The Nanoscale Organization of the Plasma Membrane and Its … · Update on Plasma Membrane Compartmentalization in Signaling The Nanoscale Organization of the Plasma Membrane and

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 subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.19.01349

1682 Plant Physiology�, April 2020, Vol. 182, pp. 1682–1696, www.plantphysiol.org � 2020 American Society of Plant Biologists. All Rights Reserved.

Dow

nloaded from https://academ

ic.oup.com/plphys/article/182/4/1682/6116401 by guest on 14 June 2021

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

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

Plant Physiol. Vol. 182, 2020 1683

Plasma Membrane Compartmentalization in Signaling

Dow



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.

1684 Plant Physiol. Vol. 182, 2020

Jaillais and Ott

Dow



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.



Dow



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,


Jaillais and Ott

Dow



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.



Dow



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


Jaillais and Ott

Dow



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



Dow



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).


Jaillais and Ott

Dow



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



Dow



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).


Jaillais and Ott

Dow



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.

LITERATURE CITED

Albers P, Üstün S, Witzel K, Kraner M, Börnke F (2019) A remorin fromNicotiana benthamiana interacts with the Pseudomonas Type-III effectorprotein HopZ1a and is phosphorylated by the immune-related kinasePBS1. Mol Plant Microbe Interact 32: 1229–1242

Ali O, Traas J (2016) Force-driven polymerization and turgor-induced wallexpansion. Trends Plant Sci 21: 398–409

Amorim-Silva V, García-Moreno Á, Castillo AG, Lakhssassi N, EstebanDel Valle A, Pérez-Sancho J, Li Y, Posé D, Pérez-Rodriguez J, Lin J,et al (2019) TTL proteins scaffold brassinosteroid signaling componentsat the plasma membrane to optimize signal transduction in Arabidopsis.Plant Cell 31: 1807–1828

Banani SF, Lee HO, Hyman AA, Rosen MK (2017) Biomolecular conden-sates: Organizers of cellular biochemistry. Nat Rev Mol Cell Biol 18:285–298

Bariola PA, Retelska D, Stasiak A, Kammerer RA, Fleming A, Hijri M,Frank S, Farmer EE (2004) Remorins form a novel family of coiled coil-forming oligomeric and filamentous proteins associated with apical,vascular and embryonic tissues in plants. Plant Mol Biol 55: 579–594

Belkhadir Y, Jaillais Y (2015) The molecular circuitry of brassinosteroidsignaling. New Phytol 206: 522–540

Bergeron-Sandoval LP, Safaee N, Michnick SW (2016) Mechanisms andconsequences of macromolecular phase separation. Cell 165: 1067–1079

Borner GH, Sherrier DJ, Weimar T, Michaelson LV, Hawkins ND,Macaskill A, Napier JA, Beale MH, Lilley KS, Dupree P (2005) Anal-ysis of detergent-resistant membranes in Arabidopsis. Evidence forplasma membrane lipid rafts. Plant Physiol 137: 104–116

Brown DA, Rose JK (1992) Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cellsurface. Cell 68: 533–544

Bücherl CA, Jarsch IK, Schudoma C, Segonzac C, Mbengue M, RobatzekS, MacLean D, Ott T, Zipfel C (2017) Plant immune and growth re-ceptors share common signalling components but localise to distinctplasma membrane nanodomains. eLife 6: e25114

Bücherl CA, van Esse GW, Kruis A, Luchtenberg J, Westphal AH, Aker J,van Hoek A, Albrecht C, Borst JW, de Vries SC (2013) Visualization ofBRI1 and BAK1(SERK3) membrane receptor heterooligomers duringbrassinosteroid signaling. Plant Physiol 162: 1911–1925

Cacas JL, Buré C, Grosjean K, Gerbeau-Pissot P, Lherminier J, RomboutsY, Maes E, Bossard C, Gronnier J, Furt F, Fouillen L, Germain V, et al(2016) Revisiting Plant Plasma Membrane Lipids in Tobacco: A Focus onSphingolipids. Plant Physiol 170: 367–384

Case LB, Ditlev JA, Rosen MK (2019a) Regulation of TransmembraneSignaling by Phase Separation. Annu Rev Biophys 48: 465–494

Case LB, Zhang X, Ditlev JA, Rosen MK (2019b) Stoichiometry controlsactivity of phase-separated clusters of actin signaling proteins. Science363: 1093–1097

Chen D, Ahsan N, Thelen JJ, Stacey G (2019) S-Acylation of plant immunereceptors mediates immune signaling in plasma membrane nano-domains. bioRxiv 720482 doi:10.1101/720482

Colin LA, Jaillais Y (2019) Phospholipids across scales: Lipid patterns andplant development. Curr Opin Plant Biol 53: 1–9

Cuevas-Velazquez CL, Dinneny JR (2018) Organization out of disorder:Liquid-liquid phase separation in plants. Curr Opin Plant Biol 45(Pt A):68–74



Dow



Cui Y, Yu M, Yao X, Xing J, Lin J, Li X (2018) Single-particle tracking forthe quantification of membrane protein dynamics in living plant cells.Mol Plant 11: 1315–1327

Dan�ek M, Angelini J, Malínská K, Andrejch J, Amlerová Z, KocourkováD, Brouzdová J, Valentová O, Martinec J, Petrášek J (2020) Cell wallcontributes to the stability of plasma membrane nanodomain organi-zation of Arabidopsis thaliana FLOTILLIN2 and HYPERSENSITIVE IN-DUCED REACTION1 proteins. Plant J 101: 619–636

Dan�ek M, Valentová O, Martinec J (2016) Flotillins, erlins, and HIRs: Fromanimal base camp to plant new horizons. Crit Rev Plant Sci 35: 191–214

Demir F, Horntrich C, Blachutzik JO, Scherzer S, Reinders Y,Kierszniowska S, Schulze WX, Harms GS, Hedrich R, Geiger D, et al(2013) Arabidopsis nanodomain-delimited ABA signaling pathwayregulates the anion channel SLAH3. Proc Natl Acad Sci USA 110:8296–8301

Dünser K, Gupta S, Herger A, Feraru MI, Ringli C, Kleine-Vehn J (2019)Extracellular matrix sensing by FERONIA and leucine-rich repeat ex-tensins controls vacuolar expansion during cellular elongation in Ara-bidopsis thaliana. EMBO J 38: e100353

Fang X, Wang L, Ishikawa R, Li Y, Fiedler M, Liu F, Calder G, Rowan B,Weigel D, Li P, et al (2019) Arabidopsis FLL2 promotes liquid-liquidphase separation of polyadenylation complexes. Nature 569: 265–269

Feng W, Kita D, Peaucelle A, Cartwright HN, Doan V, Duan Q, Liu MC,Maman J, Steinhorst L, Schmitz-Thom I, et al (2018) The FERONIAreceptor kinase maintains cell-wall integrity during salt stress throughCa21 signaling. Curr Biol 28: 666–675

Freeman SA, Goyette J, Furuya W, Woods EC, Bertozzi CR, Bergmeier W,Hinz B, van der Merwe PA, Das R, Grinstein S (2016) Integrins form anexpanding diffusional barrier that coordinates phagocytosis. Cell 164:128–140

Freeman SA, Vega A, Riedl M, Collins RF, Ostrowski PP, Woods EC,Bertozzi CR, Tammi MI, Lidke DS, Johnson P, et al (2018) Trans-membrane pickets connect cyto- and pericellular skeletons formingbarriers to receptor engagement. Cell 172: 305–317

Frescatada-Rosa M, Stanislas T, Backues SK, Reichardt I, Men S, BouttéY, Jürgens G, Moritz T, Bednarek SY, Grebe M (2014) High lipid orderof Arabidopsis cell-plate membranes mediated by sterol and DYNA-MIN-RELATED PROTEIN1A function. Plant J 80: 745–757

Furt F, König S, Bessoule JJ, Sargueil F, Zallot R, Stanislas T, Noirot E,Lherminier J, Simon-Plas F, Heilmann I, et al (2010) Poly-phosphoinositides are enriched in plant membrane rafts and form mi-crodomains in the plasma membrane. Plant Physiol 152: 2173–2187

Gerbeau-Pissot P, Der C, Grebe M, Stanislas T (2016) Ratiometric fluo-rescence live imaging analysis of membrane lipid order in Arabidopsismitotic cells using a lipid order-sensitive probe. Methods Mol Biol 1370:227–239

Ghosh R, de Campos MK, Huang J, Huh SK, Orlowski A, Yang Y,Tripathi A, Nile A, Lee HC, Dynowski M, et al (2015) Sec14-nodulinproteins and the patterning of phosphoinositide landmarks for devel-opmental control of membrane morphogenesis. Mol Biol Cell 26:1764–1781

Gronnier J, Crowet JM, Habenstein B, Nasir MN, Bayle V, Hosy E, PlatreMP, Gouguet P, Raffaele S, Martinez D, et al (2017) Structural basis forplant plasma membrane protein dynamics and organization into func-tional nanodomains. eLife 6: e26404

Gronnier J, Gerbeau-Pissot P, Germain V, Mongrand S, Simon-Plas F(2018) Divide and rule: Plant plasma membrane organization. TrendsPlant Sci 23: 899–917

Grosjean K, Der C, Robert F, Thomas D, Mongrand S, Simon-Plas F,Gerbeau-Pissot P (2018) Interactions between lipids and proteins arecritical for organization of plasma membrane-ordered domains in to-bacco BY-2 cells. J Exp Bot 69: 3545–3557

Gui J, Zheng S, Liu C, Shen J, Li J, Li L (2016) OsREM4.1 interacts withOsSERK1 to coordinate the interlinking between abscisic acid andbrassinosteroid signaling in rice. Dev Cell 38: 201–213

Gui J, Zheng S, Shen J, Li L (2015) Grain setting defect1 (GSD1) function inrice depends on S-acylation and interacts with actin 1 (OsACT1) at itsC-terminal. Front Plant Sci 6: 804

Haney CH, Long SR (2010) Plant flotillins are required for infection bynitrogen-fixing bacteria. Proc Natl Acad Sci USA 107: 478–483

Haney CH, Riely BK, Tricoli DM, Cook DR, Ehrhardt DW, Long SR(2011) Symbiotic rhizobia bacteria trigger a change in localization and

dynamics of the Medicago truncatula receptor kinase LYK3. Plant Cell 23:2774–2787

Hao H, Fan L, Chen T, Li R, Li X, He Q, Botella MA, Lin J (2014) Clathrinand membrane microdomains cooperatively regulate RbohD dynamicsand activity in Arabidopsis. Plant Cell 26: 1729–1745

Harding A, Hancock JF (2008) Ras nanoclusters: Combining digital andanalog signaling. Cell Cycle 7: 127–134

Hemsley PA, Weimar T, Lilley KS, Dupree P, Grierson CS (2013) Aproteomic approach identifies many novel palmitoylated proteins inArabidopsis. New Phytol 197: 805–814

Herger A, Dünser K, Kleine-Vehn J, Ringli C (2019) Leucine-rich repeatextensin proteins and their role in cell wall sensing. Curr Biol 29:R851–R858

Hohmann U, Lau K, Hothorn M (2017) The structural basis of ligandperception and signal activation by receptor kinases. Annu Rev PlantBiol 68: 109–137

Homann U, Meckel T, Hewing J, Hütt MT, Hurst AC (2007) Distinct flu-orescent pattern of KAT1:GFP in the plasma membrane of Vicia fabaguard cells. Eur J Cell Biol 86: 489–500

Hosy E, Martinière A, Choquet D, Maurel C, Luu DT (2015) Super-resolved and dynamic imaging of membrane proteins in plant cells re-veal contrasting kinetic profiles and multiple confinement mechanisms.Mol Plant 8: 339–342

Huang D, Sun Y, Ma Z, Ke M, Cui Y, Chen Z, Chen C, Ji C, Tran TM,Yang L, et al (2019a) Salicylic acid-mediated plasmodesmal closure viaRemorin-dependent lipid organization. Proc Natl Acad Sci USA 116:21274–21284

Huang WYC, Alvarez S, Kondo Y, Lee YK, Chung JK, Lam HYM, BiswasKH, Kuriyan J, Groves JT (2019b) A molecular assembly phase transi-tion and kinetic proofreading modulate Ras activation by SOS. Science363: 1098–1103

Hurst CH, Turnbull D, Myles SM, Leslie K, Keinath NF, Hemsley PA(2018) Variable effects of C-terminal fusions on FLS2 function: Not allepitope tags are created equal. Plant Physiol 177: 522–531

Hurst CH, Wright KM, Turnbull D, Leslie K, Jones S, Hemsley PA (2019)Juxta-membrane S-acylation of plant receptor-like kinases is likely for-tuitous and does not necessarily impact upon function. Sci Rep 9: 12818

Hutten SJ, Hamers DS, Aan den Toorn M, van Esse W, Nolles A, BücherlCA, de Vries SC, Hohlbein J, et al (2017) Visualization of BRI1 andSERK3/BAK1 nanoclusters in Arabidopsis roots. PLoS One 12: e0169905

Hyman AA, Weber CA, Jülicher F (2014) Liquid-liquid phase separation inbiology. Annu Rev Cell Dev Biol 30: 39–58

Ishikawa K, Hashimoto M, Yusa A, Koinuma H, Kitazawa Y, Netsu O,Yamaji Y, Namba S (2017) Dual targeting of a virus movement proteinto ER and plasma membrane subdomains is essential for plasmodes-mata localization. PLoS Pathog 13: e1006463

Jarsch IK, Konrad SS, Stratil TF, Urbanus SL, Szymanski W, Braun P,Braun KH, Ott T (2014) Plasma membranes are subcompartmentalizedinto a plethora of coexisting and diverse microdomains in Arabidopsisand Nicotiana benthamiana. Plant Cell 26: 1698–1711

Jarsch IK, Ott T (2011) Perspectives on remorin proteins, membrane rafts,and their role during plant-microbe interactions. Mol Plant MicrobeInteract 24: 7–12

Jin L, Millard AC, Wuskell JP, Clark HA, Loew LM (2005) Cholesterol-enriched lipid domains can be visualized by di-4-ANEPPDHQ withlinear and nonlinear optics. Biophys J 89: L04–L06

Jin L, Millard AC, Wuskell JP, Dong X, Wu D, Clark HA, Loew LM (2006)Characterization and application of a new optical probe for membranelipid domains. Biophys J 90: 2563–2575

Kf de Campos M, Schaaf G (2017) The regulation of cell polarity by lipidtransfer proteins of the SEC14 family. Curr Opin Plant Biol 40: 158–168

Kleine-Vehn J, Wabnik K, Martinière A, Łangowski Ł, Willig K,Naramoto S, Leitner J, Tanaka H, Jakobs S, Robert S, et al (2011) Re-cycling, clustering, and endocytosis jointly maintain PIN auxin carrierpolarity at the plasma membrane. Mol Syst Biol 7: 540

Konrad SS, Popp C, Stratil TF, Jarsch IK, Thallmair V, Folgmann J, MarínM, Ott T (2014) S-acylation anchors remorin proteins to the plasmamembrane but does not primarily determine their localization inmembrane microdomains. New Phytol 203: 758–769

Kusumi A, Fujiwara TK, Chadda R, Xie M, Tsunoyama TA, Kalay Z,Kasai RS, Suzuki KG (2012) Dynamic organizing principles of theplasma membrane that regulate signal transduction: Commemorating


Jaillais and Ott

Dow



the fortieth anniversary of Singer and Nicolson’s fluid-mosaic model.Annu Rev Cell Dev Biol 28: 215–250

Laloi M, Perret AM, Chatre L, Melser S, Cantrel C, Vaultier MN,Zachowski A, Bathany K, Schmitter JM, Vallet M, et al (2007) Insightsinto the role of specific lipids in the formation and delivery of lipidmicrodomains to the plasma membrane of plant cells. Plant Physiol 143:461–472

Laurent N, Der C, Simon-Plas F, Gerbeau-Pissot P (2019) Cell stage ap-pears critical for control of plasma membrane order in plant cells. PlantSignal Behav 14: 1620058

Lefebvre B, Furt F, Hartmann MA, Michaelson LV, Carde JP, Sargueil-Boiron F, Rossignol M, Napier JA, Cullimore J, Bessoule JJ, et al (2007)Characterization of lipid rafts from Medicago truncatula root plasmamembranes: A proteomic study reveals the presence of a raft-associatedredox system. Plant Physiol 144: 402–418

Lefebvre B, Timmers T, Mbengue M, Moreau S, Hervé C, Tóth K,Bittencourt-Silvestre J, Klaus D, Deslandes L, Godiard L, et al (2010) Aremorin protein interacts with symbiotic receptors and regulates bac-terial infection. Proc Natl Acad Sci USA 107: 2343–2348

Legrand A, Martinez D, Grélard A, Berbon M, Morvan E, Tawani A,Loquet A, Mongrand S, Habenstein B (2019) Nanodomain clustering ofthe plant protein remorin by solidsState NMR. Front Mol Biosci 6: 107

Li C, Wu HM, Cheung AY (2016a) FERONIA and her pals: Functions andmechanisms. Plant Physiol 171: 2379–2392

Li R, Liu P, Wan Y, Chen T, Wang Q, Mettbach U, Baluska F, Samaj J,Fang X, Lucas WJ, et al (2012) A membrane microdomain-associatedprotein, Arabidopsis Flot1, is involved in a clathrin-independent endo-cytic pathway and is required for seedling development. Plant Cell 24:2105–2122

Li X, Wang X, Yang Y, Li R, He Q, Fang X, Luu DT, Maurel C, Lin J (2011)Single-molecule analysis of PIP2;1 dynamics and partitioning revealsmultiple modes of Arabidopsis plasma membrane aquaporin regulation.Plant Cell 23: 3780–3797

Li X, Xing J, Qiu Z, He Q, Lin J (2016b) Quantification of membraneprotein dynamics and interactions in plant cells by fluorescence corre-lation spectroscopy. Mol Plant 9: 1229–1239

Liang P, Stratil TF, Popp C, Marín M, Folgmann J, Mysore KS, Wen J, OttT (2018) Symbiotic root infections in Medicago truncatula requireremorin-mediated receptor stabilization in membrane nanodomains.Proc Natl Acad Sci USA 115: 5289–5294

Lv X, Jing Y, Xiao J, Zhang Y, Zhu Y, Julian R, Lin J (2017) Membranemicrodomains and the cytoskeleton constrain AtHIR1 dynamics andfacilitate the formation of an AtHIR1-associated immune complex. PlantJ 90: 3–16

Mamode Cassim A, Gouguet P, Gronnier J, Laurent N, Germain V,Grison M, Boutté Y, Gerbeau-Pissot P, Simon-Plas F, Mongrand S(2019) Plant lipids: Key players of plasma membrane organization andfunction. Prog Lipid Res 73: 1–27

Marín M, Ott T (2012) Phosphorylation of intrinsically disordered regionsin remorin proteins. Front Plant Sci 3: 86

Marín M, Ott T (2014) Intrinsic disorder in plant proteins and phyto-pathogenic bacterial effectors. Chem Rev 114: 6912–6932

Marín M, Thallmair V, Ott T (2012) The intrinsically disorderedN-terminal region of AtREM1.3 remorin protein mediates protein-protein interactions. J Biol Chem 287: 39982–39991

Martin EW, Mittag T (2019) Dwelling at membranes promotes decisivesignaling. Science 363: 1036–1037

Martinez D, Legrand A, Gronnier J, Decossas M, Gouguet P, Lambert O,Berbon M, Verron L, Grélard A, Germain V, et al (2019) Coiled-coiloligomerization controls localization of the plasma membrane RE-MORINs. J Struct Biol 206: 12–19

Martinière A, Fiche JB, Smokvarska M, Mari S, Alcon C, Dumont X,Hematy K, Jaillais Y, Nollmann M, Maurel C (2019) Osmotic stressactivates two ROS pathways with distinct impacts on protein nano-domains and diffusion. Plant Physiol 179: 1581–1593

Martinière A, Gayral P, Hawes C, Runions J (2011) Building bridges:formin1 of Arabidopsis forms a connection between the cell wall and theactin cytoskeleton. Plant J 66: 354–365

Martinière A, Lavagi I, Nageswaran G, Rolfe DJ, Maneta-Peyret L, LuuDT, Botchway SW, Webb SE, Mongrand S, Maurel C, et al (2012) Cellwall constrains lateral diffusion of plant plasma-membrane proteins.Proc Natl Acad Sci USA 109: 12805–12810

McKenna JF, Rolfe DJ, Webb SED, Tolmie AF, Botchway SW, Martin-Fernandez ML, Hawes C, Runions J (2019) The cell wall regulates dy-namics and size of plasma-membrane nanodomains in Arabidopsis. ProcNatl Acad Sci USA 116: 12857–12862

McSwiggen DT, Mir M, Darzacq X, Tjian R (2019) Evaluating phaseseparation in live cells: Diagnosis, caveats, and functional consequences.Genes Dev 33: 1619–1634

Mongrand S, Morel J, Laroche J, Claverol S, Carde JP, Hartmann MA,Bonneu M, Simon-Plas F, Lessire R, Bessoule JJ (2004) Lipid rafts inhigher plant cells: Purification and characterization of Triton X-100-in-soluble microdomains from tobacco plasma membrane. J Biol Chem 279:36277–36286

Morel J, Claverol S, Mongrand S, Furt F, Fromentin J, Bessoule JJ, BleinJP, Simon-Plas F (2006) Proteomics of plant detergent-resistant mem-branes. Mol Cell Proteomics 5: 1396–1411

Noack LC, Jaillais Y (2017) Precision targeting by phosphoinositides: HowPIs direct endomembrane trafficking in plants. Curr Opin Plant Biol 40:22–33

Ntoukakis V, Schwessinger B, Segonzac C, Zipfel C (2011) Cautionarynotes on the use of C-terminal BAK1 fusion proteins for functionalstudies. Plant Cell 23: 3871–3878

Ott T (2017) Membrane nanodomains and microdomains in plant-microbeinteractions. Curr Opin Plant Biol 40: 82–88

Pan X, Fang L, Liu J, Senay-Aras B, Lin W, Zheng S, Zhang T, Manor U,Chen W, Yang Z (2019) Auxin-induced nanoclustering of membranesignaling complexes underlies cell polarity establishment in Arabi-dopsis. bioRxiv 734665 doi:10.1101/734665

Perraki A, Cacas JL, Crowet JM, Lins L, Castroviejo M, German-Retana S,Mongrand S, Raffaele S (2012) Plasma membrane localization of Sola-num tuberosum remorin from group 1, homolog 3 is mediated by con-formational changes in a novel C-terminal anchor and required for therestriction of potato virus X movement. Plant Physiol 160: 624–637

Perraki A, Gronnier J, Gouguet P, Boudsocq M, Deroubaix AF, Simon V,German-Retana S, Legrand A, Habenstein B, Zipfel C, et al (2018)REM1.3’s phospho-status defines its plasma membrane nanodomainorganization and activity in restricting PVX cell-to-cell movement. PLoSPathog 14: e1007378

Platre MP, Bayle V, Armengot L, Bareille J, Marquès-Bueno MDM, CreffA, Maneta-Peyret L, Fiche JB, Nollmann M, Miège C, et al (2019)Developmental control of plant Rho GTPase nano-organization by thelipid phosphatidylserine. Science 364: 57–62

Platre MP, Noack LC, Doumane M, Bayle V, Simon MLA, Maneta-PeyretL, Fouillen L, Stanislas T, Armengot L, Pejchar P, et al (2018) A com-binatorial lipid code shapes the electrostatic landscape of plant endo-membranes. Dev Cell 45: 465–480

Prior IA, Harding A, Yan J, Sluimer J, Parton RG, Hancock JF (2001) GTP-dependent segregation of H-ras from lipid rafts is required for biologicalactivity. Nat Cell Biol 3: 368–375

Raffaele S, Bayer E, Lafarge D, Cluzet S, German Retana S, Boubekeur T,Leborgne-Castel N, Carde JP, Lherminier J, Noirot E, et al (2009) Re-morin, a solanaceae protein resident in membrane rafts and plasmo-desmata, impairs potato virus X movement. Plant Cell 21: 1541–1555

Raffaele S, Mongrand S, Gamas P, Niebel A, Ott T (2007) Genome-wideannotation of remorins, a plant-specific protein family: Evolutionaryand functional perspectives. Plant Physiol 145: 593–600

Raffaele S, Vailleau F, Léger A, Joubès J, Miersch O, Huard C, Blée E,Mongrand S, Domergue F, Roby D (2008) A MYB transcription factorregulates very-long-chain fatty acid biosynthesis for activation of thehypersensitive cell death response in Arabidopsis. Plant Cell 20: 752–767

Raghupathy R, Anilkumar AA, Polley A, Singh PP, Yadav M, Johnson C,Suryawanshi S, Saikam V, Sawant SD, Panda A, et al (2015) Trans-bilayer lipid interactions mediate nanoclustering of lipid-anchoredproteins. Cell 161: 581–594

Ren H, Willige BC, Jaillais Y, Geng S, Park MY, Gray WM, Chory J (2019)BRASSINOSTEROID-SIGNALING KINASE 3, a plasma membrane-associated scaffold protein involved in early brassinosteroid signaling.PLoS Genet 15: e1007904

Roche Y, Gerbeau-Pissot P, Buhot B, Thomas D, Bonneau L, Gresti J,Mongrand S, Perrier-Cornet JM, Simon-Plas F (2008) Depletion ofphytosterols from the plant plasma membrane provides evidence fordisruption of lipid rafts. FASEB J 22: 3980–3991

Rui Y, Dinneny JR (2020) A wall with integrity: Surveillance and main-tenance of the plant cell wall under stress. New Phytol 225: 1428–1439



Dow



Simon ML, Platre MP, Marquès-Bueno MM, Armengot L, Stanislas T,Bayle V, Caillaud MC, Jaillais Y (2016) A PtdIns(4)P-driven electro-static field controls cell membrane identity and signalling in plants. NatPlants 2: 16089

Singer SJ, Nicolson GL (1972) The fluid mosaic model of the structure ofcell membranes. Science 175: 720–731

Snead WT, Gladfelter AS (2019) The control centers of biomolecular phaseseparation: How membrane surfaces, PTMs, and active processes reg-ulate condensation. Mol Cell 76: 295–305

Son S, Oh CJ, An CS (2014) Arabidopsis thaliana remorins interact withSnRK1 and play a role in susceptibility to Beet Curly Top Virus and BeetSevere Curly Top Virus. Plant Pathol J 30: 269–278

Stegmann M, Monaghan J, Smakowska-Luzan E, Rovenich H, Lehner A,Holton N, Belkhadir Y, Zipfel C (2017) The receptor kinase FER is aRALF-regulated scaffold controlling plant immune signaling. Science355: 287–289

Su C, Klein M-L, Hernández-Reyes C, Batzenschlager M, Ditengou FA,Keller J, Delaux P-M, Ott T (2019) The Medicago truncatula DREPPprotein triggers microtubule fragmentation in membrane nanodomainsduring symbiotic infections. bioRxiv 794008 doi:10.1101/794008

Szymanski WG, Zauber H, Erban A, Gorka M, Wu XN, Schulze WX(2015) Cytoskeletal components define protein location to membranemicrodomains. Mol Cell Proteomics 14: 2493–2509

Tao K, Waletich JR, Arredondo F, Tyler BM (2019) Manipulating endo-plasmic reticulum-plasma membrane tethering in plants through fluo-rescent protein complementation. Front Plant Sci 10: 635

Tian T, Harding A, Inder K, Plowman S, Parton RG, Hancock JF (2007)Plasma membrane nanoswitches generate high-fidelity Ras signaltransduction. Nat Cell Biol 9: 905–914

Tian T, Plowman SJ, Parton RG, Kloog Y, Hancock JF (2010) Mathematicalmodeling of K-Ras nanocluster formation on the plasma membrane.Biophys J 99: 534–543

Tóth K, Stratil TF, Madsen EB, Ye J, Popp C, Antolín-Llovera M,Grossmann C, Jensen ON, Schüssler A, Parniske M, et al (2012)Functional domain analysis of the Remorin protein LjSYMREM1 in Lotusjaponicus. PLoS One 7: e30817

Vaahtera L, Schulz J, Hamann T (2019) Cell wall integrity maintenanceduring plant development and interaction with the environment. NatPlants 5: 924–932

Voxeur A, Fry SC (2014) Glycosylinositol phosphorylceramides from Rosacell cultures are boron-bridged in the plasma membrane and formcomplexes with rhamnogalacturonan II. Plant J 79: 139–149

Wang L, Li H, Lv X, Chen T, Li R, Xue Y, Jiang J, Jin B, Baluška F, Šamaj J,et al (2015) Spatiotemporal dynamics of the BRI1 receptor and its reg-ulation by membrane microdomains in living Arabidopsis cells. Mol Plant8: 1334–1349

Wang Q, Zhao Y, Luo W, Li R, He Q, Fang X, Michele RD, Ast C, vonWirén N, Lin J (2013) Single-particle analysis reveals shutoff control ofthe Arabidopsis ammonium transporter AMT1;3 by clustering and in-ternalization. Proc Natl Acad Sci USA 110: 13204–13209

Wang X, Goshe MB, Soderblom EJ, Phinney BS, Kuchar JA, Li J, Asami T,Yoshida S, Huber SC, Clouse SD (2005) Identification and functionalanalysis of in vivo phosphorylation sites of the Arabidopsis BRASSI-NOSTEROID-INSENSITIVE1 receptor kinase. Plant Cell 17: 1685–1703

Wang X, Kota U, He K, Blackburn K, Li J, Goshe MB, Huber SC, ClouseSD (2008) Sequential transphosphorylation of the BRI1/BAK1 receptorkinase complex impacts early events in brassinosteroid signaling. DevCell 15: 220–235

Wei Z, Tan S-T, Liu T, Wu Y, Lei J-G, Xue H-W, Liao K (2019) Expressingremorin in animal cells forms filamentous structure unraveling themechanism of plasmodesmata biogenesis. bioRxiv 791137 doi:10.1101/791137

Yi K, Guo C, Chen D, Zhao B, Yang B, Ren H (2005) Cloning and func-tional characterization of a formin-like protein (AtFH8) from Arabi-dopsis. Plant Physiol 138: 1071–1082

Zhao X, Li R, Lu C, Baluška F, Wan Y (2015a) Di-4-ANEPPDHQ, a fluo-rescent probe for the visualisation of membrane microdomains in livingArabidopsis thaliana cells. Plant Physiol Biochem 87: 53–60

Zhao X, Zhang X, Qu Y, Li R, Baluška F, Wan Y (2015b) Mapping ofmembrane lipid order in root apex zones of Arabidopsis thaliana. FrontPlant Sci 6: 1151

Zhou Y, Hancock JF (2015) Ras nanoclusters: Versatile lipid-based signal-ing platforms. Biochim Biophys Acta 1853: 841–849


Jaillais and Ott

Dow



Documents

The Nanoscale Organization of the Plasma Membrane and Its … · Update on Plasma Membrane Compartmentalization in Signaling The Nanoscale Organization of the Plasma Membrane and