Biochimica et Biophysica Acta 1841 (2014) 1422–1430

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Inhibition of very long acyl chain sphingolipid synthesis modifiesmembrane dynamics during plant cytokinesis

Diana Molino a,b,c,d, Elisabeth Van der Giessen a,b, Lionel Gissot a,b, Kian Hématy a,b, Jessica Marion e,Julien Barthelemy a,b, Yannick Bellec a,b, Samantha Vernhettes a,b, Béatrice Satiat-Jeunemaître e, Thierry Galli c,d,David Tareste c,d, Jean Denis Faure a,b,⁎a INRA, Institut Jean-Pierre Bourgin, UMR 1318, ERL CNRS3559, Saclay Plant Sciences, RD10, F-78026 Versailles, Franceb AgroParisTech, Institut Jean-Pierre Bourgin, UMR 1318, ERL CNRS3559, Saclay Plant Sciences, RD10, F-78026 Versailles, Francec Institut Jacques Monod, UMR 7592, CNRS, Université Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, Franced Membrane Traffic in Neuronal and Epithelial Morphogenesis, INSERM ERL U950, 75013 Paris, Francee Institut des Sciences du Végétal, CNRS, Avenue de la Terrasse, F-91198 Gif-sur-Yvette Cedex, France

⁎ Corresponding author. Tel.: +33 1 30833113; fax: +E-mail address: faure@versailles.inra.fr (J.D. Faure).

http://dx.doi.org/10.1016/j.bbalip.2014.06.0141388-1981/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 January 2014Received in revised form 23 May 2014Accepted 24 June 2014Available online 5 July 2014

Keywords:SphingolipidCell plateFatty acyl chain lengthMembrane traffickingVesicle fusion

Plant cytokinesis requires intense membrane trafficking and remodeling to form a specific membrane structure,the cell plate that will ultimately separate the daughter cells. The nature and the role of lipids involved in the for-mation of the cell plate remain unclear. Plant membranes are particularly rich in sphingolipids such as glucosyl-ceramideswith long (16 carbons) or very long (24 carbons) acyl chains.We reveal here that inhibition of the syn-thesis of sphingolipidswith very long acyl chains induces defective cell plateswith persistent vesicular structuresand large gaps. Golgi-derived vesicles carrying material toward the cell plate display longer vesicle–vesicle con-tact time and their cargos accumulate at the cell plate, suggesting membrane fusion and/or recycling defects. Invitro fusion experiments between artificial vesicles show that glycosphingolipidswith very long acyl chains stim-ulate lipid bilayer fusion. Therefore we propose that the very long acyl chains of sphingolipids are essential struc-tural determinants for vesicle dynamics and membrane fusion during cytokinesis.

33 1 30833099.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Sphingolipids constitute a ubiquitous class of membrane lipidspresent in all eukaryotes as well as in several prokaryotes and virus-es [1–3]. They are characterized by a sphingoid base N-linked to afatty acid (FA). This structure, called ceramide (Cer), can be furthermodified by means of polar head substitution. In Arabidopsis, additionof glucose or glucosyl-inositol-phosphate residues leads to the forma-tion of glucosyl-ceramide (GluCer) and glucosyl-inositol-phosphoryl-ceramide (GIPC), respectively. GluCer and GIPC each represents about40–50% of the sphingolipidome of Arabidopsis seedlings [4]. The lengthof the FA chain is a hallmark of sphingolipids [5]. In both animals andplants, it can be long (LCFA: C16–18), or very long (VLCFA: C20–26),while in yeast, sphingolipids only contain VLCFA (C24–26). In plants,sphingolipids predominantly contain C16 and C24 FA chains [6]. Inmammals, ceramides are synthesized by a family of 6 enzymes displayingdifferent acyl chain specificities [7,8]. In Arabidopsis, 3 ceramide synthasesnamed LOH1-3 (for LAG One Homolog) produce LCFA and VLCFA-sphingolipids. We have recently shown that LOH1 and LOH3 null mu-tants were depleted in VLCFA-sphingolipids and were accumulating

C16-sphingolipids, whereas LOH2 null mutants were lacking C16-sphingolipids and contained exclusively VLCFA-sphingolipids. In addi-tion, LOH1 and LOH3, but not LOH2, were found to be essential forplant development and viability [9].

Sphingolipids are abundant constituents of eukaryotic membranes,where they account togetherwith sterols for 10–30% of the total plasmamembrane lipids. Synthetized in the endoplasmic reticulum (ER) andthe Golgi, animal sphingolipids are more abundant in late secretorycompartments and plasma membrane (PM), where they contribute toendocytosis and to the formation of membrane domains [10,11]. Invitro studies showed that very long and highly saturated acyl chains ofmammalian sphingolipids result in tighter packing [12]. On the con-trary, higher unsaturation, generally provided by phospholipids, tendsto reduce packing and to prevent sterol intercalation, thus leading todisordered lipid phases [13]. It has also been proposed that very longacyl chains create leaflet interdigitation reducing internal mobility.Therefore, shorter acyl chains andhigh unsaturation, generally providedby phospholipids [13], increase membrane fluidity, while longeracyl chains and higher saturation, mostly provided by mammaliansphingolipids, reduce fluidity and generate more rigid membranes[12,14]. VLCFA phosphoinositides were also reported to reduce the bi-layer to hexagonal phase transition temperature and thus to stabilizenegative curvatures in vitro [15]. This biophysical property could explain

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Fig. 1. Inhibition of VLCFA-ceramide synthases impairs cytokinesis in Arabidopsis root tip.(A) Plant sphingolipid pathway and the effect of the inhibition of VLCFA-sphingolipid syn-thesis. The effect of the doublemutant loh1/loh3 (green) or Fumonisin B1 treatment (blue)on sphingolipidmetabolites are indicated by arrows. Upward and downward arrows indi-cate respectively increase and decrease of the corresponding metabolite. The size of thearrow reflects the amplitude of the effect. The scheme was made from previously pub-lished data [9]. (B) FM4-64 staining of membranes of control (left) and FB1-treated(right) root tips. Incomplete (asterisks) or tilted (arrow)division planes could be observedin FB1-treated root tips. The inset shows FB1-treated cells with characteristic cell wallstub. (C) FB1-treated cell (bottom) compared to control cell (top); the FB1-treated cellis binucleated. Membranes are labeled by Lti6-GFP (green) and nuclei with H2B-RFP(red). Bars are 10 μm.

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the importance of yeast VLCFA in stabilizing the nuclear pore struc-ture [16] as well as their role in vesicle trafficking [17] where importantmembrane bending is required. In another work, the glycosphingolipidGM1 with longer acyl chains was shown to facilitate plasma membraneinvagination and internalization of the virus SV40 [18]. Highly curvedmembrane structures are also formed during animal cytokinesis at thecleavage site between the two daughter cells. In Drosophila, VLCFAswere found to be required for efficient cleavage furrow ingression duringcytokinesis [19].

Plant cytokinesis requires vesicle trafficking for membranesorting and recycling toward and from the cell plate, a complexneo-synthetized double-bilayer. The cell plate is assembled by a seriesof rapid centripetal vesicle fusion events occurring at the cell center dur-ing anaphase [20,21]. Vesicular fusions first generate a tubulovesicularstructure that develops into a more tubular network within a few mi-nutes. The incoming material supports cell plate growth, and cell plateexpansion is guided by a complex structure composed of microtubules,microfilaments, polysaccharides and ER [22,23]. Cell plate expansionends with its anchoring and fusion with the lateral PM. Finally, cellplate maturation involves remodeling of lipids and proteins but alsothe assembly of cell wall components between the two newly formedmembranes. A callose-based cell wall is transiently accumulated duringcell plate formation, probably to stabilize and rigidify the young growingcell plate [22]. VLCFAswere shown to be essential for cell plate expansionduring plant cytokinesis [24]. However, the nature and the role of theVLCFA-containing lipids involved in this process remain unknown.

In Arabidopsis, inhibition of VLCFA-sphingolipid synthesis causedthe aggregation of a specific class of trans-Golgi network (TGN)-earlyendosomal compartment containing the small GTPase RabA2a [9]. RabGTPases are evolutionarily conserved regulators of membrane traffick-ing machineries that switch from an inactive GDP-bound to an activeGTP-bound state to regulate the transport and docking to target mem-branes [25]. RabA2a, the plant ortholog of the animal Rab11, is requiredfor sorting vesicles toward the cell plate, and RabA2a-containing vesi-cles are potential precursors of cell plate components [26]. Inhibitionof VLCFA-sphingolipid synthesis was also found to alter cell divisionand cell shape in a cytoskeleton-independent manner in tobacco BY2cells [27]. However, the role of sphingolipids in cell plate biogenesis isstill unknown. Here, we show that inhibition of VLCFA-Cer synthase al-ters cell plate biogenesis and structure in planta. It also impairs the dis-tribution of specific cell plate markers like callose and clathrin andcauses the ectopic localization of the TGN-early markers RabA2a andVHAa1 at the cell plate, suggesting a defect in vesicle dynamics. Inhibi-tion of VLCFA-sphingolipid synthesis also extends the interaction timebetween RabA2a and VHAa1-labeled vesiclesmost probably bymodify-ing their docking/fusion properties. Finally, in vitro fusion experimentsbetween artificial vesicles demonstrate that glycosphingolipids withvery long acyl chains facilitate membrane fusion. Our study thus sug-gests that sphingolipids with very long acyl chains are essential lipidsfor vesicle dynamics during plant cytokinesis.

2. Results

2.1. VLCFA-ceramide synthase inhibition induces cytokinesis defects

The synthesis of ceramides and hydroxyceramides with very longfatty acid chains (VLCFA-Cer and VLCFA-hCer respectively) could bespecifically inhibited by Fumonisin B1 (FB1), a mycotoxin produced bythe plant parasite Fusarium moniliforme (Fig. 1A) [9,28,29]. FB1 treat-ment caused a reduction of VLCFA-Cer in Arabidopsis similar to whatwas found in VLCFA-ceramide synthase double mutants loh1/loh3 [9].Ceramides with very long fatty acid chains decreased from 4.3 ± 0.2to 3.5 ± 0.1 nmol/g fresh weight after 24 h of FB1 treatment. A similar20% decrease could be observed with VLCFA-hCer (from 2.7 ± 0.1to 2.2 ± 0.1 nmol/g after FB1 treatment). Long term exposition(36 h) or high concentration (10 μM) of FB1 also induced the

accumulation of short chain-sphingolipids and free sphingoid bases(substrates for ceramide synthesis) that were described to induce celldeath in plants (Fig. 1A) [30,31]. In the present work, FB1 concentrationand duration of application was thus carefully adjusted like in our pre-vious study to prevent potential cell death [9]. One of themain develop-mental effects was the inhibition of primary root growth [9]. The roottip is divided in three successive developmental zones. First, an activedivision zone, followedby an elongation zone,where cells have stoppeddividing, and finally a differentiation zone, characterized by the devel-opment of root hairs (Fig. S1A). FB1-treated roots showed a shorteningof the division zone, caused by a reduction of both total number of cellsand cell size (Fig. S1B and C). A shorter elongation zone was also ob-served (Fig. S1B). These data thus suggest that FB1-treated root cellswere impaired in both cell division and cell elongation.

FB1 treatment of 12–16 h with 2.5 μM also induced abnormal divi-sion planes consisting of tilted, incomplete and unanchored cell plates,as well as cell wall stubs, suggesting incomplete cytokinesis in the dif-ferent root cell types but in particular in the epidermis (Fig. 1B, asterisksand arrow). The presence of binucleated epidermal cells confirmed thatFB1 affects cell division (Fig. 1C). Abnormal cell plates and cell divisionplanes were also observed in the double mutant loh1/3 (Fig. S1E).Three-dimensional reconstruction of cell plates from FB1-treated cellsrevealed unusually large and swollenmembrane structures surroundedby large vesicles compared to control cell plates (Fig. S1G). Microtubuleorganization did not seem to be altered by FB1 since cell plate specificmicrotubule assembly could still be observed around abnormal cellplates (Fig. S3A, B and C).

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2.2. Inhibition of VLCFA-sphingolipid synthesis modifies cell plate structure

Cell plate formation initiates with the gathering of vesicles in theequatorial plan of the cell into a filamentous ribosome-free andcytoskeleton-based cell plate assembly matrix (CPAM) [32]. These ves-icles then fuse massively to form a micrometer scale tubulovesicularnetwork (TVN) at the center of cells that expands centrifugally. On elec-tron microscope pictures, the TVN is characterized by a wavy structureof dark filled interconnected vesicles and tubules [22,32]. While the cellplate expands, the TVN rapidly evolves into a flatter tubule network(TN) with clear filled tubules and fewer mostly clear filled vesicles(Fig. 2A). The TN finally matures into a planar membrane sheet withseveral open fenestrae (Fig. 2D). The TN and planar fenestrated mem-brane stages are characterized by callose deposition [22].

Root tip cells treated with FB1 showed several abnormal featuresduring cell plate formation. As observed previously, we could confirmthe presence of misaligned cell plates (Fig. 2B). Interestingly, a persis-tent dark filled vesicular network could be observed along the entirecell plate and not only at the center, suggesting a delay in TN formation.At late telophase, when nuclei are interphasic, FB1-treated cells werestill displaying a highly vesiculated cell plate with an intermediateTVN/TN structure (Fig. 2C). On the contrary, at the same stage, untreat-ed cells displayed the characteristic planar fenestrated structure of amature cell plate (Fig. 2D). The open fenestrae are usually membrane

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Fig. 2. FB1 induces persistence of vesiculated membrane structures at the cell plate. (A) ControNote the tubular networkwith large clearfilledmembranes (open arrow) and the presence of gathe presence of a large number of dark filled vesicles (closed arrow) and the absence of any tubplate (open arrow). (C) FB1-treated cell at the telophase stage showing the occurrence of the twand unusually thick membrane (closed arrow) near a residual TVN (open arrow). (D) Controlarrow). Note the small gaps (fenestrae) remaining (open arrow). (E) FB1-treated cell showinglarge amount of vacuolar structure around the gap (closed arrow). (F) FB1-treated cells showinof themembranes (closed arrow). Selected pictures are representative of observations perform(F); 1 μm in (B) and (E) and 200 μm in (C).

gaps ranging from 50 to few 100 nm that allow the ER to pass through.In FB1-treated cells, mature fenestrae gaps could extend over severalmicrometers, allowing even nuclei to pass through (Fig. 2E). Finally,these large gaps were occasionally associated with unusually thickand twisted membranes (Fig. 2F). Often, numerous vacuole-like struc-tures were gathered around membrane gaps, suggesting intense vesic-ular traffic (Fig. 2E, filled arrows).

Ultra-structural analysis of the cell plate thus shows that FB1 treat-ment delays cell plate maturation with the persistence of vesicularstructures at all intermediate stages of cell plate formation (transitionfrom TVN to TN, from TN to planar fenestrated membrane, and evenduring closure of the plate fenestrae). This suggests that the inhibitionof VLCFA-sphingolipid synthesis is required for the transition fromvesicular to tubular and planar structures and for closing the platefenestrae.

2.3. FB1 treatment impairs late cytokinesis steps

The cell plate is a novel compartment whose formation strongly de-pends on membrane trafficking processes. RabA2a is a major player incell plate biogenesis and its association at the cell plate follows a dy-namic localization pattern [26]. First, it is transported through vesiclestoward the cell plate, where it labels the entire expanding cell plate.Then it becomes restricted to the edges, when expansion is almost

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l cell with a cell plate at the TN stage growing linearly and perpendicularly to the cell axis.ps (closed arrow);mt:mitochondria. (B) FB1-treated cellwith a large tilted cell plate. Noteular network like in (A). Large aggregation of material could also be observed near the cello new nuclei (nu) set apart from the new cell surface. Note the presence of an unfinishedcell in telophase with two interphasic nuclei (nu) and the characteristic linear TN (closedincomplete cytokinesis with a large gap remaining and misaligned nuclei (nu). Note theg an incomplete cytokinesis with a thick cell plate. Note thewavy and unsmooth structureed on 20 sections issued from 2 independent experiments. Bars are 0.5 μm in (A), (D) and

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completed and presumably when anchoring and fusion to lateral mem-branes occur. Finally, it is completely removed from the cell plate at theend of cytokinesis [26]. As previously observed in interphasic cells [9],FB1 treatment induced defects in YFP-RabA2a vesicles that appearedto coalesce in large membrane structures by optical microscopy (re-ferred therein as FB1 compartment). These FB1 compartments werecaused by VLCFA-sphingolipid depletion and not by the resulting in-crease in C16-sphingolipids since they could still be observed in theC16-specific ceramide synthase mutant loh2-1 (Figs. 1A and S1H). FB1compartments labeled by YFP-RabA2a were also present in dividing

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cells (Figs. 3A, inset, and S1F) and could still target the cell plate(Fig. S1F). In addition, FB1 specifically impacts YFP-RabA2a populationsince vesicles labeled with the late endosome marker GFP-RabF2bwere not modified by FB1 treatment (Fig. S2D). Strikingly, the totalnumber of YFP-RabA2a-labeled cell plates also increased in thepresenceof FB1 in both primary and lateral roots (Figs. 3A, right and S1D, right).Similarly, root tips stained with DAPI and anti-KNOLLE antibodiesshowed a higher number of cells in late stages of cytokinesis, i.e. withfully expanded KNOLLE labeled cell plates and interphasic nuclei inFB1-treated seedlings (Figs. S2B and S3E). The syntaxin KNOLLE is ex-clusively synthesized during cytokinesis; it first accumulates in vesiclesthen relocates to the growing cell plate, a transport pathway that par-tially overlaps with that of RabA2a [26,33]. But contrary to RabA2a,KNOLLE was not associated to FB1 compartments (Fig. S2B). Also, con-trary to very long chain fatty acid or sterol mutants [24,34] we did notobserve any ectopic localization of KNOLLE in the presence of FB1 orin double loh1/3mutant (Fig. S2B and C).

The higher number of cell plates could be the consequence of eitheran increase in cytokinesis entry or a delay in cytokinesis exit. However,the total number of cells per root presenting mitotic figures visualizedwith microtubule binding protein (MBD) that labels early stages ofcell division was not modified by FB1 (Fig. S3A–C). This observation isconsistent with a specific defect on cytokinesis exit rather than cytoki-nesis entry.

Higher occurrence of cell plates could also result from slow cell plateexpansion, as previously described in fatty acid elongation mutant pas2[24]. We thus monitored cell plate expansion in seedlings expressingLti6-GFP/H2B-RFP treated or not with FB1 (Fig. S3F). Cell plates werefollowed fromyoung plates (about 40%of the total cell length) to almostcomplete plates (about 90% final length) and no difference in mem-brane expansion rate was found between control and FB1-treated cells.

Altogether these results indicate that the inhibition of VLCFA-sphingolipid synthesis impaired late steps of cell plate formation.

2.4. FB1 treatment modifies the dynamics of late cytokinesis markers

Marker removal during late cytokinesis is a hallmark of cell platemat-uration [35]. Besides the transient localization of membrane markers,such as RabA2a or KNOLLE, cell plate formation is also characterized bythe transient accumulation of cell wall material such as callose [22,36].In control cells, aniline blue (AB) staining showed that callose started toaccumulate at the center of expanding cell plates, while residual YFP-RabA2a was at the edges (Fig. 3B, top). In fully expanded cell plates, AB

Fig. 3. FB1 induces ectopic labeling of the cell plate. (A) FB1 treatment increases thenumberof YFP-RabA2a-labeled cell plates per root. Primary roots tips expressing YFP-RabA2a andtreated (+FB1) or not (−FB1) with FB1. Seedlings were treated with 2.5 μM FB1 for16 h. The graph shows the relative number of cell plates per root (mean and standarderror, n = 11 and 9 respectively). Note the presence of YFP-RabA2a aggregation near thecell plate in FB1-treated root (inset). Significance was determined by paired t-test, *** indi-cates P b 0.001. (B) Aniline blue (AB) staining of callose− or+FB1 in YFP-RabA2a express-ing root cell seedlings. The asterisks indicate the cell plates. The panels − or +FB1 showtwo cell plateswith different AB labeling. In the absence of FB1, the top plate displays resid-ual RabA2a-YFP labeling at the edges and AB staining at the center, and the twomarkers re-main separated. The bottom plate displays AB staining at the edges and no more YFP-RabA2a labeling. In the presence of FB1, the bottom plate displays full AB staining and nomore YFP-RabA2a labeling. The top cell plate displays residual YFP-RabA2a labeling at theedges and AB staining at the center. Note on this cell plate, the juxtaposition of AB andYFP-RabA2a markers (open arrows). (C) CLCB-GFP distribution in non-treated cytokineticcell. CLCB-GFP (left), cell membranes stained with FM4-64 (middle) and merged channel(right) are shown. Note that CLCB-GFP labels only late cell plate stages and the centerzone of the expanding cell plate (between arrows). (D) CLCB-GFP distribution in cytokineticcells treated with FB1. CLCB-GFP (left), cell membranes stained with FM4-64 (middle) andmerged channel (right) are shown. CLCB-GFP labels cell plates even at early stages (topcell). (E) CLCB-GFP distribution at later stages in FB1-treated cell. CLCB-GFP (left), cellmem-branes stained with FM4-64 (middle) and merged channel (right) are shown. Note the ac-cumulation of CLCB-GFP around the swollen cell plate. (F) VHAa1-RFP/YFP-RabA2acolocalization in dividing cells in control condition (top row) or after treatment with FB1(bottom row). YFP-RabA2a (left), VHAa1-RFP (middle) and merged channel (right) areshown. Bars are 2 μm in (A), (B), (C), (D) and (E), and 5 μm in (F).

1426 D. Molino et al. / Biochimica et Biophysica Acta 1841 (2014) 1422–1430

stainingwas either spread all along the plate (Fig. S2E) ormore restrictedto the edges (Fig. 3B, bottom), while YFP-RabA2a was completely re-moved. Although cell plates in late expansion phases could contain bothYFP-RabA2a and AB in distinct regions (center versus edges), the twomarkers never colocalized. Cell plate maturation thus seems to proceedwith the transient accumulation of YFP-RabA2a followed by callose, in-dicating the sequential presence of both markers during cell plate mat-uration (Fig. S2E). In FB1-treated cells, AB and YFP-RabA2a co-labeledexpanding cell plates in a non-overlapping pattern: callose was mostlyaccumulatedwithin the cell plate center and YFP-RabA2awas restrictedto the edges (Figs. 3B and S2E). This juxtaposition of both labeling couldbe the consequence of a delay in YFP-RabA2a retrieval. Indeed, in someextreme cases, YFP-RabA2a could form small patches in mature cellplates (Fig. S2A).

In plants, clathrins and dynamins are involved in internalization aswell as intracellular trafficking of membrane proteins [37]. These pro-cesses were found to be particularly important during cytokinesis [38,39]. The dynamin DRP1C-GFP labeled the different stages of cell plateformation from initiation tomaturation and no FB1 effect was observed(Fig. S4A and B). On the contrary, the clathrin light chain B CLCB-GFP la-beled only expanding and mature cell plates and was never found to beassociated with young cell plates (Fig. 3C). In the expanding cell plate,CLCB-GFP was more specifically associated with the plate center wherematuration begins (Fig. 3Cmiddle, between arrows). FB1 treatmentmod-ified CLCB-GFP localization since young cell plates were clearly labeledeven at the earliest visible stage (Fig. 3D). At later stages, swollen or in-complete cell plateswere associatedwith evenmore intenseCLCB-GFP la-beling (Fig. 3D). Extensive recruitment of CLCB-GFP at the cell plate couldalso be observed in wild type cells undergoing spontaneous cytokinesisarrest after long imaging time under a confocal microscope (Movie S1).

In conclusion, the recruitment of clathrin during the early stages ofcell plate formation and the altered distribution dynamics of cell platemarkers (RabA2a and callose) point toward defective membrane dy-namics in cells with limited VLCFA-Cer synthesis.

2.5. FB1 treatment perturbs vesicle dynamics at the cell plate

In non-dividing cells, RabA2a vesicles extensively colocalizewith theTGN resident vacuolar ATPase VHAa1 [26,40]. In plant, the TGN com-partment appears as a population of small isolated and independentvesicles. When division starts, RabA2a vesicles, but not VHAa1 vesicles,move to the cell plate, indicating that these two compartments have a

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Fig. 4. FB1 lengthens homotypic and heterotypic interactions of YFP-RabA2a and VHAa1-RFPVHAa1-RFP (red) vesicles in a control cell. Colocalization is shown in the yellow channel. Topwhile the bottom row shows an example of heterotypic colocalization. Time scale is in seconds. (ence of FB1. Dots are vesicles from independent replicates (3, 3 and 2 for RabA2a/RabA2a, VHAain control and FB1 conditions are respectively: 78 and 62 (RabA2a/RabA2a), 96 and 80 (VHAa1determined by Mann–Whitney test, and P values resulted b0.0001 for the 3 populations. Bars

different outcome during cell division. Hence, in control conditions,while YFP-RabA2a strongly labeled the cell plate during cytokinesis,VHAa1-RFP or VHAa1-GFP did not (Figs. 3F and S4C–D). Eventually, atlate cytokinesis, concomitant with the removal of RabA2a from thecell plate, colocalization of YFP-RabA2a and VHAa1-RFP at the TGN re-sumes [40]. Interestingly, FB1 treatment induced ectopic labeling ofVHAa1-GFP at the cell plate (Fig. S4C), and co-expression of YFP-RabA2a and VHAa1-RFP demonstrated that both markers colocalizedat the cell plate in FB1-treated seedlings (Fig. 3F). This observationwas confirmed by introgression of both VHAa1-RFP and YFP-RabA2ain the double mutant loh1-1/loh3-1 (Fig. S4D). We thus reasoned thatthe presence of VHAa1 at the cell plate in FB1-treated cells could resultfrom the rerouting of VHAa1 into the secretory pathway associatedwithcell plate formation (for example through interactions between VHAa1and RabA2a vesicles), or alternatively from the persistence of a pre-existing transient interaction of VHAa1 vesicles with the cell plate, orboth. The latter hypothesis is supported by the fact that VHAa1-labeledvesicles showed transient colocalization with YFP-RabA2a at the cellplate in control conditions (Fig. S4E).

The interaction dynamics between YFP-RabA2a and VHAa1-RFP ves-icles was therefore analyzed by high-speed live imaging. We chose tofocus on the interaction between vesicles because the interaction be-tween vesicles and the cell plate suffered several technical limitations.The high density of fast moving vesicles surrounding the cell plate andthe low spatial resolution in the fast imaging mode provided too muchuncertainty in vesicle tracking and colocalization to capture interactionevents. YFP-RabA2a and VHAa1-RFP-labeled vesicles were found to co-localize for several seconds. The fact that vesicle colocalization wasmaintained in mobile vesicles supports direct vesicle interactions(Fig. 4A). We reasoned that the time during which two vesicles werecolocalized could provide an estimation of their interaction time.Homotypic and heterotypic interactions were thus defined by thecolocalization time of vesicles labeled with identical or different markers.In control conditions, the duration of homotypic YFP-RabA2a and VHAa1-RFP interactions, and of heterotypic YFP-RabA2a/VHAa1-RFP interactionsranged from 5 to 25 s with a mean of respectively 10, 11 and 13 s(Fig. 4B). FB1 treatment significantly increased vesicle interactiontimes by a factor of 2 for homotypic interactions (mean of 23 and20 s), and by a factor of 4 for heterotypic interactions (mean of 45s). Since colocalization time may depend on the overall vesicle mo-bility, we also measured the speed of each vesicle population (Fig. S4Fand G). YFP-RabA2a vesicles were on average faster than VHAa1-RFP

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vesicles. (A) Homotypic and heterotypic colocalization between YFP-RabA2a (green) andand middle rows show homotypic colocalizations of YFP-RabA2a and VHAa1-RFP vesiclesB) Distribution of homotypic and heterotypic colocalization times in the absence and pres-1/VHAa1 and RabA2a/VHAa1 interactions, respectively). The numbers of vesicles analyzed/VHAa1), and 96 and 83 (RabA2a/VHAa1). The mean ± SEM is provided. Significance wasare 5 μm in (A).

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C16:0

C24:1

C24:0

A

B

Fig. 5.Glycosphingolipidswith very long acyl chain promotemembrane fusion. (A) Effect ofglycosphingolipid acyl chain length on PEG-mediated liposome fusion. Lipidmixing ismea-sured bymonitoring the dequenching of NBD lipid probes resulting from their dilution intothe fused liposomemembrane; the fluorescence signal is normalized to the maximal valueat infinite dilution (obtained upon lysis of the liposomes at the end of the reaction). Lipo-somes harboring VLCFA-glycosphingolipids (C24:1 and C24:0) are more fusogenic thanthose lacking glycosphingolipids (No GS) or containing LCFA-glycosphingolipids (C16:0).(B) Effect of glycosphingolipids acyl chain length on the initial fusion rate, measured5 min after starting the reaction (slope of the tangent line at t = 5 min of the curve in A).Means and standard errors are from n= 4 independent experiments (4 independent lipo-some preparations). Significance was determined by oneway ANOVA and a post-hoc t-testwith Bonferroni correction; ** indicates P b 0.01 and n.s. (non significant) P N 0.05.

1427D. Molino et al. / Biochimica et Biophysica Acta 1841 (2014) 1422–1430

vesicles, but no significant effect of FB1 could be observed, suggesting thatthe enhanced colocalization time was indeed caused by longer vesicle–vesicle contacts (Fig. S4G).

Altogether these results show that inhibition of VLCFA-Cer synthaseswithholds together closely associated vesicles. Such extended vesicleassociation could even lead to the formation of large aggregates. Time-lapse imaging in fact identified co-labeled YFP-RabA2a/VHAa1-RFP ag-gregates displaying intensive activity of vesicles moving in and out ofthe aggregate (Movie S2) confirming the dynamic nature of these inter-actions. The synthesis of VLCFA-Cer is thus required for vesicle dynamicsby modifying vesicle interaction time.

2.6. VLCFA-Cer promotes membrane fusion

FB1-treated cells display strong membrane dynamics defects illus-trated by (i) a persistence of vesicular structures at the cell plate thatdo not transform into the normal tubular and planar structures (Fig. 2)and (ii) an increase of the interaction time between endosomal vesicles(Fig. 4). Such defects strongly suggest a role of sphingolipids in the regu-lation of vesicle–vesicle and/or vesicle–cell plate exchanges throughmembrane fusion.

To investigate the role of sphingolipid acyl chain length in membranefusion,we used an in vitro fusion assay between liposomesmade of a con-stant base of phospholipids and sitosterol mimicking the lipid composi-tion of plant membranes [41], and containing or not ceramides withvarious chain lengths. We chose to work with glycosphingolipids harbor-ing a common (C18:1) sphingoid base (sphingosine) and either long(C16:0) or very long (C24:1, C24:0) acyl chains to mirror the most abun-dant FA configurations found in sphingolipid backbones of plant mem-branes [9,42,43]. In vitro fusion between protein-free membranes can betriggeredwith polyethylene glycol (PEG)which induces very closemem-brane apposition via depletion forces [44], thus giving the opportunity toprobe for the specific effect of lipids in membrane fusion.

For each condition to be tested, we generated two sub-populationsof liposomes with the same lipid composition except that one sub-populationwas labeledwith the Fluorescence Resonance Energy Transfer(FRET) pair of fluorescent lipids PE-NBD (donor) and PE-RHO (acceptor).Fusionwas triggered by the addition of 5% (w/v) PEG8000, andmeasuredas the increase of theNBDfluorescence (dequenching) caused by dilutionof thefluorescent lipids into the fused liposomemembrane [45]. Thepres-ence of C24:1 or C24:0 acyl chains into liposomes enhanced both thekinetics and extent of lipid mixing compared to liposomes devoid ofglycosphingolipids or containing glycosphingolipids with C16:0 acylchains (Fig. 5A). Notably, the initial rate of lipid bilayer fusion (measured5min after initiating the reaction)was doubled in the presence of VLCFA-glycosphingolipids (Fig. 5B). To rule out any potential effect of liposomesize on fusion [46], wemeasured the size distribution of our different li-posome populations by dynamic light scattering (DLS) (Fig. S4H). Thepresence of glycosphingolipids did not have any effect on the liposomesize: all liposome preparations displayed similar average diameter(~65 nm) and polydispersity (~30 nm). Stimulation of fusion wastherefore due to an intrinsic property of the very long acyl chain ofVLCFA-glycosphingolipidsmost likely through the stabilization of high-ly curved intermediatemembrane structures during fusion (see below).

3. Discussion

FB1 treatment, like null mutations in both LOH1 and LOH3 genes,caused cytokinesis defectswith tilted, swollen and unfinished cell platesdisplaying persistent vesicular structures and large remaining gaps. Al-though we cannot completely exclude that FB1 could (like any otherdrug) target other cellular processes, thepresence of similar phenotypesbetween the drug-treated plants and the double mutant loh1/loh3 indi-cate that ceramide synthesis inhibition is directly responsible for the ob-served cytokinesis defects. Ceramide synthase inhibition also leads to anincrease in C16-sphingolipids and free sphingoid bases that were both

involved in cell death induction in cultured plant cells [30,47], plant seed-lings and leaves [31,48], as well as in animal cells [49,50] and yeast [51].The present study was carried out under controlled conditions with anexperimental setting preventing cell death as monitored by a fluoresceindiacetate (FDA) hydrolysis assay [9]. The role of free sphingoid bases incytokinesis is also rather unlikely sincewepreviously showed that the ac-cumulation of free sphingoid bases was not causing defective membranetrafficking [9]. Finally, the induction of YFP-RabA2a aggregation by FB1 inthe loh2 mutant indicates that long acyl chains (C16) are not in-volved in endomembrane defects associated with the inhibition ofVLCFA-sphingolipid synthesis.

In vitro studieswith artificial vesicles directly demonstrated the pos-itive role of VLCFA-glycosphingolipids in membrane fusion. The pres-ence of glycosphingolipids with an acyl chain of 24 carbons, saturatedor not, was sufficient to promote membrane fusion. The specific func-tion of lipid acyl chain length inmembrane fusion is difficult to deciphersince it canmodify at once lipid packing, membrane fluidity, membranethickness and membrane curvature that can all impact fusion [5,55,56].In the currentmodels,membrane fusion proceeds through a hemifusionintermediate allowing outer leaflets, but not inner leaflets, lipid mixing.In this intermediate state, the lipid assembly forms an hourglass-shaped

1428 D. Molino et al. / Biochimica et Biophysica Acta 1841 (2014) 1422–1430

structure named stalk (Fig. 6). The stalk structure was experimentallyobserved demonstrating the presence of highly bent inner leafletswith hydrophobic voids below the outer leaflets [57]. The presence ofstrong curvatures and voids in the stalk implies that a considerableamount of energy must be provided for its formation [58]. An attractivehypothesis would be that the presence of lipids with acyl chains longerthan the canonical phospholipid monolayer would fill these hydropho-bic voids to lower the energy of stalk formation and thus facilitate fusion(Fig. 6). A depletion of VLCFA-sphingolipids at fusion sites would thusdelay or prevent hemifusion/fusion leading to an increased number ofvesicles blocked in a docked state.

This hypothesis would provide an explanation for the modified dy-namics of RabA2a and VHA1 vesicles during late cytokinesis observedwhen VLCFA-sphingolipid synthesis was inhibited. RabA2a is one ofthe major actors in vesicular delivery of material toward the growingcell plate. VHAa1 is a member of the V-ATPase family that is specificallylocalized to the TGN; it is involved in both endocytic and secretory path-ways and it is also essential for cell expansion [52]. Reduced levels ofVLCFA-sphingolipids caused aggregation of RabA2a vesicles, retentionof RabA2a at the cell plate, and delocalization of VHAa1 from the TGNto the cell plate. Interestingly, both RabA2a vesicle aggregation andVHAa1 ectopic localization at the cell plate were also observed in theechidna mutant, an essential member of the TGN required for secretionof cell wall polysaccharides [53,54]. In control conditions, partial andtransient co-localizations were observed between YFP-RabA2a andVHAa1-RFP vesicles but also between those vesicles and the cell plate.In the absence of VLCFA-sphingolipids, these transient co-localizationsbetween vesicles lasted longer, which could explain the presence ofVHAa1 at the cell plate.

The effect of VLCFA-sphingolipids during hemifusion/fusion wouldalso explain the enhanced interaction times of YFP-RabA2a and VHAa1-RFP vesicles as well as their longer association with the cell plate. Thepresence of large and dynamic aggregates of YFP-RabA2a and VHAa1-RFP vesicles with single YFP-RabA2a and VHAa1-RFP vesicles coming inand getting out (Movie S2) would also be consistent with a blockage ata docked state. Interestingly, in yeast, the VLCFA-phospholipid C26-phosphatidylinositol (C26-PI), which can structurally mimic and func-tionally replace C26-sphingolipids, was shown to stabilize highly curvedmembrane structures such as those involved in nuclear pore formation[15]. The authors proposed that C26-PI could occupy, with its very longacyl chain, the void volume present in the hydrophobic core of curvedmembrane domains. This model for C26-PI would be in agreement witha possible role of VLCFA-sphingolipids in stabilizing the highly curvedstalk structure. This could also explain the defective retrieval of mem-branes by inefficient membrane bending during the formation ofendocytic vesicles from the cell plate.

Plant cell plate biogenesis provides a simple and efficient system to in-vestigate the role of sphingolipids inmembrane dynamics. It is particular-ly interesting that the effect of VLCFA-sphingolipids could be revealed in

Approach Contact

VLCF

Fig. 6.Model for the role of VLCFA-sphingolipids in vesicle fusion. Membrane fusion involvescharacterized by an hourglass-shaped structure displaying zones of high membrane curvaturVLCFA-Cer (in red), which would stabilize the stalk structure and facilitate fusion.

the absence of any membrane fusion proteins (notably the solubleN-ethylmaleimide sensitive factor attachment protein receptors orSNAREs) thus suggesting the direct importance of lipid acyl chainlength in fusion rather than in the regulation of a protein fusion ma-chinery. We thus anticipate that the results shown here are relevantto events occurring downstream of SNARE dependent steps. Bio-physical studies on reconstituted liposomes containing recombinantplant SNARE proteins will clarify the role of sphingolipid acyl chainlengths but also headgroups on SNARE regulation versusmembrane fu-sion. Several other questions remain to be addressed and in particularthe role of GIPC, the other plant family of complex sphingolipids, andtheir relative contribution tomembrane trafficking during cell plate for-mation. Indeed sphingolipid headgroups, and not only sphingoid basesor acyl chains, may be important for membrane fusion. For example,sphingomyelin (SM), which contains a non-sugar based polar head,was shown to inhibit membrane fusion but also to limitmembrane rup-ture, therefore favoring non-leaky fusion, whereas specific SM/sterolratio provided optimal conditions to minimize rupture and maximizefusion [55]. More in depth biophysical analysis of the effect of differentsphingolipids on membrane bending, lipid packing and membrane flu-idity should shed light on the different roles of these essential lipids inmembrane dynamics. Finally, the impact of very long chain fatty acidsin membrane fusion and cytokinesis in other organisms includingmammalian cells remains an important question to explore in orderto generalize the principle unraveled here.

4. Materials and methods

4.1. Plant material and growth condition

Seedlingswere grown inArabidopsis agarmediamodified fromEstelleand Somerville [59]. Double mutant loh1-1/1-3 from the WisconsinT-DNA collection and loh1-2/3-2 allele from Salk T-DNA collectionwere already described in [9]. Fluorescent transgenic lines used forthis work were: YFP-RabA2a [26], VHAa1-GFP, VHAa1-RFP [40],and YFP-RabF2b (wave2) [60]. FB1-treatments were performed asdescribed previously and their effect was monitored in every exper-iment by the presence of YFP-RabA2a vesicle aggregation and en-hanced cell plate numbers as previously observed [9].

4.2. Live imaging and particle tracking

Root tip imaging was carried out with seedlings mounted in mediaandhighmagnification imageswere takenwith 63×objective (water im-mersion) with a line averaging of 8. Roots were stained with 2 μg/mlFM4-64 directly on the microscope slide to visualize cell membranesbut also to check for cell viability. All the imaging analysis was carriedout in the division zone of epidermal cells. Immunolocalization was per-formed as in [24]. The primary antibodies used were rabbit anti-Knolle

Stalk Fusion

A

close contact between lipid bilayers followed by a transient state called stalk. The stalk ise and hydrophobic voids. These voids could be filled with the long hydrophobic chain of

1429D. Molino et al. / Biochimica et Biophysica Acta 1841 (2014) 1422–1430

(from Gerd Jurgens) and mouse anti-α-tubuline (Invitrogen). Secondaryantibodies were Alexa 488 anti-rabbit and Alexa 647 anti-mouse. Forcallose staining, seedlings were previously fixed for 1 min in parafor-maldehyde 4%/MTSB 1/2 bufferwashed 3 timeswithMTSB 1/2 then im-merged in aniline blue staining solution (10% acetic acid in EtOH, 50mMKPO4 buffer (4.17 ml 1 M K2HPO4 + 0.83 ml 1 M KH2PO4 + 995ml H2O,pH 7.5), 0.5% aniline blue) for 15min. Seedlingswerewashedwithwaterbefore mounting.

Spinning disc confocal microscopy was used with sequential imaging(0,870ms) for 2min.Microscopeparameterswere the following: laser in-tensity at 3% for the GFP (488 nm) and 15% for the RFP (561 nm); gain60%, frequency 400 ms, exposition time 500 ms, objective 100× (oil im-mersion). Manual tracking plug-in of Image J software was used forspeed calculation.

For statistical validation, data were first processed with a normalitytest (Kolmogorov–Smirnov test, the Shapiro–Wilk test and D'Agostinoand Pearson's omnibus normality test). Data with normal distributionswere validated with t-tests; non-normal distributed sets of data wereevaluated with Mann–Whitney and ANOVA.

4.3. Electron microscopy preparation and observations

High-pressure freezing, freeze substitution and embedding processeswere carried out as previously described [61]. 80 nm ultrathin sections(Ultracut UC6, Leica) were collected on formwar coated copper grids.Theywere post stainedwith aqueous 2%uranyl acetate/lead citrate as de-scribed in Hawes and Satiat-Jeunemaitre [62]. They were examined witha JEOL 1400 transmission electronmicroscope (Croissy, France) operatingat 120 kV. Images were acquired using a post-column high resolution(11 megapixels) high speed camera (SC1000 Orius, Gatan).

4.4. Liposome reconstitution and fusion

4.4.1. Liposome reconstitutionThe lipids used in this studywere purchased fromAvanti Polar Lipids:

1,2-dioleoyl-sn-glycero-3-phosphocholine (PC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (PE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (PS), 22,23-dihydrostigmasterol (SITO), C16 glucosyl(β) cer-amide (d18:1/16:0) (C16:0-Cer), C24:1 glucosyl(β) ceramide (d18:1/24:1(15Z)) (C24:1-Cer), C24:0 galactosyl(β) ceramide (d18:1/24:0)(C24:0-Cer), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (PE-NBD), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (PE-RHO).Except otherwise noted, chemicals were purchased from Merck withthe Molecular Biology grade. All aqueous solutions were prepared using18.2 MΩ ultra-pure water and filtered through 0.2 μm hydrophilicmembranes.

Liposomes were formed using the detergent-assisted methodand consisted of 45 mol% PC, 25 mol% PE, 10 mol% PS, 10 mol%SITO ± 10 mol% Cer (white liposomes) or 42 mol% PC, 25 mol%PE, 10 mol% PS, 10 mol% SITO, 1.5 mol% PE-NBD, and 1.5 mol% PE-RHO±10 mol% Cer (red liposomes). 0.9 μmol of the appropriatelipid mixture (in chloroform) was dried in a glass tube for 10 minunder a gentle stream of nitrogen, and for 1 h under vacuum. Thedried lipid film was resuspended in 300 μl buffer A [25 mM HEPES/KOH (pH 7.5), 150 mM KCl, 10% (v/v) glycerol, 1% (w/v) n-octyl-β-D-glucopyranoside], under vigorous vortexing for 1 h at room tempera-ture. The detergent concentration was next reduced to 0.33% (w/v) byrapid dilution in buffer B [25 mM HEPES/KOH (pH 7.5), 150 mM KCl,10% (v/v) glycerol], and then removed by overnight flow dialysisagainst 4 l of buffer B. Liposomes (1 mM lipid final) were kept on icefor up to 2 weeks. Liposome size was measured by dynamic light scat-tering (DLS) using the Zetasizer Nano ZS (Malvern Instruments)with li-posomes diluted 10 fold in buffer B (100 μM lipid final).

4.4.2. FRET-based lipid mixing assay18 μl ofwhite liposomes and 2 μl of red liposomeswere added as two

separate drops to 96-well FluoroNunc plates (Fisher Scientific). The fu-sion reaction was initiated by adding 40 μl of polyethylene glycol (PEG)8000 (Sigma BioUltra) at 7.5% (w/v) in buffer B. Lipid mixing was mea-sured by following the fluorescence dequenching of PE-NBD lipidsresulting from their dilution into the fused liposomes. The NBD fluo-rescence was monitored at 1 min intervals for 60 min (excitation at485 nm; emission at 535 nm) by the Wallac 1420 Victor2 (PerkinElmer) plate reader equilibrated to 37 ºC. After 60 min, 10 μl of2.5% (w/v) n-dodecyl-β-D-maltoside was added to completely dissolvethe liposomes and thus measure the NBD fluorescence at infinite dilu-tion; the data were then normalized by setting this maximal NBD fluo-rescence signal to 100% and the lowest NBD fluorescence signal to 0%.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbalip.2014.06.014.

Acknowledgements

D.M. and this work was supported by the ANR program blancSphingopolaR (07-BLAN-202), Marie Curie Fellowship FP7 (MEST-CT-2004-7576 VERT) and postdoctoral fellowships from Région Ile-de-France (DIM Nerf) and the Ecole des Neurosciences de Paris (ENP).D.T. is funded by the ANR Jeunes Chercheurs (JCJC) grant ANR-09-JCJC-0062-01.We are grateful to the Galli Laboratory for helpful discus-sions. We thank Lydia Danglot for the critical reading and help with thestatistical validation of data.We thank Carlos Kikuti and Florent Carn forthe help with the DLS, and we are indebted to the French Labex SEAM(Sciences and Engineering for Advanced Materials and devices) sup-ported by CGI (Commissariat Général à l'Investissement). We thankMalcolm Bennett and Ranjan Swarup for the gift p35S:H2B-RFP; p35S:Lti6-GFP lines and for helpful discussion. We are grateful to Ian Moorefor providing the different pRab-A2:YFP-Rab-A2a lines and to GerdJürgens for the KNOLLE antibodies. We thank Bruno Letarnec for takingcare of the plants. We are really grateful to Marie-Cécile Caillaud for theimmunolocalization protocol. This work has used the cytology and im-aging facility of the plateforme de Cytologie et Imagerie Végétale (sup-ported by Région Ile de France and Conseil Général des Yvelines) andthe electronmicroscopy facilities and cell biology unit of the Imagif plat-form (CNRS), supported by the Conseil Général de l'Essonne (www.imagif.cnrs.fr).

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