et al., 2019).The underlying molecular basis
is not fully under-
stood, but it is likely that remorin oligomerization isimportant
to remodelmembrane properties and perhapsalso induce local
phosphoinositide clustering (Gronnieret al., 2018; Legrand et al.,
2019; Martinez et al., 2019).Indeed, if each C-terminal anchor of
StREM1.3 bindsPI4P, and considering that StREM1.3 is present as
atrimer, this could induce local clustering of PI4P(Legrand et al.,
2019). Similar mechanisms were pro-posed for the Sec14-Nodulin
protein AtSfh1, which iscritical for the focal accumulation of
PI(4,5)P2 at the tip ofgrowing root hairs (Ghosh et al., 2015).
Local PI(4,5)P2accumulation requires both PI(4,5)P2 binding by a
pol-ycationic peptide at the C terminus of AtSfh1 and
thehomo-oligomerization activities of its nodulin domain.In the
case of AtSfh1, this could act as part of a self-organizing system,
since AtSfh1 stimulates PI(4,5)P2synthesis, either directly or by
inducing PI4P production(Ghosh et al., 2015; Kf de Campos and
Schaaf, 2017).
PLASMA MEMBRANE COMPARTMENTALIZATION:REVISITING THE ANCHORED
PICKETFENCE CONCEPT
One of the most unifying models to explain the regu-lation of
plasmamembrane organization is the anchored
picket fence model (Kusumi et al., 2012). In this model,the
cortical cytoskeleton, made of actin microfilaments,defines
membrane domains ;40–300 nm in size byacting as a fence that
restricts lateral diffusion of pro-teins and lipids within these
domains (Fig. 3). As such,the cortical actin network is seen as a
“membraneskeleton” that is key for plasma membrane organiza-tion
(Fig. 3). Because lipids in both inner and outerleaflets are fenced
by this membrane skeleton, themodel additionally postulates the
existence of trans-membrane proteins that act as pickets. These
pickets arethen anchored either by the cytoskeleton in the
cytosolor the extracellular matrix (Fig. 3; Kusumi et al.,
2012).The diffusion of lipids, even on the outer leaflet, isthereby
physically hindered by these anchored picketsand lipids may also
transiently interact with them. Al-though the picket fence model is
useful when thinkingabout the organization of the plant plasma
membrane,it fails to include some plant-specific features that
mayhave a profound impact on the organization of the
cellsurface.
The Role of the Cytoskeleton inNanodomain Organization
In addition to cortical actin, plants also have
corticalmicrotubules, and it is possible that microtubules
couldadditionally act as a membrane skeleton (Fig. 3). In-deed, a
number of membrane nanodomain proteinsalign with or directly bind
to intact actin or microtubulefilaments (Homann et al., 2007;
Jarsch et al., 2014; Guiet al., 2015; Szymanski et al., 2015; Liang
et al., 2018). Inaddition, a recent preprint suggested that
stimulus-dependent microtubule fragmentation by a memberof theM.
truncatulaDEVELOPMENTALLYREGULATEDPLASMAMEMBRANEPOLYPEPTIDE (DREPP)
family
Figure 3. The picket fence model inanimals and possible revision
of themodel in plants. A, Picket fence modelfor plasma membrane
compartmental-ization in animal cells, with a prom-inent influence
of cortical actin as amembrane skeleton (Kusumi et al.,2012). B,
The picket fence model inplants is likely to involve both
intra-(actin and microtubules) and extracel-lular (cell wall)
skeletons (i.e. fences),which may explain the limited diffu-sion of
plasma membrane proteins.Lines with arrowheads represent ex-amples
of diffusion trajectories of therespective plasma membrane
proteins.
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occurswithin SYMREM1/FLOT4-containingmembranenanodomains (Su et
al., 2019).
In several cases, chemical disruption of these fila-ments was
shown to cause a reduction or loss ofnanodomain localization even
though the used markerproteins themselves mostly resided at the
plasmamembrane (e.g. Raffaele et al., 2008; Jarsch et al.,
2014;Konrad et al., 2014; Szymanski et al., 2015; Bücherl et
al.,2017; Lv et al., 2017). This indicates an organizationalrole of
the cytoskeleton during nanodomain and/orprotein complex assembly,
which is supported bystudies of Arabidopsis HYPERSENSITIVE
INDUCEDREACTION 1 (HIR1; Lv et al., 2017). Nonetheless, itwas
recently shown that HIR1 as well as FLOT2-containing nanodomains,
remain intact upon chemicaldisruption of the cytoskeleton (Dan�ek
et al., 2020). Inaddition, FRAP and single-particle analyses showed
aminor effect of the cytoskeleton on lateral diffusion
oftransmembrane proteins such as the FLS2 receptor(McKenna et al.,
2019). Given the size of these proteinassemblies in the nanometer
range, the usual width ofthe cytoskeleton network, and the
restricted dataavailability, a general mechanism of how the
cytoskel-eton supports or even mediates nanodomain assemblycannot
be proposed to date. Overall, there is strongevidence that cortical
cytoskeleton components partic-ipate in the dynamics and spatial
repartitioning ofplasma membrane nanodomains, but they may play
aless prominent role in plasma membrane compart-mentalization
compared to animal cells.
The Cell Wall-Plasma Membrane Continuum in Shapingthe Plasma
Membrane Landscape
In addition to the cytoskeleton in the cytosol, a keycomponent
in understanding the nanoscale organiza-tion of the plasma membrane
landscape in plants is theextracellular cell wall. Indeed, plant
cells have a highturgor pressure that physically presses the
plasmamembrane to the rigid, yet porous, cell wall. This has
astrong impact on the dynamic behavior of lipids andproteins at the
plasmamembrane-cell wall contacts andsets apart the plasma membrane
from any othermembranes of the cell. Most importantly, the cell
wall isa barrier to the free diffusion of plasma membranemolecules
that are sticking out into the wall (Fig. 3;Martinière et al.,
2012). Using FRAP and particletracking, it was demonstrated that
synthetic reporterswith an extracellular domain exhibit limited
lateraldiffusion at least on the minute timescale (Martinièreet
al., 2012; McKenna et al., 2019). This is in great con-trast to
soluble cytosolic synthetic reporters that interactwith the inner
leaflet of the plasma membrane anddiffuse significantly faster
(Martinière et al., 2012).Disruption of the plasma membrane/cell
wall contin-uum, by digestion of the cell wall, induction of
plas-molysis, or chemical perturbations of cell wallsynthesis, have
a profound impact on the diffusion ofcell surface proteins and the
formation of nanodomains
(Martinière et al., 2012; McKenna et al., 2019; Dan�eket al.,
2020).
Whereas the full extent of the importance of theplasma
membrane/cell wall continuum for organiza-tion of the cell surface
is not understood and has barelybeen challenged experimentally, its
existence pointstoward several predictions and hypotheses, as well
asdifferences from the canonical view of membrane or-ganization
derived from animal systems. Animaltransmembrane proteins that
stably and directly inter-act with extracellular matrix components
are excellentcandidates as anchored pickets (Freeman et al.,
2016,2018), whereas proteins without interaction with
theextracellular matrix are expected to diffuse within theboundary
imposed by the membrane skeleton. How-ever, in plants, the anchored
picket situation seems tobe pushed to the extreme, since virtually
every proteinwith an extracellular domain could be seen as an
an-chored picket. Indeed, even the diffusion of proteinswith few
extracellular residues appears to be impactedby the presence of the
cell wall (Martinière et al., 2012).Pushing this reasoning further,
GIPCs, which have avery large head group sticking out of the outer
leaflet ofthe plasma membrane, should also encounter
limiteddiffusion because of the cell wall (Fig. 2; Cacas et
al.,2016; Gronnier et al., 2018). Given their high abun-dance, both
membrane proteins with an extracellulardomain and GIPCs may have a
very strong impact onthe diffusion of plasma membrane
constituents.
It is even possible that components of the outermembrane
leaflets are barely diffusing. This could ex-plain the observation
in plants that membrane proteinsare basically “fixed” (i.e.
nondiffusing), a feature that israrely seen in animal systems
(Martinière et al., 2012).However, if receptors are fixed and
unable to diffuse,one may wonder how protein complexes are
formedupon ligand binding. It is possible that receptor com-plexes
are preformed but inhibited, and that ligandbinding triggers their
activation rather than oligomeri-zation. Such a view is supported
by in vivo interactiondata using FRET-FLIM between BRI1 and BAK1
re-ceptors (Bücherl et al., 2013). Indeed, such analysessuggest
that BRI1/BAK1 heterodimers are presenteven in the absence of
brassinosteroid and that theproportion of dimers is not induced by
exogenoustreatment with this ligand (Bücherl et al., 2013).
Theresults of these experiments have recently been con-firmed using
selective surface observation FILM, wherethe confocal spot is
placed perpendicular to the surfaceof the observed cells to reduce
signal detection fromstructures below the plasma membrane (Hutten
et al.,2017). However, they contradict results obtained
bycoimmunoprecipitation, and also models based oncrystallographic
data, which indicate that brassinoste-roids act as a molecular glue
to bridge BRI1 and BAK1extracellular domains (Belkhadir and
Jaillais, 2015;Hohmann et al., 2017).
The idea that membrane proteins with an extracel-lular domain do
not diffuse in plants because of thepresence of the cell wall is
perhaps too strong. Indeed, it
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is possible that we perceive an apparent lack of diffu-sion due
to the resolution limits of the techniques used,notably FRAP and
total internal reflection fluorescencemicroscopy. The plant cell
wall is to some extent po-rous, so it is possible that proteins are
able to diffusewithin the boundaries of this porous material. In
such aview, the cell wall could constitute a membrane skele-ton,
but it would act as an exoskeleton rather than acytoplasmic
cortical actin skeleton (Fig. 3).From a regulatory point of view,
this implies that
modifications of the cell wall, rather than (or in parallelwith)
the cytoskeleton could impact protein diffusion.In addition, the
cell wall may form membrane domainsthat could be much smaller than
the 300 nm delimitedby the actin membrane skeleton. If so, this
could per-haps reconcile data obtained from FRET-FLIM
andcoimmunoprecipitation of receptor kinases (Wanget al., 2005,
2008; Bücherl et al., 2013; Hutten et al.,2017). Indeed, FRET is a
proximity technique. Conse-quently, constitutive FRET can be
observed even in theabsence of physical interaction if receptors
are enclosedin small membrane domains. If they exist,
determiningthe size of such cell wall-delimited membrane domainsand
their composition would provide critical informa-tion for future
studies.According to the scenario highlighted above, recep-
tor proximity could be achieved by preformation ofnanodomains
during the insertion of transmembraneproteins into the endoplasmic
reticulummembrane andthus prior to their exocytosis. Here, but also
subse-quently, scaffolding could be executed by proteins likethe
peptide-binding receptor FERONIA, which facili-tates complex
formation of EFR and FLS2 with theircoreceptor BAK1 (Stegmann et
al., 2017). Interestingly,FER itself, or its protein partners,
directly bind cell wallcomponents (Li et al., 2016a; Feng et al.,
2018; Dünseret al., 2019), which could be an additional way to
inte-grate the cell wall information (i.e. composition,
re-modeling, peptide signaling, etc.) into nanodomainformation.
However, whether FER also controls nano-domain patterningmust be
further tested. Interestingly,soluble scaffold proteins localizing
to the inner mem-brane leaflet only access the receptor complex
uponexocytotic vesicle fusion with the plasma membrane.This
spatiotemporal association seems highly specificfor scaffolds like
remorins, as the great majority ofstudies on remorins did not
observe any labeling ofintracellular membrane, even though these
proteinswere often strongly overexpressed. In fact, remorins donot
use the secretory pathway but are rather directlysynthesized as
soluble proteins in the cytosol and sub-sequently inserted in the
inner plasma membraneleaflet via their REM-CA anchor (Fig. 1;
Gronnier et al.,2017).In addition, some proteins or lipids may act
as real
anchored pickets through direct interaction with wallcomponents
(Voxeur and Fry, 2014; Herger et al., 2019;Vaahtera et al., 2019;
Rui and Dinneny, 2020). This isexemplified by extensin-like formins
that bind the cellwall via a long extracellular domain or a
comparably
short Pro- and Ser-rich amino acid stretch in their Nterminus,
which resembles an extensin-like motif(Martinière et al., 2011;
Herger et al., 2019). These do-mains are connected via a
single-span transmembranehelix with an actin- or
microtubule-binding forminhomology domain. As such, members of the
forminfamily were shown to act as actin nucleation facilitatorsin
tip growing systems such as root hairs (Yi et al.,2005). Therefore,
it is impossible to fully uncouple thecell wall from the
cytoskeleton. Furthermore, there is acontinuum between cortical
microtubules inside thecell, cellulose synthases in the plasma
membrane, andcellulose microfibrils in the cell wall. This
three-wayconnection may impact membrane partitioning, but ithas so
far remained largely unexplored.To conclude, the relative impact of
the cytoskeleton
and the cell wall on plasma membrane organization ispoorly
understood, but it is likely that they are differentfrom animal
systems.
THE PLASMA MEMBRANE AS A DIGITAL SCREENFOR SIGNAL INTEGRATION:
ATWO-DIMENSIONAL PERSPECTIVE
What is the function of plasma membrane compart-mentalization in
signal perception and integration?There is neither a single
response to this question norsatisfying exhaustive answers. During
signaling, ex-tracellular information is transmitted through
theplasmamembrane inside the cell. The signals perceivedby single
activated receptors have to be amplified. Itbecomes increasingly
evident that such amplificationsteps often happen directly at the
plasma membrane.Indeed, whereas the initial view of signaling
“cascades”would place receptors at the plasma membrane andtheir
downstream components in the cytosol, this isoften not the case.
For example, in the brassinosteroidpathway, nearly all downstream
components of BRI1signaling, including the transcription factors
BES1/BZR1, have recently been shown to localize to theplasma
membrane at some point during signal trans-duction (Amorim-Silva et
al., 2019; Ren et al., 2019).This is achieved through the action of
scaffolds thattransiently recruit kinases/phosphatases and
othersignaling molecules (Amorim-Silva et al., 2019). It islikely
that the mechanisms of lateral membrane orga-nization discussed
above contribute to the formationand scaffolding of active receptor
complexes.In addition to signal amplification, clustering may
also increase sensitivity. For example, clustered recep-tors are
more likely to transduce information carried byligandswithweak
binding properties. Furthermore, thelocalization of receptors in
laterally segregated nano-domains may also increase signaling
specificity byunmixing downstream signaling components. This
isparticularly relevant when different receptors share thesame
amplifying downstream components. For exam-ple, spatial separation
of BRI1 and FLS2 in distinctnanoclusters may ensure robust
signaling from these
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receptors, even though they share several kinases, suchas BAK1
or BSKs (Bücherl et al., 2017). Another possiblerole of receptor
clustering is their connection with in-tracellular trafficking,
notably endocytosis, which isoften associated with signal
downregulation.
Modeling suggests that the plasma membrane mayalso act as a
digital two-dimensional screen, allowingthe integration of analog
information and its conversioninto digital pixels before this
information is relayedwith high fidelity inside the cell (Tian et
al., 2007, 2010).This has been mainly studied at experimental and
the-oretical levels for Ras signaling in animal cells (Hardingand
Hancock, 2008). Ras is a small GTPase from theRho/Ras superfamily
that is involved in canonicalmitogen-activated protein kinase
(MAPK) signalingdownstream of growth factor receptors (i.e.
receptorTyr kinase; Harding and Hancock, 2008). The idea be-hind
digital signaling is that it relies on bistableswitches that can
only be in two states, namely offand on (Fig. 4). This is the
opposite of analog circuits,which transmit continuous information
(input) intoa proportional continuous output. Most informationin
biology is of analog nature, including the varyingamounts of growth
factors experienced by the cell(Fig. 4). The challenge is to
transmit such analog signalwith high fidelity across the plasma
membrane into aproportional output signal. Thus, Ras signaling
worksas an analog-digital-analog converter, which is onewayto
convey the signal across the membrane with highfidelity (Fig. 4;
Tian et al., 2007). Indeed, membrane-localized Ras may be in two
states, diffusing or innanoclusters, which correspond to its
inactive state (off)
and active state (on), respectively (Prior et al., 2001; Tianet
al., 2007). As such, each Ras nanocluster can be seenas a digital
pixel at the membrane, which relays theinformation to downstream
MAPK. Ultimately, thisdigital information is processed in an analog
(i.e. con-tinuous) output signal, which is the amount of
MAPKphosphorylation (Fig. 4; Tian et al., 2007).
Whereas it has been validatedmathematically for Rassignaling, it
is likely that such analog-digital-analogrelays are widespread in
signal transduction processesacross the plasma membrane. One of the
prerequisitesfor such a system is that it indeed behaves as a
binarysystem with nonclustered molecules that are fully un-able to
transduce the signal. This is the case for the plantRho GTPase
ROP6, which belongs to the same GTPasesuperfamily as Ras and, like
Ras, is not able to signalwhen it is not stably localized in
nanoclusters, evenwhen it is in a constitutive active GTP-loaded
confor-mation (Platre et al., 2019).
One of the interesting features of analog-digital-an-alog
converters is that it is possible to adjust their“digital gain”
(Harding and Hancock, 2008). In otherwords, it is possible to tune
the sensitivity of the systemby boosting or restricting the
formation of the nano-clusters (Fig. 4). This is well exemplified
for ROP6,whose nanoclustering is regulated by PS (Platre et
al.,2019). In the absence of PS, ROP6 is not stabilized
innanoclusters and does not signal, whereas in the pres-ence of low
amounts of PS, it can be stabilized innanoclusters but not without
an increased input signal(i.e. auxin; Platre et al., 2019). By
contrast, in plants withelevated PS levels, ROP6 nanoclustering is
boosted and
Figure 4. The plasma membrane may act as an
analog-digital-analog converter for high-fidelity signal
transduction. A, Duringsignaling both the input (i.e. ligand) and
output (i.e. cellular response such as phosphorylation) signals are
analog by nature.However, small GTPases, such as ROPs, act in a
binary fashion akin to a digital signal by beingOFFwhen they are
freely diffusing,and being ON when they are immobilized in
nanodomains (Harding and Hancock, 2008). B, As such, molecules
(such as PS inroots; Platre et al., 2019) that favor or dampen the
OFF (diffusing) or ON (nanodomains) state of the GTPase may act as
a digitalgain, which may modify the magnitude of the output signal
even though the input signal is constant (Zhou and Hancock,
2015).
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requires less input signal. Therefore, variations in PSlevel act
as a molecular rheostat that adjusts the digitalgain of the system
and as such may modulate thestrength of the output signal while
keeping the inputsignal constant (Fig. 4; Platre et al., 2019).
Given that PSlevels at the plasma membrane vary during root
epi-dermis differentiation, such variations are one wayto modify
cellular output according to the develop-mental context of the cell
(Colin and Jaillais, 2019; Platreet al., 2019).The comparison
between the plasma membrane and
a two-dimensional digital screen is useful, as it allowsthe
conceptualization of how different membrane pa-rameters may
participate together in signaling. How-ever, it should also be
emphasized that this is asimplification, as the plasma membrane
should not beseen as isolated from its environment, but rather as
partof a three-dimensional continuum that includes theextracellular
cell wall and the cytoplasm.
LIQUID-LIQUID PHASE SEPARATION IN PLASMAMEMBRANE
NANO-ORGANIZATION: HOW MUCHTHREE-DIMENSIONAL IS IN A
NANODOMAIN?
At present, we define nanodomains purely as mem-brane
compartments, which only extend into the cyto-sol by proteins
interacting with the inner membraneleaflet or proteins within the
complex. However, giventhe time scales of reactions, the lack of
free diffusion inthe cytosol due to tight packing, the high net
charges oflipids and proteins, and the necessity to
compartmen-talize cellular processes in order to avoid
unwantedcross talk between pathways, a concept that
spatiallyextends nanodomains significantly into the cytosol
isfavored.Recently, liquid-liquid phase separations (LLPSs)
have been proposed as a novel organization concept(Hyman et al.,
2014; Banani et al., 2017; McSwiggenet al., 2019). These LLPSs are
dynamic cellular com-partments that lack a membrane envelope and
are alsoreferred to as “membraneless organelles.” Similar
toreactions inside the nucleolus, proteins and nucleicacids can be
sequestered into LLPSs that have so farmostly been found in the
nucleus or the cytosol andtermed e.g. “stress granules” or “liquid
droplets.” Incontrast to lipid droplets, LLPSs rely on a reversible
andprotein-induced liquid unmixing providing almostviscoelastic
properties to these compartments (Hymanet al., 2014). As such, four
major characteristics ofLLPSs have been proposed: (1) they are
spheres; (2)they have a fluid content; (3) they fuse upon
contact;and (4) they can be actively reshaped by shear
flow(Cuevas-Velazquez and Dinneny, 2018).They are most often
organized by highly oligomeric
proteins with long stretches of intrinsic disorder (ID).LLPSs
only occur under specific conditions, and dis-sociation of proteins
maintaining this phase separationresults in total disintegration of
the structure. Whereastheoretically most ID proteins could
contribute to LLPS
formation, some proteins have a higher tendency toself-coalesce
at critical pH or salt concentrations or tobind nucleic acids or
other proteins (Bergeron-Sandovalet al., 2016). In contrast to
protein aggregates, LLPS-associated proteins can maintain native
folding andactivity upon liquid mixing and dissociation.
Thesefeatures are well exemplified by the nuclear Arabi-dopsis
RNA-binding protein FCA that interacts withRNA processing
components (Fang et al., 2019).To date, there is no convincing
experimental evi-
dence demonstrating the existence and functionalimportance of
LLPSs at or near the plant plasmamembrane. However, the plasma
membrane, as anymembrane surface, restricts the molecular diffusion
to atwo-dimensional plane, which reduces the concentra-tion
threshold required for phase separation (Fig. 5;Case et al., 2019a;
Snead and Gladfelter, 2019). In ad-dition, recent advances in
mammalian systems suggestthat LLPSs may play a crucial role in cell
surface sig-naling (Case et al., 2019a, 2019b; Martin and
Mittag,2019). Indeed, several examples were found in
whichmultivalent interactions between transmembrane re-ceptors and
their downstream partners trigger phasetransitions (Fig. 5; Case et
al., 2019b; Huang et al.,2019b). In this scenario, the clustered
receptor-effectorcomplexes separate from the rest of the cytosol in
amembraneless liquid compartment, which significantlyenhances the
residence time of the adaptors at theplasma membrane (Fig. 5). The
induction of the enzy-matic activity of effector molecules is slow,
and theirrecruitment to unclustered receptors is too transient
toallow signal transduction (Huang et al., 2019b).By contrast,
prolonged residence time in the biomo-
lecular condensate at the receptor nanocluster phasefavors
signal activation (Fig. 5). This model was coined“kinetic
proofreading,” as it directly relates signalingactivity to the
dwell time of membrane association ofdownstream receptor components
(Fig. 5). This dwelltime is regulated by LLPS, which can be rapidly
mod-ulated by posttranslational modification such as
Tyrphosphorylation (Huang et al., 2019b). Given that (1)the
N-terminal regions of remorin proteins are intrin-sically
disordered (Marín et al., 2012; Marín and Ott,2014), (2) their
C-terminal regions are able to formhigher-order oligomers (Bariola
et al., 2004; Marín et al.,2012; Legrand et al., 2019; Martinez et
al., 2019), (3) theyactively bind polyanions, including anionic
lipids(Gronnier et al., 2017; Legrand et al., 2019), and (4)
theyare phosphorylated (Perraki et al., 2018), remorin pro-teins
are promising candidates to induce LLPSs at theplasma membrane
interface and would support andextend a recently proposed model for
liquid-like re-ceptor clustering compartments (Cuevas-Velazquezand
Dinneny, 2018).Furthermore, multivalent association with the
ex-
tracellular matrix in animal cells also potentiatesLLPSs at the
extracellular surface of animal cells (Caseet al., 2019a).
Accordingly, the extent of phase separa-tions within the plant cell
wall could also profoundlyimpact signaling, and reciprocally,
plasma membrane
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organization could impact phase separation within theplant cell
wall. For example, cellulose may be present ineither of two states:
amorphous or crystalline. In addi-tion, cellulose microfibrils are
embedded in a matrixcomposed of water, polysaccharides, proteins,
andions. This matrix is a biphasic mixture between a po-rous solid
phase and a liquid phase (Ali and Traas,2016). One may envision
that a change in the equilib-rium between these different states
may impact lateraldiffusion of plasma membrane proteins/lipids.
Con-versely, the presence of anchored pickets could locallyimpact
the equilibrium between the cell wall poroussolid phase and the
liquid phase.
CONCLUDING REMARKS AND PERSPECTIVES
Altogether, accumulating evidence over the last yearsclearly
demonstrates that the plant plasma mem-brane, like the plasma
membrane in other eukaryoticcells, is highly compartmentalized.
However, mostof our knowledge originates either from
biochemical
fractionation of detergent-resistant membranes or
fromdiffraction-limited microscopy techniques. It is in-creasingly
clear that these techniques lack the resolu-tion to properly
address the challenges underlying thestudy of plasma membrane
compartmentalization, asthe size of nanodomains are typically below
the reso-lution limit of light microscopy.Nonetheless, in the
pastfew years, superresolution microscopy was used inseveral
studies to address this problem in planta, andwe expect that it
will be used more frequently in fu-ture studies. So far, such
techniques have mainly beenused to localize one protein at a time,
and an importantfuture development of superresolution microscopyin
plants will be to set up pipelines for colocalizationanalysis.
As described above, the cell wall plays a crucial rolein plasma
membrane organization and the diffusion ofcell surface proteins and
lipids. This highlights the im-portance of studying the cell in its
native state insideits organism rather than in isolated cellular
systemssuch as protoplasts. It also exemplifies the
differencesbetween plants and animals with respect to plasma
Figure 5. Liquid-liquid phase separation: concepts and roles in
signaling at the plasmamembrane/cytosol interface. A,
Schematicrepresentation of ideal liquids and solids. In liquids
(left), molecules diffuse to distances greater than their size. In
solids (right),molecules are confined by their neighbors.
Furthermore, for crystalline solids, positional order exists over
long distances and it ispossible to draw straight lines alongwhich
particles are equally spaced (dashed lines; Hyman et al., 2014). In
LLPS, two liquids areunmixed, similar to a water/oil solution. This
limits the diffusion of molecules in and out of each liquid phase
and allows specificreactions to occur in each of these
“membraneless” compartments. B, LLPS in the cytosol may be coupled
with lipid phaseseparation in membranes (Snead and Gladfelter,
2019). C, Phase separation may also occur upon clustering of
receptors andadaptormolecules at the plasmamembrane. The resulting
biomolecular condensate will locally limit the diffusion of
downstreamsignaling components, increasing their dwell time at the
plasma membrane and thereby triggering signaling. This process
wascoined “kinetic proofreading” because it allows the filtering
out of noise (i.e. uncontrolled recruitment of effectors at the
plasmamembrane) from real activation (i.e. induction of LLPS upon
receptor nanoclustering; Huang et al., 2019b).
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membrane compartmentalization. A clear challenge forthe coming
years will be to better understand the po-tential coupling between
the plasma membrane andcell wall organization (see Outstanding
Questions).This will notably require experimental approaches
tocharacterize their respective physical states and tomonitor the
evolution of these states in both space andtime. In that respect,
new developments in the field ofatomic force microscopy, Raman
microscopy, Brillouinmicroscopy, andmass spectrometry imaging may
opennew opportunities to challenge our current under-standing of
the cell wall/plasma membrane contin-uum. Since both systems are
highly complex, weenvision that such studies will likely require
mathe-matical and/or physical modeling together with
re-constitution experiments using minimal membrane/cell wall
components.Alongside extracellular matrices, the differences
be-
tween plants and animals also include differences intheir use of
cytoskeleton components and the pres-ence of different lipid
species, notably sphingolipids,phytosterols, and very long-chain
phosphatidyl-Sers.Therefore, although models based on animal or
yeaststudies are useful, they should not be taken at face valuewhen
thinking about the plant plasma membrane. It islikely that the
plasma membrane greatly contributesto the phenotypic plasticity of
plants.We therefore needto understand it on a functional level and
address localand temporal specifications of the bilayer in
responseto the ever-changing environment (see Outstanding
Questions). In comparison to many other model orga-nisms, the
ability to combine genetic, biochemical, andcell biological
approaches at a tissue, organ, and evenorganismic level provides
outstanding potential tomechanistically understand signal
transduction acrossmultiple scales.
ACKNOWLEDGMENTS
We thank Julien Gronnier for his membrane lipid template and
AlexandreMartinière and Matthieu Platre for critical comments on
the manuscript.
Received October 30, 2019; accepted December 9, 2019; published
December 19,2019.
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