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The laterally heterogeneous plant plasma membrane (PM) is organized into finely controlled specialized areas that include membrane-ordered domains. Recently, the spatial distribution of such domains within the PM has been iden- tified as playing a key role in cell responses to environmental challenges. To examine membrane order at a local level, BY-2 tobacco suspension cell PMs were labelled with an environment-sensitive probe (di-4-ANEPPDHQ). Four experimental models were compared to identify mechanisms and cell components involved in short-term (1 h) main- tenance of the ordered domain organization in steady-state cell PMs: modulation of the cytoskeleton or the cell wall integrity of tobacco BY-2 cells; and formation of giant vesicles using either a lipid mixture of tobacco BY-2 cell PMs or the original lipid and protein combinations of the tobacco BY-2 cell PM. Whilst inhibiting phosphorylation or disrupt- ing either the cytoskeleton or the cell wall had no observable effects, we found that lipids and proteins significantly modified both the abundance and spatial distribution of ordered domains. This indicates the involvement of intrinsic membrane components in the local physical state of the plant PM. Our findings support a major role for the ‘lipid raft’ model, defined as the sterol-dependent ordered assemblies of specific lipids and proteins in plant PM organization. Keywords: di-4-ANEPPDHQ, cell wall, cytoskeleton, lipid raft, membrane organization, ordered domains, protein-lipid interactions. Introduction Plant cells are delimited by a plasma membrane (PM) that and also plays a major role in transducing various signals into protects them against the external environment, and that also the appropriate adaptive responses. Since the advent of the regulates what (and how much) enters the cell. The PM defines classic ‘Fluid Mosaic Model’, consisting of a homogeneous the boundary between the intracellular and extracellular space lipid bilayer with embedded proteins arranged as mosaic-like Abbreviations: DOPC, 1,2- dioleoyl -sn-glycero-3- phosphocholine; DPPC, 1,2- dipalmitoyl -sn-glycero-3- phosphocholine; GUV, giant unilamellar vesicle; GVPM, giant vesicles of native plasma membrane; GPMV, giant plasma membrane vesicles; RGM, red-to-green ratio of membrane fluorescence; RGR, red-to-green ratio for a specific region of interest. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/jxb/article/69/15/3545/4990806 by DeepDyve user on 20 July 2022 3546 | Grosjean et al. structures (Singer and Nicolson, 1972), multiple lines of evi- While lipid-driven segregation can be coupled with add- dence have suggested the presence of a nanoscale lateral het- itional actin cytoskeleton-based processes to spatially organ- erogeneity with regards to the composition and biophysical ize the dynamic status of membrane proteins in animal cells properties of plant PMs. (Lenne et al., 2006), no similar actin-dependent mechanisms One of the original concepts developed within this frame- have yet been fully described in plant cells. Nevertheless, work is the ‘lipid raft’ model (Simons and Ikonen, 1997; interactions and crosstalk between microtubules and micro- Simons and Gerl, 2010; Nicolson, 2014), which is based on filaments have been reported in plant cells (Petrásek and the sub-division of membranes into regions further defined Schwarzerova, 2009), revealing the role of microtubule as ‘small (10–200 nm), heterogeneous, highly dynamic, sterol- organization in modulating plant protein motility (Szymanski and sphingolipid-enriched domains’ (Pike, 2006). According to et al., 2015; Lv et al., 2017). Furthermore, the pattern of cel- this model, preferential interactions between cholesterol and lulose deposition in the cell wall has been shown to strongly sphingolipids generate liquid-ordered (Lo) phase separation affect the trajectory and speed of PM protein diffusion in (Phillips et al., 1970; Shimshick and McConnell, 1973; Grant Nicotiana tabacum leaves (Martinière et al., 2012), suggesting et al., 1974; Lentz et al., 1976), in which the resulting mem- the involvement of the cell wall in regulating plant PM lateral brane domains are highly ordered and tightly packed relative organization. to the surrounding regions (Simons and Sampaio, 2011). The These previous reports indicate that the plant PM con- similar ability of plant-specific sterols to form an Lo phase has sists of heterogeneously distributed components, in which been reported in PM-purified fractions (Roche et al., 2008) grouped localization has been demonstrated for some lipid as well as in living tobacco cells (Gerbeau-Pissot et al., 2014), raft markers, including proteins and lipids. However, few with diffing capabilities to stabilize the lipid bilayer depending studies have characterized the spatial distribution of these on the phytosterol structure (Rujanavech et al., 1986; Schuler clusters at the whole-cell scale. Nonetheless, these data have et al., 1990, 1991; Hartmann, 1998; Halling and Slotte, 2004). independently described specific ways that, either alone or This capacity of phytosterols to modulate the size and pro- in combination, can account for PM spatial organization in portion of the Lo phase in the model membrane has been plant cells (Ovecka et al., 2010; Li et al., 2012; Gerbeau- associated with their ability to interact with plant sphingolipids Pissot et al., 2014; Sandor et al., 2016), and they justify a (Grosjean et al., 2015). comprehensive analysis of the global mechanisms responsible Enrichment in sterols and sphingolipids has been identified for the distribution of ordered domains. In order to obtain for detergent-resistant membrane (DRM) fractions from the a snapshot evaluation of the respective influence of each of PMs of tobacco (Mongrand et al., 2004; Moscatelli et al., 2015), these different molecular and cellular elements on the fea- Arabidopsis thaliana (Borner et al., 2005; Minami et al., 2009), tures of PM lateral organization, we have compared the Medicago truncatula (Lefebvre et al., 2007), leek (Laloi et al., biophysical properties of several experimental systems with 2007), maize and bean (Carmona-Salazar et al., 2011), con- decreasing complexity, all of which correspond to tobacco firming their association within specific membrane regions. In BY-2 cells in a resting state. We used di-4-ANEPPDHQ, the tobacco PM, use of immunogold electron microscopy has a lipid packing-sensitive dye capable of assessing different revealed localization of glycosyl inositol phosphorylceramide levels of membrane order, coupled with confocal micros- (GIPC), the major class of sphingolipids in plants, within 35-nm copy. This approach allowed us to measure the global level diameter domains (Cacas et al., 2016). Phosphatidylinositol of membrane order of the entire PM together with the spa- 4,5-bisphosphate (PIP2) is similarly found in ~25-nm diam- tial distribution of ordered domains in either intact tobacco eter clusters (Furt et al., 2010), suggesting that there is a het- cells, cells with a disrupted cytoskeleton, or cells devoid of a erogeneous spatial distribution of the lipids on a nanometre cell wall. The same parameters were also measured in model scale. Furthermore, various proteins have also shown a grouped membranes including giant unilamellar vesicles (GUVs) distribution (about 70 nm in size) in tobacco leaf (Raffaele composed of lipids extracted from the tobacco PM, and et al., 2009), tobacco BY-2 suspension (Noirot et al., 2014), giant vesicles of native PMs (GVPMs) from tobacco cells and Arabidopsis (Li et al., 2012) cells. Plant cell PMs are thus (see Fig. 1 for rationale). Our comprehensive comparisons covered with different types of domains enriched in specific of the individual ability of membrane and cell elements to membrane-resident proteins (Jarsch et al., 2014). Interestingly, modify membrane order revealed a major role for lipids in the integrity of some of these protein clusters is sensitive to promoting the formation of ordered domains, and the cap- the amount of sterol, as observed after methyl-β-cyclodextrin acity of proteins to limit this formation. Our results suggest (MβCD)-induced sterol depletion (Vereb et al., 2000; Raffaele that PM components appear to act together to regulate plant et al., 2009), which suggests sterol-driven formation of these PM heterogeneity, whilst neither the cytoskeleton nor the protein-enriched domains. Furthermore, a close relationship cell wall seem to play a significant role in the short-term between the sterol amount and polar localization of auxin control of ordered domain distribution within steady-state efflux carriers has been proposed, since sterol methyltrans- cell PMs. Phosphorylation events also failed to regulate PM ferase 1 (SMT1) and sterol-dependent endosomal recycling organization of tobacco cells in a resting state. Finally, we are essential in maintaining the polar localization of PIN1 and discuss how our results may support the ‘lipid raft’ model in PIN3 (Willemsen et al., 2003), in addition to PIN2 (Men et al., our understanding of the mechanisms that control the spatial 2008; Titapiwatanakun and Murphy, 2009). distribution of ordered domains within the plant PM. Downloaded from https://academic.oup.com/jxb/article/69/15/3545/4990806 by DeepDyve user on 20 July 2022 Cellular determinants of tobacco cell plasma membrane organization | 3547 Materials and methods Cell growth conditions Wild-type BY-2 (Nicotiana tabacum cv. Bright Yellow-2) cells were grown in Murashige and Skoog (MS) modified medium (basal salt mixture, –1 M0221, Duchefa) at pH 5.6, supplemented with 1 mg l thiamine-HCl, –1 –1 0.2 mg l 2,4 dichlorophenylacetic acid, 100 mg l myo-inositol, 30 g –1 –1 –1 l sucrose, 200 mg l KH PO , and 2 g l MES. Cell suspensions were 2 4 –2 –1 maintained under continuous light conditions (200 µE m s ) on a rotary shaker (140 rpm) and diluted (4:80) weekly into fresh medium. Chemicals treatments BY-2 cells were equilibrated according to Gerbeau-Pissot et al. (2014). After a 2-h cell incubation period, concentrated stock solutions (1000× in DMSO) of the cytoskeleton inhibitors cytochalasin D, latrunculin B, nocodazole, and oryzalin (Sigma-Aldrich), were individually added to cell suspensions at a final concentration of 50 µM, 10 µM, 20 µM, and 10 µM, respectively. Control cells were incubated with the same dilu- tion of DMSO. Cells were treated for 1 h on a rotary shaker (120 rpm) at 25 °C before observation. Cells were subsequently plasmolysed in I2 (0.5 mM CaCl , 0.5 mM K SO , and 2 mM MES, pH 5.9) containing 2 2 4 400 mM mannitol (instead of 175 mM used by Gerbeau-Pissot et al., 2014) for several minutes before observation. Staurosporine (Sigma- Aldrich) was added to the cell suspension from concentrated stock solu- tions in DMSO, taking care not to exceed the final DMSO concentration (0.5 % v/v). Protoplast preparation and cell wall regeneration The protoplast preparation protocol was adapted from (Zaban et al., 2013). All steps were performed in sterile conditions. The BY-2 cells were collected and centrifuged at 100 g. Cells were washed in 0.4 M mannitol at pH 5.5 and centrifuged again, then resuspended in an enzymatic solu- tion (Pectolyase Y23 0.1 % w/v, cellulose Onozuka RS 1 % w/v in 0.4 M mannitol at pH 5.5) and digested for 4–5 h at 25 °C, under shaking at 65 rpm in Petri dishes. Protoplasts were harvested after centrifugation (500 g for 5 min) and washed three times in FMS wash medium (4.3 g –1 –1 –1 –1 l MS salts, 100 mg l myo-inositol, 0.5 mg l nicotinic acid, 0.5 mg l –1 –1 pyridoxine-HCl, 0.1 mg l thiamine, 10 g l sucrose in 0.25 M man- nitol, pH 5.8). For cell wall regeneration, protoplasts were transferred to –1 FMS-store medium (FMS with 0.1 mg l 1-naphthaleneacetic acid and –1 1 mg ml benzylaminopurin) and incubated at 26 °C in the dark, with shaking in Petri dishes. Protoplasts were observed at 0, 24 h, 48 h, and 5 d after digestion. Preparation of GUVs Giant unilamellar vesicles (larger than 10µm) were prepared as follows. Tobacco PM isolation PM fractions were obtained from BY-2 cells by membrane partitioning in an aqueous polymer two-phase system with polyethylene glycol 3350/ dextran T-500 (6.6% each), according to Mongrand et al. (2004). Protein Fig. 1. Different models used to characterize the cellular determinants of content was quantified using the Bradford method, in order to obtain an plasma membrane order. (A) Intact whole BY-2 suspension cells in which –1 aliquoted solution of 10 mg ml final concentration. the plasma membrane, composed of ordered domains (in orange), is tightly connected with the underlying filaments of the cytoskeleton network and/or the surrounding fibres of the cell wall meshwork. (B) Treatment Purification and quantification of tobacco PM lipids of BY-2 suspension cells with chemicals disrupts the cytoskeletal Polar lipids were extracted according to three independent methods components. (C) Protoplasts obtained by enzymatic digestion of BY-2 detailed in Cacas et al. (2016) and based on different extraction solv- suspension cells. (D) Giant vesicles were either formed from the whole ent mixtures, namely chloroform/methanol/HCl (200/100/1, v/v/v), diversity of tobacco PM lipids (giant unilamellar vesicles, GUVs) or from methyl tert-butyl ether (MTBE)/methanol/water (100/30/25, v/v/v), or the direct electrofusion of purified PM vesicles, thus containing both a lower phase of propan-2-ol/hexane/water (55/20/25, v/v/v). GIPCs proteins and lipids in their original amounts (giant vesicles of native plasma were purified according to a method adapted from Carter and Koob membrane, GVPMs). Samples were observed by microscopy, using either (1969) to obtain sufficient amounts for GUV production and lipid quan- differential interference contrast (DIC; in grey-scale), or fluorescence after tification, as described in Buré et al. (2011). Extracted lipids were dis- di-4-ANEPPDHQ labelling (excitation at 488 nm; emission acquired in a solved in chloroform/methanol/water (30/60/8, v/v/v) for storage and 520–680 nm band-pass, in yellow). Scale bars are 5 µm. further quantified by GC-MS according to Buré et al. (2011). Downloaded from https://academic.oup.com/jxb/article/69/15/3545/4990806 by DeepDyve user on 20 July 2022 3548 | Grosjean et al. GUV production measuring the distribution of the liquid-ordered/liquid-dis- GUVs were prepared by electroformation in a flow chamber (ICP-25 Perfusion ordered (Lo/Ld) phases (Gerbeau-Pissot et al., 2014). In order Imaging Chamber, Dagan) connected to a function generator (PCGU1000, to identify key players that govern this PM lateral organiza- Velleman) and a temperature controller (TC-10, Dagan). Tobacco PM frac- tion, we exploited the fluorescence properties of the environ- tions (2 µg of proteins) or a mixture of tobacco PM lipids corresponding to a ment-sensitive dye di-4-ANEPPDHQ (Jin et al., 2005), the final phospholipid/sphingolipid/sterol composition of 4/4/1.5 (w/w/w, 2 µg final) were deposited on two microscope slides (18 × 18 mm) coated with red/green ratio of which is inversely correlated with the level electrically conductive indium tin oxide (resistivity 8–12 ohms). Lipid-coated of membrane order (Jin et al., 2006). BY-2 suspension cells slides were placed under a vacuum and away from light for at least 2 h until a were stained for 2 min with di-4-ANEPPDHQ (3 µM) and thin film was obtained. Cover slips were set up in the flow chamber, and the observed by confocal microscopy. We then measured the fluor- lipid layer was rehydrated with 200 µl of swelling solution (25 mM HEPES, escence intensities of the entire PM and evaluated the mem- 10 mM NaCl, and 100 mM sucrose) pre-heated to 40 °C for lipid GUVs. A voltage of 3.5 V (adjustable during the experiment) at 10 Hz and a tem- brane order using the RGM parameter (red-to-green ratio of perature of 40 °C were applied for a 2-h minimum period in a light-protected the membrane, RGM=I /I ). To characterize the spatial 660 550 environment. After lipid swelling, the temperature of the chamber was slowly distribution of PM ordered domains, we calculated the RGM cooled to 22 °C (2 h minimum cooling time). value of the PM in tobacco cell regions, hereafter referred to as RGR for ‘red-to-green ratio of the ROI’, where ROI is a Fluorescence labelling ‘region of interest’ corresponding to a 300 × 300 nm square. To examine cytoskeleton integrity, rhodamine-phalloidin (Invitrogen, To test for the contribution of active pathways in the con- –1 TM 0.1 mg ml , 30 min) and Tubulin Tracker (Invitrogen, 50 µM, 45 min) trol of these parameters, tobacco suspension cells were treated were used to detect actin filaments and microtubules, respectively. To with staurosporine, a broad-spectrum protein-serine/threonine determine whether the cell wall was present samples were examined after kinase inhibitor (Suzuki and Shinshi, 1995). Addition of stauro- staining with calcofluor-white (Sigma-Aldrich, 0.01 %, w/v) for several minutes. The resulting fluorescence signal of this product reveals cellulose sporine (2.5 µM) failed to modify the RGM, regardless of the and chitin structures. To determine the membrane order, GUV suspensions incubation time (from 10 min to 3 h, Fig. 2). In contrast, we were or tobacco cells were labelled with the fluorescent probe 1-[2-Hydroxy- able to determine a significant modification in membrane order 3-(N,N-di-methyl-N-hydroxyethyl)ammoniopropyl]-4-[β-[2-(di-n- using the same experimental set-up under conditions known butylamino)-6-napthyl]vinyl] pyridinium dibromide (di-4-ANEPPDHQ; to induce an increase PM order (Supplementary Fig. S1 at JXB Invitrogen, stock solution in DMSO, 3 µM final, 1–2 min). online). This indicates that the technique is sensitive enough to detect minor variations, and that the conditions are suffi- Confocal fluorescence microscopy cient to inhibit signalling events (Bonneau et al., 2010; Sandor Labelled cells were deposited between the slide and the cover slip and et al., 2016), demonstrating that the maintenance of membrane observed with a Leica TCS SP2-AOBS laser scanning microscope (Leica order is independent of phosphorylation events. These results Microsystems) coupled to a HCPL Apochromat CS 63× (N.A. 1.40) suggested that the signalling pathway was not involved in the oil immersion objective. Fluorescence excitations were obtained using either the 543-nm line of a helium-neon laser (rhodamine-phalloidin), control of PM order during the course of the experiment. The the 488-nm line of an argon laser (tubulin tracker), or a 405-nm diode results prompted us to address the issue of short-term regulation (calcofluor). Fluorescence emissions were recorded between 555–700 nm of ordered domain organization via other mechanisms, espe- (rhodamine-phalloidin), 500–600 nm (tubulin tracker), and 410–480 nm cially the contribution of physical interactions. (calcofluor). For di-4-ANEPPDHQ observation, after excitation at 488 nm, emission intensities were acquired between 540–560 nm (green image) and between 650–670 nm (red image). Ratiometric imaging was performed using the ImageJ software (http://imagej.nih.gov/ij/). Fluorescence spectroscopy Sample solutions (1 ml) were placed in a 10-mm special optic path glass cuvette filled in a thermoelectric cooler (24 °C, Wavelength Electronics, Inc.). Fluorescence measurements were performed using a Fluorolog-3 FL3-211 spectrometer (Jobin-Yvon, Horiba Group). The emission spec- trum was monitored by one photomultiplier (520–700 nm). A xenon arc lamp (488 nm) was used as the light source. Data acquisitions were per- formed using the Datamax software (Jobin-Yvon/Thermo Galactic, Inc.). Statistical tests Statistical analyses were based on non-parametric tests (Mann–Whitney), since we observed that our data exhibited a non-Gaussian distribution. Fig. 2. Effect of a protein kinase inhibitor on membrane order of living tobacco cells. After incubation (for 10 min, 1 h, or 3 h) with the kinase Results inhibitor staurosporine (Stau; 2.5 µM), cells were labelled for 2 min with di-4-ANEPPDHQ (3 µM). Cells were subsequently analysed by Phosphorylation does not regulate the level of PM spectrofluorimetry, and membrane order was quantified using the red- order or the distribution of ordered domains to-green membrane fluorescence ratio (RGM, =I /I ). The RGM of 660 550 PMs from treated cells is shown as a relative value compared to control We previously described the heterogeneity of tobacco PM cells (Ctl, with equivalent volume of DMSO). Data are means (±SD), n=26 independent experiments. lateral organization with respect to local membrane order by Downloaded from https://academic.oup.com/jxb/article/69/15/3545/4990806 by DeepDyve user on 20 July 2022 Cellular determinants of tobacco cell plasma membrane organization | 3549 Cytoskeleton remodelling does not modify the cause disturbance of the cytoskeleton (Fig. 3C) without affect- organization of PM-ordered domains ing cell viability (Supplementary Fig. S2). Spectrofluorimetry measurements, which can assess PM order in batches of thou- To analyse the potential relationship between the cytoskel- sands of live tobacco cells, confirmed that PM order was inde- eton and BY-2 cell PM order, we measured the evolution of pendent of actin filament or microtubule polymerization PM order in response to different compounds that affect cyto- (Supplementary Fig. S3). Simultaneous addition of latrunculin skeleton components. Brief incubations were performed to B and oryzalin also did not result in any significant differences exclude any long-term metabolic regulation. Latrunculin B between the RGM of control and treated cells (Supplementary (Spector et al., 1983) and cytochalasin D (Cooper, 1987) bind Fig. S3), indicating the independence of this parameter with to actin monomers and prevent their polymerization; their regards to cytoskeleton integrity in living tobacco cells. application led to the disruption of actin filaments (Fig. 3A), However, modifications to the lateral organization of mem- without any effect on plant cell viability during the 1-h brane order could occur at the nanometre scale without affect- experiment (Supplementary Fig. S2). Monitoring single cells ing the global RGM. Indeed, the RGM represents the mean by confocal microscopy showed that these treatments failed value of a multitude of small areas (ROIs) that exhibit different to significantly change BY-2 cell RGM (Fig. 3B). No sig- levels of local membrane order (RGR values): the distribution of nificant differences in RGM were observed between control individual values may be different even though the overall aver- cells and cells treated with nocodazole (Samson et al., 1979) age remains the same. To test this possibility, the emission signals or oryzalin (Morejohn and Fosket, 1984) (Fig. 3B), both of of fluorescently labelled cells were acquired on a tangential plane corresponding to membrane surfaces of 100–500 µm which can interfere with microtubule polymerization and , and the Fig. 3. Influence of the cytoskeleton on the membrane order of tobacco cell PMs. (A, C) Effect of pharmacological treatments on cytoskeleton integrity. BY-2 cells were incubated for 1 h with either latrunculin B (Lat, 10 µM), cytochalasin D (Cyt, 50 µM), nocodazole (Noc, 20 µM), or oryzalin (Ory, 10 µM), or with the same concentration of DMSO (control, Ctl). The cytoskeleton was observed by fluorescence microscopy using (A) rhodamine-phalloidin for –1 actin colouration (0.1 mg ml ) and (C) tubulin tracker for microtubule staining (50 µM). After 1 h, patches were detected on treated cells, in comparison to the intact network of filaments observed on control cells. Scale bars are 20 µm. (B) Effect of cytoskeleton integrity on global membrane order. BY-2 cells were exposed to pharmacological treatments that disrupted the actin or tubulin meshwork (as detailed above), or both (Lat, 10 µM and Ory, 10 µM). After 1 h of incubation, cells were labelled for 2 min with di-4-ANEPPDHQ (3 µM). Cells were then observed by confocal microscopy, and membrane order was quantified using the red-to-green membrane fluorescence ratio (RGM, =I /I ). The RGM values obtained for treated cells are shown relative to the 660 550 values of untreated cells (Ctl, in DMSO). Data are means (SEM), n=96–428 from at least five independent experiments. (D) Effect of cytoskeleton integrity on the organization of ordered domains. The distribution of red-to-green membrane fluorescence values (RGR) of individual regions of interest (ROIs, 300 × 300-nm squares) of the PM (control conditions, Ctl) is not influenced by latrunculin B, 10 µM, 1 h). The x-axis corresponds to the class of RGR values, and only the maximal value of each class is indicated on the graph. The y-axis corresponds to the ROI percentage of each class. Data are means (±SEM), n=111–428 cells from at least five independent experiments. Downloaded from https://academic.oup.com/jxb/article/69/15/3545/4990806 by DeepDyve user on 20 July 2022 3550 | Grosjean et al. relative abundance of Lo/Ld phases was evaluated. We did not The cell wall does not affect the organization of observe any significant difference in the distribution of RGR PM-ordered domains values between control and latrunculin B-treated cells (Fig. 3D). To investigate the influence of the cell wall on PM order, we Furthermore, all of the microtubule- and actin-depolymerizing compared the RGM of freshly prepared protoplasts devoid of agents that we tested failed to modify the distribution of RGR cell walls (1–3 h after enzymatic digestion) and after 24 h of values (Supplementary Fig. S4). A granulometric analysis char- cell wall regeneration (Fig. 5A). The presence of a newly syn- acterizing the aggregation of the most ordered domains was then thesized cell wall, visualized by staining with calcofluor-white performed, with a focus on ROIs exhibiting a value in the first (Fig. 5A), did not modify the RGM value (Fig. 5B), suggesting quartile of RGR values (Fig. 4–C). This approach did not reveal cellulose deposition has no role in the control of PM order. any significant difference between the size of ordered domains Correspondingly, no modifications in RGM were measured in cells treated with cytoskeletal disruptors and the control cells between control and plasmolysed cells when plasmolysis was (Fig. 4D). Taken together, our data suggest that cytoskeleton induced and protoplast shrinkage kept the PM away from the integrity has no short-term effect on the level of membrane cell wall (Supplementary Fig. S5), suggesting no role in mem- order, either globally (across the entire PM surface) or locally brane packing for the bounded regions between the cell wall (within small areas at our scale of observation). and the PM. Furthermore, the absence of a direct effect of the cell wall suggests that there is no direct contribution from cell wall–PM connections on the regulation of PM order. A characterization of the abundance of ordered domains was then performed, although the spherical shape of protoplasts limited the size of the tangential area that could be analysed (10–100 µm ). No significant difference was observed between the freshly prepared protoplasts and protoplasts in which the cell wall had been regenerated (Fig. 5C), with both conditions displaying the same distribution of RGR values. Live ratiomet- ric imaging also indicated a similar size for ordered domains in protoplasts, before and after cell wall regeneration (Fig. 5D). Thus, the cell wall does not seem to influence the spatial dis- tribution of ordered domains within the tobacco PM. Production of giant vesicles from tobacco PMs reveals the involvement of lipids and proteins in the control of the spatial distribution of ordered domains The data presented above highlight the possibility of a crucial role for intrinsic PM components with regards to PM-ordered domains. To assess the involvement of a wide diversity of PM lipids in the regulation of membrane order, we prepared giant unilamellar vesicles (GUVs) using a mixture of the different classes of lipids in their relative proportions as found in native BY-2 cell PMs. A careful lipidomic analysis highlighted the Fig. 4. Influence of the cytoskeleton on the spatial distribution of ordered large diversity of BY-2 PMs (Supplementary Fig. S6), with a domains. (A) Observation of di-4-ANEPPDHQ-labelled membrane surfaces phospholipid/sphingolipid/sterol ratio of 4/4/1.5 (w/w/w). (excitation at 488 nm; emission corresponds to a 520–680 nm band-pass) The RGM value of these GUVs labelled with di-4-ANEPP- with the grey-scale representing fluorescence intensity. (B) The subsequent DHQ was 0.89 ± 0.16 (±SD, n=29). Taking into account the ratiometric image describing the red-to-green fluorescence ratio of the vast array of lipids that comprise PMs, we then characterized PM region of interest (RGR) of di-4-ANEPPDHQ within 300 × 300-nm membrane areas displayed in a grey-scale colour-coded representation. the ability of fatty acid saturation to increase membrane order. (C) A binarized image, representing a detail extracted from the membrane The results showed that RGM significantly decreased from surface focused on the most ordered domains. Recorded regions of 2.47 ± 0.24 (n=38) to 1.49 ± 0.16 (n=20) for GUVs consisting interest (ROIs) exhibiting an RGR value within the first quartile of lower of only 1,2 dioleoylphosphatidylcholine (DOPC) or DOPC/ RGR values (the most ordered ones) are represented as black pixels. DPPC (DPPC: 1,2 dipalmitylphosphatidylcholine; 1/1, mol/ (D) A granulometric approach was then used to compare the spatial distribution of ordered domains on the membrane surface under the mol), respectively (Supplementary Fig. S7). The complexity different experimental conditions. Tobacco suspension cells were treated level of different lipid combinations (in terms of both num- for 1 h with a cytoskeletal-active compound (latrunculin B, Lat, 10 µM; ber and composition of the different lipid families) similarly cytochalasin D, Cyt, 50 µM; nocodazole, Noc, 20 µM; or oryzalin, Ory, modified the GUV membrane order (Supplementary Fig. S7). 10 µM) or a control (Ctl) before labelling with di-4-ANEPPDHQ (3 µM, Overall, this suggests that the diversity of lipid molecules rep- 2 min). The mean area of the black ROI groups is shown. Data are means (±SD), n>27 cells from five independent experiments. No significant resented in BY-2 cell PMs, together with their specific ability differences were observed (P>0.05). to organize the membrane (Grosjean et al., 2015), could be Downloaded from https://academic.oup.com/jxb/article/69/15/3545/4990806 by DeepDyve user on 20 July 2022 Cellular determinants of tobacco cell plasma membrane organization | 3551 Fig. 5. Influence of the cell wall on PM order of BY-2 protoplasts. (A) Cell morphology (differential interference contrast, DIC) and cellulose deposition (fluorescence imaging after calcofluor-white coloration) were analysed for protoplasts, either freshly prepared (3 h) or after cell wall regeneration (1 d after protoplast preparation). (B–D) Confocal microscopy was used to characterize di-4-ANEPPDHQ-labelled protoplasts (3 µM, 2 min), either freshly prepared (FP) or after cell wall regeneration (PCW). (B) Global membrane order was quantified using the red-to-green membrane fluorescence ratio (RGM, =I /I ). (C) Local membrane order was estimated in each region of interest (ROI) of the fluorescent PM, and the distribution of RGM values of 660 550 individual ROIs of corresponding PMs (RGR) is shown. The x-axis corresponds to the class of RGR values, and only the maximal value of each class is indicated on the graph. The y-axis corresponds to the ROI percentage of each class. (D) The size of ordered domains was measured as the mean area of groups of pixels corresponding to ROIs exhibiting an RGR value belonging to the first quartile of lower values. Data are means (±SD), n>66 cells from five independent experiments. Fig. 6. Influence of lipids and proteins on the global and local order of tobacco PMs. Giant unilamellar vesicles (GUVs) were produced by mixing extracts of purified lipid classes according to their relative amounts within the membrane (phospholipids/phytosphingolipids/phytosterols: 4/4/1.5 w/w/w) or by fusing isolated PM vesicles (giant vesicles of native plasma membrane, GVPMs). Vesicles were labelled with di-4-ANEPPDHQ (3 µM, 2 min). (A) Transverse (top) and tangential (below) views of fluorescent vesicles were made using confocal microscopy (excitation at 488 nm; emission corresponds to the sum of fluorescence intensities acquired in a 520–680 nm band-pass) of GUVs and GVPMs. (B) The red-to-green fluorescence ratio (RGM, =I /I ) of fluorescent vesicles was measured using confocal microscopy (dividing the red, 545–565 nm, by the green, 635–655 nm, emission band- 660 550 passes). Data are means (±SD), n>24 experiments; significant difference: *P<0.05. (C) Individual regions of interest (ROIs, 300 × 300 nm) of the surface of fluorescent vesicles were classified according to their RGM values (i.e. giving RGR values). The RGR distribution of a representative GUV composed of a PM lipid mixture is compared to the distribution of GVPMs. The x-axis represents the class of RGR values; only the maximal value of each class is indicated on the graph. The y-axis represents the percentage of each class of ROI values. (D) The size of ordered domains was measured as the mean area of groups of pixels corresponding to ROIs exhibiting an RGR value belonging to the first quartile of lower values, and is compared for giant vesicles composed of either PM lipids (GUV) or PM lipids and proteins (GVPM). Data are means (±SD), n>20 vesicles from five independent experiments. responsible for the high membrane order reported here for mechanisms involved in PM vesicle fusion induced by deter- GUVs composed of tobacco PM lipids. gents cause significant artefacts, e.g. bilayer–micelle transi- In addition to lipid–lipid interactions, protein–lipid interac- tion (Alonso et al., 1982). Furthermore, technical limitations tions are essential contributors to PM organization (van den restrict the protein levels included in these proteoliposomes Bogaart et al., 2011). Hence it is of primary interest to compare to 5% (Kahya et al., 2005), far below the level of native PMs, the characteristics of GUVs composed of tobacco PM lipids (as which is estimated to be 50% by weight; consequently, the use detailed above) with GUVs containing lipids and proteins from of these approaches is restricted. Giant PM vesicles (GPMVs), tobacco PMs in their native amounts. However, the molecular corresponding to PM blebs detached from cells, have a protein Downloaded from https://academic.oup.com/jxb/article/69/15/3545/4990806 by DeepDyve user on 20 July 2022 3552 | Grosjean et al. and lipid diversity mirroring the native PM (Scott, 1976) and Fig. S9A), confirming that the reduction in ordered domain separate into co-existing Lo/Ld phases, which enables the abundance was predominantly involved in the low membrane investigation of the structural determinants of ordered domain order reported for GVPMs (Fig. 6B). To determine the lat- association (Baumgart et al., 2007; Kaiser et al., 2009; Levental eral organization of these ordered domains within PM vesi- et al., 2011). However, the cell wall surrounding the plant cell cles, we analysed their degree of clustering. The group size of prevents the use of this procedure for tobacco cells. We there- ordered domains revealed a protein-dependent decrease, with fore developed a new protocol to produce giant vesicles of the presence of ordered domains exhibiting a mean size of 2 2 native PMs (GVPMs) by electrofusing small vesicles of puri- ~0.8 µm in GUVs composed of only PM lipids and ~0.6 µm fied PM fractions. However, GUV formation using purified in GVPMs (Supplementary Fig. S9B). We observed a linear PM fractions was extremely difficult due to the presence of correlation between the size of ordered domains characterizing proteins and the obstacle they presented to fusion, and so the GUVs composed of different lipid mixtures and the membrane electroformation method was modified by varying time, tem- order (Supplementary Fig. S9C). This correlation disappeared perature, osmolarity, voltage, and frequency in order to enable in GVPMs (Supplementary Fig. S9C), suggesting that the pres- a high yield of GVPM formation (Supplementary Fig. S8A). ence of proteins decreases the number of ordered domains, but By comparing the different protocols, we were able to effi- concomitantly induces another mechanism that can reduce the ciently produce GVPMs (10 vesicles starting from 2 µg lipids) propensity of ordered domains to lie within clusters. with a size amenable to observation by confocal microscopy Taken together, these results support the ability of PM lipids (Supplementary Fig. S8B). Under optimized conditions, 20% and proteins to finely govern PM order, and to subsequently of the GVPM population had a diameter larger than 15 µm. control the tenuous organization of the PM. The procedure (described in detail the Methods section) can now be used routinely to efficiently prepare GVPMs directly Discussion from purified PMs. As has been previously reported (Takahashi et al., 2013), Lipids and proteins can account for the structuring of protein abundance can modify the contours of vesicles, allow- plant PM ordered domains ing the formation of soft GVPMs with a non-spherical shape (Fig. 6A). Moreover, GVPMs containing proteins and lipids In this study we determined a high level of membrane order for exhibited a higher RGM than GUVs formed with the same GUVs mimicking the native composition of tobacco BY-2 PM lipid mixture (Fig. 6B), indicating that proteins tend to limit lipids. This included 38% GIPCs, 8% free phytosterols, and 10% the packing of the membrane. conjugated phytosterols, which were associated with a large and To compare local membrane order at the surface of these continuous distribution of individual levels of membrane order GUVs and GMPVs, the emission signals of fluorescently exhibited by different membrane regions of these vesicles. One labelled vesicles were acquired on a tangential plane corre- possible explanation is that the ‘lipid raft’ model is also applicable sponding to membrane surfaces of 100–350 µm (Fig. 6A), to plants. This model places the local interactions between lipid and the distribution of RGR values was determined (Fig. 6C). species (Ramstedt and Slotte, 2006; Quinn, 2010) as the first The distribution for PM lipid GUVs was centred towards low level of membrane organization (Lingwood and Simons, 2010). values (from 0.6–1.1; Fig. 6C), consistent with the high mem- Consistent with this hypothesis, plant-specific conjugated sterols brane order of these GUVs (Fig. 6B). In contrast, the RGR display a striking ability to induce ordered domain formation in value of GVPMs exhibited a more peaked distribution shifted the membrane that acts in synergy with the similar ability of free towards higher RGR values (from 1.1–1.4; Fig. 6C), suggest- phytosterols (Grosjean et al., 2015). Indeed, phytosterols increase ing that the presence of protein induced a shift towards fewer membrane stiffness and the inclusion of other lipids, depending ordered domains. To investigate a possible concomitant adjust- on their structure and shape (Shahedi et al., 2006), resulting in ment of the spatial organization of ordered domains, a gra- macromolecular assemblies and lipid bilayer ordering (Mannock nulometric analysis was performed by focusing on ROIs that et al., 2015; Shaghaghi et al., 2016). Furthermore, formation of exhibit an RGR value in the first quartile of values, in order to sterol-dependent membrane domains is modified by the add- eliminate any density effects. We measured an ordered domain ition of GIPCs (Grosjean et al., 2015). This adds weight to a size of 0.342 µm for GVPMs (Fig. 6D); interestingly this was model in which the combination of multiple molecular species similar to the value for BY-2 cells (i.e. 0.350 µm , Fig. 4D). of phytosphingolipids and phytosterols present in living tobacco Moreover, this size tended to be lower than the size of GUVs cell PMs may induce membrane ordering and enhance the for- composed of a PM lipid mixture (Fig. 6D), suggesting a nega- mation of various ordered domains. The high diversity of plant tive impact of the presence of protein on both the quantity and PM lipids could then increase opportunities for local interac- size of ordered domains at our scale of observation. To better tions with different intensities, and consequently bring about understand the dissolution of the ordered domains in native heterogeneity at the local membrane compaction level, provid- PM conditions, ratiometric images were further segmented ing an explanation for the wide range of RGR values measured to only take into account ROIs exhibiting red/green ratios for GUVs consisting of tobacco PM lipid components. RGR below a RGR value of 1.2, which corresponds to one popu- values, which corresponded to average ratios of the smallest lation of ordered domains (Grosjean et al., 2015). This ordered area optically possible to analyse (a 300 × 300 nm ROI), might domain fraction was calculated, and a significant decrease was reflect the mean membrane order of sub-population domains observed for GVPMs in comparison to GUVs (Supplementary co-existing in varying proportions within these areas. Such a Downloaded from https://academic.oup.com/jxb/article/69/15/3545/4990806 by DeepDyve user on 20 July 2022 Cellular determinants of tobacco cell plasma membrane organization | 3553 complex model assumes a multitude of nanodomains with lev- of proteins to emulsify ordered domains (Bhatia et al., 2016). els of order between (rather than strictly corresponding to) the This PM organization should be based on hydrogen bonds and Lo and Ld phases (Bagatolli et al., 2010). In agreement with this van der Waals interactions operating on a time scale represen- new degree of complexity of PM organization, the diversity of tative of what we observed, and should show, in a coherent mammalian lipid mixtures has been speculated to favour the manner, an insensitivity to protein kinase inhibitors. formation of ultra-nanodomains (Pathak and London, 2015). In accordance with this, the formation of distinct nanodomains The cell wall–PM–cytoskeleton continuum is not a has been simulated in the outer leaflet of an idealized mamma- common hallmark of domain assembly lian PM consisting of a complex mixture of 63 different lipid species (Ingólfsson et al., 2014). Besides local membrane composition, domain formation could To investigate the influence of protein–lipid interac- also be related to a limited lateral diffusion of PM components. tions on PM lateral organization, we characterized GVPMs The cortical cytoskeleton in particular has been proposed to produced from tobacco BY-2 cell PMs using a novel pro- generate barriers that constrain movement in the membrane, cedure. These GVPMs, which contain hundreds of integral as revealed by the restricted diffusion of PM proteins in certain membrane proteins and lipids (corresponding to native PM membrane compartments (Kusumi et al., 2005; Umemura et al., amounts), provide a very interesting system, and yet they are 2008). Furthermore, the hindered diffusion of phospholipids rarely used due to the difficulty in obtaining them. In con- and sphingolipids was similarly abolished in actin cytoskele- trast to GPMVs, which are isolated after chemical treatment ton-free cell-derived GPMVs, as measured using super-reso- of animal cells that induces formation of detachable PM blebs lution stimulated emission depletion microscopy combined (Levental and Levental, 2015), the electrofusion procedure uti- with fluorescence correlation spectroscopy (Schneider et al., lized here allows the initial characterization of isolated PM 2017). These observations support the ‘picket fence’ model, in organization in a native resting state. In agreement with our which transmembrane proteins, like pickets, are anchored to observations on tobacco BY-2 cells, GPMVs from animal cells and lined up along a ‘fence’ of cytoskeletal proteins surround- have shown a clear segregation into Lo/Ld phases, with a lat- ing the confinement zones (Kusumi et al., 2005). In plants, eral distribution that depends on the overall protein content single-particle tracking analysis has recently revealed that cyto- (Baumgart et al., 2007). Using a cell-swelling procedure to iso- skeleton integrity, especially microtubules, restricts the lateral late PM spheres, the cholera toxin B subunit-dependent for- mobility of plant innate immunity proteins such as AtHIR1 at mation of ordered domains has also previously been reported the PM surface of Arabidopsis cells (Lv et al., 2017). However, (Lingwood et al., 2008). Here, by comparing the GUVs of PM quantifying protein diffusion using fluorescence recovery after lipids and the GVPMs, we further demonstrated the involve- photobleaching experiments has previously demonstrated that ment of PM proteins in the modulation (especially loosen- the cytoskeleton is not responsible for the relative immobil- ing) of plant PM order. Indeed, when proteins were positioned ity of plant PM proteins (Martinière et al., 2012). This appar- between lipid molecules, they increased the membrane line ent contradiction could be explained by different sensitivities tension and modified the mean size of the ordered domains in to microtubule disturbance depending on the particular pro- the tobacco PM, which has also been reported for lung sur- tein that is observed (Szymanski et al., 2015). This shows that factant monolayers (Dhar et al., 2012). Peptides with a short multiple types of membrane domains (which are specifically hydrophobic transmembrane domain accordingly decrease the enriched in one or the other of these proteins) co-exist at the affinity of sterols for neighbouring phospholipids (Nyström same time in plants (Jarsch et al., 2014). Some of these domains et al., 2010; Nyholm et al., 2011; Ijäs et al., 2013), suggesting may reflect the formation of specific areas enriched in PM that proteins could modify membrane lateral organization by components that are temporarily trapped by the cytoskeleton excluding certain lipids from the closest surrounding bilayer. network within distinct PM sub-regions, as has been proposed Moreover, lipids in direct interaction with proteins result in in animal cells (Murase et al., 2004). Among these domains, the areas that are much less compact in the immediate vicinity of composition of DRM-associated domains has been shown to the proteins (Brannigan and Brown, 2006), since protein–lipid be under the control of the cytoskeleton network, since micro- interactions depend on the size and the charge of chemical tubules regulate the dynamics of DRM-marker proteins such groups present at the protein surface (Honig et al., 1986). The as Arabidopsis Flot1 (Li et al., 2012) or Arabidopsis remorin anchoring of transmembrane proteins into ordered domains (Szymanski et al., 2015). is also hypothesized to redistribute ordered domains (Epand In plants, these domains may co-exist with other domains et al., 2004; Epand, 2008). whose formation has been proposed to be directly subject We are thus able to propose a final model in which lipid–lipid to lipid and protein composition (Urbanus and Ott, 2012), interactions may control the formation of ordered domains at and that have been revealed in our present study. Indeed, by the plant PM surface (whereas protein–lipid and/or protein– comparing the membrane organization of living tobacco cells protein interactions contribute to drive the whole-membrane affected (or not) in the structure of their actin and/or tubu- organization). With this model in mind, we noticed a remark- lin networks, we have shown that the cytoskeleton does not able limitation in the size of ordered domains within GVPMs, impact on the short-term regulation of the distribution of which could undoubtedly be attributed to the presence of pro- PM-ordered domains in tobacco. To the best of our know- teins. Indeed, GVPMs and living cells both exhibit similar small ledge, only one previous study has reported that alteration of ordered domains, and this small size originates from the ability either actin content or its association with the PM affects the Downloaded from https://academic.oup.com/jxb/article/69/15/3545/4990806 by DeepDyve user on 20 July 2022 3554 | Grosjean et al. physical properties of the PM in animal cells, e.g. disruption of diffusion, and the second corresponds to ordered domains with the cortical cytoskeleton coinciding with specific limitations of a sterol-enriched composition. Thus, microscale domains of the ordered domain fraction (Dinic et al., 2013). However, this restricted diffusion proteins might be distinct from nanoscale actin-dependent formation of ordered domains was demon- ordered domains (observed here using a lipid packing-sensitive strated to occur at 37 °C. At this temperature, lipid–lipid inter- probe), since the mechanisms responsible for their formation actions reduce the formation of ordered domains in GPMVs are most probably different. One hypothesis in animals pos- (Baumgart et al., 2007), giving prominence to interactions its that PM organization synthesizes a multiscale organization between the membrane and intracellular filaments. In line with of: (i) plasma membrane compartments (i.e. microscale-sized this, it has been proposed that ordered domains are too small domains) partitioned through membrane component entrap- to be optically detected at high temperature, regardless of the ment that depends on the actin-based cytoskeleton; (ii) lipid lipid composition (Pathak and London, 2015). In order to be raft domains (2–20 nm) created via sphingolipid–sterol inter- able to compare our different systems, we performed all of our actions; and (iii) protein complexes (3–10 nm) composed of experiments at room temperature (24 °C), as it is convenient dimer/oligomer assemblies (Nicolson, 2014). In addition to for cultivating plant cells. At this temperature, we observed that the cytoskeleton-dependent formation of PM sub-regions the presence and characteristics of ordered domains essentially with restricted lateral diffusion of their components mentioned depended on the lipid and protein mixtures present within the above, immuno-electron microscopy experiments in plants membrane. have revealed that the lipid PIP2 is partitioned into 25-nm In this study, we attempted to identify all the cellular ele- clusters (Furt et al., 2010). Furthermore, the protein remorin is ments underlying the heterogeneity of PM biophysical proper- similarly aggregated into 70-nm domains in the cytosolic leaf- ties in plant cells at a specific time, using as a system a tobacco let of tobacco leaf PMs (Raffaele et al., 2009). These two lines cell PM that had already been synthesized and was in a steady of evidence argue in favour of the existence of integrated levels state. The results we obtained in characterizing the membrane of PM organization in plants similar to that found in animals. order of protoplasts indicated that the cell wall does not play In the PM of tobacco cells in a resting state, domains of a key role in the maintenance of membrane order, or in the restricted lateral diffusion may thus co-exist with ordered spatial organisation of ordered domains in resting tobacco domains that are compatible with the size investigated here, cells. Even though direct interactions between PM proteins and for which we have demonstrated that lipid–lipid and/or and cell wall components are known to constrain the lateral protein–lipid interactions are the major driving forces of their diffusion of PM proteins (Martinière and Runions, 2013), no formation. Interestingly, both of these domains have recently involvement of cell wall organization in PM packing has been been implicated in plant stress responses, and notably in plant reported to date, and our results tend to exclude such a hypoth- immunity (Bucherl et al., 2017). Future work should aim to esis. Furthermore, the absence of an effect of short-term cyto- unravel the mechanisms used to regulate the number of ordered skeleton disorganization also questions the relevance of the domains, the dynamics of which are known to be involved in ‘picket fence’ hypothesis in plant cells. Instead, our data suggest the early stages of defense responses (Liu et al., 2009; Gerbeau- that lipids and proteins are the main determinants involved in Pissot et al., 2014; Sandor et al., 2016). the organization of membrane order in tobacco BY-2 cells. In agreement with our model, studies on the brassinosteroid binding receptor and its co-receptor have shown that the for- Supplementary data mation of nanoclusters within the PM of Arabidopsis seedlings Supplementary data are available at JXB online. is mainly subject to biophysical restraints, whereas cytoskel- Fig. S1. Effect of staurosporine on RGM of control and eton disruption does not have any effect on this parameter cryptogein-elicited BY-2 cells. (Hutten et al., 2017). The complex mechanisms by which the Fig. S2. Influence of cytoskeletal-active compounds on the PM is synthesized and renewed might be a way to control viability of tobacco cells. the targeting of the different membrane components (Zarský Fig. S3. Influence of the cytoskeleton on the membrane et al., 2009; Kleine-Vehn et al., 2011), and consequently to con- order of BY-2 PMs measured by spectrofluorimetry. trol the interactions between neighbouring lipids and proteins. Fig. S4. Comparison of the distribution of RGR values In accordance with this, modifications of PM composition in between control cells and cells treated with cytoskeletal-active an Arabidopsis mutant have been reported to alter membrane compounds. order at the resting state (Sena et al., 2017). According to the Fig. S5. Influence of cell wall–PM connections on the level limited data available, such a metabolic turnover of membrane of tobacco cell PM order. components might occur over a time frame of several hours Fig. S6. Lipid composition of PMs isolated from tobacco (Shi et al., 2008; Stanislas et al., 2009; Chalbi et al., 2015), which suspension cells. far exceeds our experimental conditions. Fig. S7. Effect of different lipid compositions on the mem- brane order of giant vesicles. Ordered domains are part of PM lateral heterogeneity Fig. S8. Influence of swelling-solution composition on Our results highlight the possible co-existence of distinct GVPM size. domains: the first corresponds to PM domains, in which pro- Fig. S9. Formation of large ordered domains in giant vesicles teins or oligomers of proteic complexes exhibit restricted lateral made up of tobacco PM lipids and proteins. Downloaded from https://academic.oup.com/jxb/article/69/15/3545/4990806 by DeepDyve user on 20 July 2022 Cellular determinants of tobacco cell plasma membrane organization | 3555 Epand RM. 2008. Proteins and cholesterol-rich domains. Biochimica et Acknowledgements Biophysica Acta 1778, 1576–1582. This work was partially supported by grants from the ’Région Epand RM, Epand RF, Sayer BG, Melacini G, Palgulachari MN, Bourgogne’ and the Bordeaux Metabolome Facility-MetaboHUB Segrest JP, Anantharamaiah GM. 2004. An apolipoprotein AI mimetic (grant no. ANR–11–INBS–0010) to SM. We wish to thank the peptide: membrane interactions and the role of cholesterol. Biochemistry Microscopy Centre INRA/Université de Bourgogne Franche-Comté 43, 5073–5083. of the DImaCell facility for technical assistance in confocal microscopy. Furt F, König S, Bessoule JJ, et al. 2010. Polyphosphoinositides are We also acknowledge the Metabolome-Fluxome-Lipidome facility of enriched in plant membrane rafts and form microdomains in the plasma Bordeaux (http://www.biomemb.cnrs.fr) for their contribution to the membrane. Plant Physiology 152, 2173–2187. lipid analysis. We thank Brandon Loveall of Improvence for for provid- Gerbeau-Pissot P, Der C, Thomas D, Anca IA, Grosjean K, Roche Y, ing English language editing for this paper. Perrier-Cornet JM, Mongrand S, Simon-Plas F. 2014. Modification of plasma membrane organization in tobacco cells elicited by cryptogein. Plant Physiology 164, 273–286. Grant CW, Wu SH, McConnell HM. 1974. Lateral phase separations in References binary lipid mixtures: correlation between spin label and freeze–fracture electron microscopic studies. Biochimica et Biophysica Acta 363, 151–158. Alonso A, Sáez R, Goñi FM. 1982. The interaction of detergents with phospholipid vesicles: a spectrofluorimetric study. FEBS Letters 137, Grosjean K, Mongrand S, Beney L, Simon-Plas F, Gerbeau-Pissot P. 141–145. 2015. Differential effect of plant lipids on membrane organization: specificities of phytosphingolipids and phytosterols. The Journal of Biological Chemistry Bagatolli LA, Ipsen JH, Simonsen AC, Mouritsen OG. 2010. An outlook 290, 5810–5825. on organization of lipids in membranes: searching for a realistic connection with the organization of biological membranes. Progress in Lipid Research Halling KK, Slotte JP. 2004. Membrane properties of plant sterols in 49, 378–389. phospholipid bilayers as determined by differential scanning calorimetry, resonance energy transfer and detergent-induced solubilization. Biochimica Baumgart T, Hammond AT, Sengupta P, Hess ST, Holowka DA, Baird et Biophysica Acta 1664, 161–171. BA, Webb WW. 2007. Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles. Proceedings of the National Hartmann MA. 1998. Plant sterols and the membrane environment. Trends Academy of Science, USA 104, 3165–3170. in Plant Science 3, 170–175. Bhatia T, Cornelius F, Ipsen JH. 2016. Exploring the raft-hypothesis by Honig BH, Hubbell WL, Flewelling RF. 1986. Electrostatic interactions probing planar bilayer patches of free-standing giant vesicles at nanoscale in membranes and proteins. Annual Review of Biophysics and Biophysical resolution, with and without Na,K-ATPase. Biochimica et Biophysica Acta Chemistry 15, 163–193. 1858, 3041–3049. Hutten SJ, Hamers DS, Aan den Toorn M, van Esse W, Nolles A, Bonneau L, Gerbeau-Pissot P, Thomas D, Der C, Lherminier J, Bücherl CA, de Vries SC, Hohlbein J, Borst JW. 2017. Visualization of Bourque S, Roche Y, Simon-Plas F. 2010. Plasma membrane sterol BRI1 and SERK3/BAK1 nanoclusters in Arabidopsis roots. PLoS ONE 12, complexation, generated by filipin, triggers signaling responses in tobacco e0169905. cells. Biochimica et Biophysica Acta 1798, 2150–2159. Ijäs HK, Lönnfors M, Nyholm TK. 2013. Sterol affinity for phospholipid Borner GH, Sherrier DJ, Weimar T, et al. 2005. Analysis of detergent- bilayers is influenced by hydrophobic matching between lipids and resistant membranes in Arabidopsis. Evidence for plasma membrane lipid transmembrane peptides. Biochimica et Biophysica Acta 1828, 932–937. rafts. Plant Physiology 137, 104–116. Ingólfsson HI, Melo MN, van Eerden FJ, et al. 2014. Lipid organization Brannigan G, Brown FL. 2006. A consistent model for thermal fluctuations of the plasma membrane. Journal of the American Chemical Society 136, and protein-induced deformations in lipid bilayers. Biophysical Journal 90, 14554–14559. 1501–1520. Jarsch IK, Konrad SS, Stratil TF, Urbanus SL, Szymanski W, Braun Bucherl CA, Jarsch IK, Schudoma C, Segonzac C, Mbengue M, P, Braun KH, Ott T. 2014. Plasma membranes are subcompartmentalized Robatzek S, MacLean D, Ott T, Zipfel C. 2017. Plant immune and into a plethora of coexisting and diverse microdomains in Arabidopsis and growth receptors share common signalling components but localise to Nicotiana benthamiana. The Plant Cell 26, 1698–1711. distinct plasma membrane nanodomains. eLIFE 6. Jin L, Millard AC, Wuskell JP, Clark HA, Loew LM. 2005. Cholesterol- Buré C, Cacas JL, Wang F, Gaudin K, Domergue F, Mongrand enriched lipid domains can be visualized by di-4-ANEPPDHQ with linear S, Schmitter JM. 2011. Fast screening of highly glycosylated plant and nonlinear optics. Biophysical Journal 89, L04–L06. sphingolipids by tandem mass spectrometry. Rapid Communications in Jin L, Millard AC, Wuskell JP, Dong X, Wu D, Clark HA, Loew LM. Mass Spectrometry 25, 3131–3145. 2006. Characterization and application of a new optical probe for membrane Cacas JL, Buré C, Grosjean K, et al. 2016. Revisiting plant plasma lipid domains. Biophysical Journal 90, 2563–2575. membrane lipids in tobacco: a focus on sphingolipids. Plant Physiology Kahya N, Brown DA, Schwille P. 2005. Raft partitioning and dynamic 170, 367–384. behavior of human placental alkaline phosphatase in giant unilamellar Carmona-Salazar L, El Hafidi M, Enríquez-Arredondo C, Vázquez- vesicles. Biochemistry 44, 7479–7489. Vázquez C, González de la Vara LE, Gavilanes-Ruíz M. 2011. Isolation Kaiser HJ, Lingwood D, Levental I, Sampaio JL, Kalvodova L, of detergent-resistant membranes from plant photosynthetic and non- Rajendran L, Simons K. 2009. Order of lipid phases in model and plasma photosynthetic tissues. Analytical Biochemistry 417, 220–227. membranes. Proceedings of the National Academy of Science, USA 106, Carter HE, Koob JL. 1969. Sphingolipids in bean leaves (Phaseolus 16645–16650. vulgaris). Journal of Lipid Research 10, 363–369. Kleine-Vehn J, Wabnik K, Martinière A, et al. 2011. Recycling, Chalbi N, Martínez-Ballesta MC, Youssef NB, Carvajal M. 2015. clustering, and endocytosis jointly maintain PIN auxin carrier polarity at the Intrinsic stability of Brassicaceae plasma membrane in relation to changes plasma membrane. Molecular Systems Biology 7, 540. in proteins and lipids as a response to salinity. Journal of Plant Physiology Kusumi A, Nakada C, Ritchie K, Murase K, Suzuki K, Murakoshi 175, 148–156. H, Kasai RS, Kondo J, Fujiwara T. 2005. Paradigm shift of the plasma Cooper JA. 1987. Effects of cytochalasin and phalloidin on actin. The membrane concept from the two-dimensional continuum fluid to the Journal of Cell Biology 105, 1473–1478. partitioned fluid: high-speed single-molecule tracking of membrane molecules. Annual Review of Biophysics and Biomolecular Structure 34, Dhar P, Eck E, Israelachvili JN, Lee DW, Min Y, Ramachandran A, 351–378. Waring AJ, Zasadzinski JA. 2012. Lipid–protein interactions alter line tensions and domain size distributions in lung surfactant monolayers. Laloi M, Perret AM, Chatre L, et al. 2007. Insights into the role of specific Biophysical Journal 102, 56–65. lipids in the formation and delivery of lipid microdomains to the plasma membrane of plant cells. Plant Physiology 143, 461–472. Dinic J, Ashrafzadeh P, Parmryd I. 2013. Actin filaments attachment at the plasma membrane in live cells cause the formation of ordered lipid Lefebvre B, Furt F, Hartmann MA, et al. 2007. Characterization of lipid domains. Biochimica et Biophysica Acta 1828, 1102–1111. rafts from Medicago truncatula root plasma membranes: a proteomic study Downloaded from https://academic.oup.com/jxb/article/69/15/3545/4990806 by DeepDyve user on 20 July 2022 3556 | Grosjean et al. reveals the presence of a raft-associated redox system. Plant Physiology subcellular distribution of the tobacco ROS-producing enzyme RBOHD 144, 402–418. in response to the oomycete elicitor cryptogein. Journal of Experimental Botany 65, 5011–5022. Lenne PF, Wawrezinieck L, Conchonaud F, Wurtz O, Boned A, Guo XJ, Rigneault H, He HT, Marguet D. 2006. Dynamic molecular confinement in Nyholm TK, van Duyl B, Rijkers DT, Liskamp RM, Killian JA. 2011. the plasma membrane by microdomains and the cytoskeleton meshwork. Probing the lipid–protein interface using model transmembrane peptides The EMBO Journal 25, 3245–3256. with a covalently linked acyl chain. Biophysical Journal 101, 1959–1967. Lentz BR, Barenholz Y, Thompson TE. 1976. Fluorescence depolarization Nyström JH, Lönnfors M, Nyholm TK. 2010. Transmembrane peptides studies of phase transitions and fluidity in phospholipid bilayers. 2. Two- influence the affinity of sterols for phospholipid bilayers. Biophysical Journal component phosphatidylcholine liposomes. Biochemistry 15, 4529–4537. 99, 526–533. Levental I, Grzybek M, Simons K. 2011. Raft domains of variable Ovecka M, Berson T, Beck M, Derksen J, Samaj J, Baluska F, properties and compositions in plasma membrane vesicles. Proceedings of Lichtscheidl IK. 2010. Structural sterols are involved in both the initiation the National Academy of Science, USA 108, 11411–11416. and tip growth of root hairs in Arabidopsis thaliana. The Plant Cell 22, 2999–3019. Levental KR, Levental I. 2015. Giant plasma membrane vesicles: models for understanding membrane organization. Current Topics in Membranes Pathak P, London E. 2015. The effect of membrane lipid composition 75, 25–57. on the formation of lipid ultrananodomains. Biophysical Journal 109, 1630–1638. Li R, Liu P, Wan Y, et al. 2012. A membrane microdomain-associated protein, Arabidopsis Flot1, is involved in a clathrin-independent endocytic Petrásek J, Schwarzerová K. 2009. Actin and microtubule cytoskeleton pathway and is required for seedling development. The Plant Cell 24, interactions. Current Opinion in Plant Biology 12, 728–734. 2105–2122. Phillips MC, Kamat VB, Chapman D. 1970. The interaction of cholesterol Lingwood D, Ries J, Schwille P, Simons K. 2008. Plasma membranes with the sterol free lipids of plasma membranes. Chemistry and Physics of are poised for activation of raft phase coalescence at physiological Lipids 4, 409–417. temperature. Proceedings of the National Academy of Science, USA 105, Pike LJ. 2006. Rafts defined: a report on the keystone symposium on lipid 10005–10010. rafts and cell function. Journal of Lipid Research 47, 1597–1598. Lingwood D, Simons K. 2010. Lipid rafts as a membrane-organizing Quinn PJ. 2010. A lipid matrix model of membrane raft structure. Progress principle. Science 327, 46–50. in Lipid Research 49, 390–406. Liu P, Li RL, Zhang L, Wang QL, Niehaus K, Baluska F, Samaj J, Lin Raffaele S, Bayer E, Lafarge D, et al. 2009. Remorin, a Solanaceae JX. 2009. Lipid microdomain polarization is required for NADPH oxidase- protein resident in membrane rafts and plasmodesmata, impairs Potato dependent ROS signaling in Picea meyeri pollen tube tip growth. The Plant virus X movement. The Plant Cell 21, 1541–1555. Journal 60, 303–313. Ramstedt B, Slotte JP. 2006. Sphingolipids and the formation of sterol- Lv X, Jing Y, Xiao J, Zhang Y, Zhu Y, Julian R, Lin J. 2017. Membrane enriched ordered membrane domains. Biochimica et Biophysica Acta 1758, microdomains and the cytoskeleton constrain AtHIR1 dynamics and 1945–1956. facilitate the formation of an AtHIR1-associated immune complex. The Plant Roche Y, Gerbeau-Pissot P, Buhot B, Thomas D, Bonneau L, Gresti Journal 90, 3–16. J, Mongrand S, Perrier-Cornet JM, Simon-Plas F. 2008. Depletion Mannock DA, Benesch MG, Lewis RN, McElhaney RN. 2015. A of phytosterols from the plant plasma membrane provides evidence for comparative calorimetric and spectroscopic study of the effects of cholesterol disruption of lipid rafts. FASEB Journal 22, 3980–3991. and of the plant sterols β-sitosterol and stigmasterol on the thermotropic Rujanavech C, Henderson PA, Silbert DF. 1986. Influence of sterol phase behavior and organization of dipalmitoylphosphatidylcholine bilayer structure on phospholipid phase behavior as detected by parinaric acid membranes. Biochimica et Biophysica Acta 1848, 1629–1638. fluorescence spectroscopy. The Journal of Biological Chemistry 261, Martinière A, Lavagi I, Nageswaran G, et al. 2012. Cell wall constrains 7204–7214. lateral diffusion of plant plasma-membrane proteins. Proceedings of the Samson F, Donoso JA, Heller-Bettinger I, Watson D, Himes RH. National Academy of Science, USA 109, 12805–12810. 1979. Nocodazole action on tubulin assembly, axonal ultrastructure and Martinière A, Runions J. 2013. Protein diffusion in plant cell plasma fast axoplasmic transport. The Journal of Pharmacology and Experimental membranes: the cell-wall corral. Frontiers in Plant Science 4, 515. Therapeutics 208, 411–417. Men S, Boutté Y, Ikeda Y, Li X, Palme K, Stierhof YD, Hartmann Sandor R, Der C, Grosjean K, Anca I, Noirot E, Leborgne-Castel N, MA, Moritz T, Grebe M. 2008. Sterol-dependent endocytosis mediates Lochman J, Simon-Plas F, Gerbeau-Pissot P. 2016. Plasma membrane post-cytokinetic acquisition of PIN2 auxin efflux carrier polarity. Nature Cell order and fluidity are diversely triggered by elicitors of plant defence. Journal Biology 10, 237–244. of Experimental Botany 67, 5173–5185. Minami A, Fujiwara M, Furuto A, Fukao Y, Yamashita T, Kamo M, Schneider F, Waithe D, Clausen MP, Galiani S, Koller T, Ozhan G, Kawamura Y, Uemura M. 2009. Alterations in detergent-resistant plasma Eggeling C, Sezgin E. 2017. Diffusion of lipids and GPI-anchored proteins membrane microdomains in Arabidopsis thaliana during cold acclimation. in actin-free plasma membrane vesicles measured by STED-FCS. Molecular Plant & Cell Physiology 50, 341–359. Biology of the Cell 28, 1507–1518. Mongrand S, Morel J, Laroche J, et al. 2004. Lipid rafts in higher Schuler I, Duportail G, Glasser N, Benveniste P, Hartmann MA. plant cells: purification and characterization of Triton X-100-insoluble 1990. Soybean phosphatidylcholine vesicles containing plant sterols: microdomains from tobacco plasma membrane. The Journal of Biological a fluorescence anisotropy study. Biochimica et Biophysica Acta 1028, Chemistry 279, 36277–36286. 82–88. Morejohn LC, Fosket DE. 1984. Inhibition of plant microtubule Schuler I, Milon A, Nakatani Y, Ourisson G, Albrecht AM, Benveniste polymerization in vitro by the phosphoric amide herbicide amiprophos- P, Hartman MA. 1991. Differential effects of plant sterols on water methyl. Science 224, 874–876. permeability and on acyl chain ordering of soybean phosphatidylcholine Moscatelli A, Gagliardi A, Maneta-Peyret L, et al. 2015. Characterisation bilayers. Proceedings of the National Academy of Science, USA 88, of detergent-insoluble membranes in pollen tubes of Nicotiana tabacum (L.). 6926–6930. Biology Open 4, 378–399. Scott RE. 1976. Plasma membrane vesiculation: a new technique for Murase K, Fujiwara T, Umemura Y, et al. 2004. Ultrafine membrane isolation of plasma membranes. Science 194, 743–745. compartments for molecular diffusion as revealed by single molecule Sena F, Sotelo-Silveira M, Astrada S, Botella MA, Malacrida L, techniques. Biophysical Journal 86, 4075–4093. Borsani O. 2017. Spectral phasor analysis reveals altered membrane Nicolson GL. 2014. The Fluid–Mosaic Model of Membrane Structure: still order and function of root hair cells in Arabidopsis dry2/sqe1-5 drought relevant to understanding the structure, function and dynamics of biological hypersensitive mutant. Plant Physiology and Biochemistry 119, 224–231. membranes after more than 40 years. Biochimica et Biophysica Acta 1838, Shaghaghi M, Chen MT, Hsueh YW, Zuckermann MJ, Thewalt JL. 1451–1466. 2016. Effect of Sterol structure on the physical properties of 1-Palmitoyl- Noirot E, Der C, Lherminier J, Robert F, Moricova P, Kiêu K, Leborgne- 2-oleoyl-sn-glycero-3-phosphocholine membranes determined using H Castel N, Simon-Plas F, Bouhidel K. 2014. Dynamic changes in the nuclear magnetic resonance. Langmuir 32, 7654–7663. Downloaded from https://academic.oup.com/jxb/article/69/15/3545/4990806 by DeepDyve user on 20 July 2022 Cellular determinants of tobacco cell plasma membrane organization | 3557 Shahedi V, Orädd G, Lindblom G. 2006. Domain-formation in DOPC/SM Takahashi T, Nomura F, Yokoyama Y, Tanaka-Takiguchi Y, Homma M, bilayers studied by pfg-NMR: effect of sterol structure. Biophysical Journal Takiguchi K. 2013. Multiple membrane interactions and versatile vesicle 91, 2501–2507. deformations elicited by melittin. Toxins 5, 637–664. Shi Y, An L, Zhang M, Huang C, Zhang H, Xu S. 2008. Regulation of the Titapiwatanakun B, Murphy AS. 2009. Post-transcriptional regulation plasma membrane during exposure to low temperatures in suspension-cultured of auxin transport proteins: cellular trafficking, protein phosphorylation, cells from a cryophyte (Chorispora bungeana). Protoplasma 232, 173–181. protein maturation, ubiquitination, and membrane composition. Journal of Experimental Botany 60, 1093–1107. Shimshick EJ, McConnell HM. 1973. Lateral phase separation in phospholipid membranes. Biochemistry 12, 2351–2360. Umemura YM, Vrljic M, Nishimura SY, Fujiwara TK, Suzuki KG, Kusumi A. 2008. Both MHC class II and its GPI-anchored form undergo Simons K, Gerl MJ. 2010. Revitalizing membrane rafts: new tools and hop diffusion as observed by single-molecule tracking. Biophysical Journal insights. Nature Reviews. Molecular Cell Biology 11, 688–699. 95, 435–450. Simons K, Ikonen E. 1997. Functional rafts in cell membranes. Nature 387, 569–572. Urbanus SL, Ott T. 2012. Plasticity of plasma membrane compartmentalization during plant immune responses. Frontiers in Plant Simons K, Sampaio JL. 2011. Membrane organization and lipid rafts. Science 3, 181. Cold Spring Harbor Perspectives in Biology 3, a004697. van den Bogaart G, Meyenberg K, Risselada HJ, et al. 2011. Singer SJ, Nicolson GL. 1972. The fluid mosaic model of the structure of Membrane protein sequestering by ionic protein–lipid interactions. Nature cell membranes. Science 175, 720–731. 479, 552–555. Spector I, Shochet NR, Kashman Y, Groweiss A. 1983. Latrunculins: Vereb G, Matko J, Vamosi G, et al. 2000. Cholesterol-dependent clustering novel marine toxins that disrupt microfilament organization in cultured cells. of IL-2Rα and its colocalization with HLA and CD48 on T lymphoma cells Science 219, 493–495. suggest their functional association with lipid rafts. Proceedings of the Stanislas T, Bouyssie D, Rossignol M, Vesa S, Fromentin J, Morel National Academy of Science, USA 97, 6013–6018. J, Pichereaux C, Monsarrat B, Simon-Plas F. 2009. Quantitative Willemsen V, Friml J, Grebe M, van den Toorn A, Palme K, Scheres proteomics reveals a dynamic association of proteins to detergent- B. 2003. Cell polarity and PIN protein positioning in Arabidopsis resistant membranes upon elicitor signaling in tobacco. Molecular & Cellular require STEROL METHYLTRANSFERASE1 function. The Plant Cell 15, Proteomics 8, 2186–2198. 612–625. Suzuki K, Shinshi H. 1995. Transient activation and tyrosine phosphorylation of a protein kinase in tobacco cells treated with a fungal Zaban B, Maisch J, Nick P. 2013. Dynamic actin controls polarity induction elicitor. The Plant Cell 7, 639–647. de novo in protoplasts. Journal of Integrative Plant Biology 55, 142–159. Szymanski WG, Zauber H, Erban A, Gorka M, Wu XN, Schulze WX. Zárský V, Cvrcková F, Potocký M, Hála M. 2009. Exocytosis and cell 2015. Cytoskeletal components define protein location to membrane polarity in plants – exocyst and recycling domains. New Phytologist 183, microdomains. Molecular & Cellular Proteomics 14, 2493–2509. 255–272.
Journal of Experimental Botany – Oxford University Press
Published: Jun 27, 2018
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