Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complexes in Arabidopsis thaliana.

Plant and Cell Physiology Advance Access published December 15, 2014

Title: Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complex in Arabidopsis thaliana

Running head: Phosphoinositide requirements in CESA trafficking

Corresponding author: T. Ueda

7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Telephone: +81-3-5841-4579 Fax: +81-3-5841-7613 E-mail: [email protected]

Subject areas: Membrane and transport

Number of black and white figures: 0

Number of color figures, tables: 8

Type and number of supplementary material: 1

© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: [email protected]

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Downloaded from http://pcp.oxfordjournals.org/ at texas christian university on January 12, 2015

Address: Department of Biological Sciences, Graduate School of Science, The University of Tokyo,

Title: Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of the cellulose synthase complex in Arabidopsis thaliana

Running head: Phosphoinositide requirements in CESA trafficking

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Authors: Masaru Fujimoto , Yasuyuki Suda , Samantha Vernhettes , Akihiko Nakano , and

Affiliations: 1 Laboratory of Developmental Cell Biology, Department of Biological Sciences, Graduate 2

School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; INRA, UMR1318, Institut Jean-Pierre Bourgin, Saclay Plant Sciences, F-78000 Versailles, France; AgroParisTech, Institut Jean-Pierre Bourgin, RD10, F-78000 Versailles, France;

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RIKEN Center for

Advanced Photonics, Live Cell Molecular Imaging Research Team, Extreme Photonics Research Group, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan;

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Japan Science and Technology Agency

(JST), PRESTO, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan

Keywords: Cellulose synthase, CESA, Membrane trafficking, Endocytosis, Phosphoinositides

Abbreviations: CESA, cellulose synthase; CGA, CGA 325'615; CLC, clathrin light chain; CLSM, confocal laser scanning microscopy; CME, clathrin-mediated endocytosis; CSC, cellulose synthase complex; DMSO, dimethylsulfoxide; GFP, green fluorescent protein; MASC, microtubule-associated cellulose

synthase

compartment;

MS,

Murashige-Skoog;

PAO,

phenylarsine

oxide;

PI,

phosphoinositide; PI3K, phosphatidylinositol 3-kinase; PI4K, phosphatidylinositol 4-kinase; PM, plasma membrane; PtdIns, phosphatidylinositol; PVC, prevacuolar compartments; SCLIM, super-resolution

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Takashi Ueda1,5

confocal live imaging microscopy; SIM, structured illumination microscopy; SmaCC, small CESA-containing compartment; ST, sialyltransferase; SYP43, syntaxin of plants 43; TEM, transmission electron microscopy; TGN, trans-Golgi network; VHAa1, vacuolar ATPase a1 subunit; VIAFM, variable incidence angle fluorescence microscopy; Wm, wortmannin;

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Present Address: Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life

Molecular Cell Biology, Graduate School of Comprehensive Human Sciences and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan

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Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; 7Department of

Abstract The oriented deposition of cellulose microfibrils in the plant cell wall plays a crucial role in various plant functions such as cell growth, organ formation, and defense responses. Cellulose is synthesized by cellulose synthase complexes (CSCs) embedded in the plasma membrane, which comprise the cellulose synthases (CESAs). The abundance and localization of CSCs at the plasma membrane (PM) should be strictly controlled for precise regulation of cellulose deposition, which

transport of CSCs is still poorly understood. In this study, we explored requirements for phosphoinositides (PIs) in CESA trafficking by analyzing the effects of inhibitors of PI synthesis in Arabidopsis thaliana expressing green fluorescent protein-tagged CESA3 (GFP-CESA3). We found that a shift to a sucrose-free condition accelerated re-localization of PM-localized GFP-CESA3 into the periphery of the Golgi apparatus via the clathrin-enriched trans-Golgi network (TGN). Treatment with wortmannin (Wm), an inhibitor of phosphatidylinositol 3- (PI3K) and 4- (PI4K) kinases, and phenylarsine oxide (PAO), a more specific inhibitor for PI4K, inhibited internalization of GFP-CESA3 from the PM. In contrast, treatment with LY294002, which impairs the PI3K activity, did not exert such an inhibitory effect on the sequestration of GFP-CESA3, but caused a predominant accumulation of GFP-CESA3 at the ring-shaped periphery of the Golgi apparatus, resulting in the removal of GFP-CESA3 from the PM. These results indicate that PIs are essential elements for localization and intracellular transport of CESA3 and that PI4K and PI3K are required for distinct steps in secretory and/or endocytic trafficking of CESA3.

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strongly depends on the membrane trafficking system. However, the mechanism of the intracellular

Introduction Load-bearing cellulose microfibrils confer structural and mechanical rigidity to the plant cell wall, which allows the polarized cell expansion underlying organ formation and growth of plants (Baskin 2005; Green 1962). In land plants, cellulose is synthesized by hexameric membrane-bound complexes comprising three distinct cellulose synthase catalytic subunits (CESAs), which are referred to as cellulose synthase complexes (CSCs) (Kimura et al. 1999). CESA members constitute a large gene

member of CESA6-like clade are responsible for primary cell wall synthesis (Desprez et al. 2007; Persson et al. 2007; Persson et al. 2005; Wang et al. 2008), and three other isoforms, CESA4, CESA7, and CESA8, are implicated in secondary cell wall synthesis (Brown et al. 2005; Taylor et al. 2003; Taylor et al. 1999). Cellulose synthesis by CSCs is assumed to occur solely at the plasma membrane (PM) (Cosgrove 2005; Oikawa et al. 2013; Reiter 2002; Scheible and Pauly 2004). However, live- and fixed-cell imaging with confocal laser scanning microscopy (CLSM) or transmission electron microscopy (TEM) have shown that the localization of CESAs is not restricted to the PM; CESAs are also detected in several intracellular compartments including the Golgi apparatus, the trans-Golgi network (TGN), and the distinctive organelles referred to as microtubule-associated cellulose synthase compartments (MASCs) and/or small CESA-containing compartments (SmaCCs) (Crowell et al. 2009; Desprez et al. 2007; Gu et al. 2010; Gutierrez et al. 2009; Haigler and Brown 1986; Li et al. 2012; Paredez et al. 2006). This multiple localization of CESAs has an implication on the intracellular trafficking system, which could be responsible for the regulated deposition and abundance of CSCs on the PM in response to developmental and environmental cues (Crowell et al. 2010; Wightman and Turner 2010). Recent research has identified a few proteins involved in intracellular trafficking of the CSC. The µ2 subunit of the adaptor protein complex 2 (AP-2), which is involved in clathrin-mediated

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family, which is classified into two subgroups (Richmond and Somerville 2000). CESA1, CESA3, and a

endocytosis (CME) (Bashline et al. 2013; Di Rubbo et al. 2013; Gadeyne et al. 2014; Kim et al. 2013; Yamaoka et al. 2013), has been shown to interact with multiple CESA proteins and to be required for efficient sequestration of CESA6 from the PM (Bashline et al. 2013). GFP-CESA endocytosis from the cell plate and from the plasma membrane has also been described in dividing cells (Miart et al. 2014). More recently, KORRIGAN1, which is a membrane bound endo-1,4-β-D-glucanase and a component of the CSC, was also shown to have a role in intracellular trafficking of the CSC (Lei et al. 2014; Ueda

unknown. Although membrane lipids are known to play pivotal roles in membrane trafficking (Hanzal-Bayer and Hancock 2007; Sprong et al. 2001), possible roles of lipids in CSC trafficking has not been explored thus far. Phosphoinositides (PIs) are a class of membrane lipids comprising phosphatidylinositol (PtdIns) and its phosphorylated derivatives, which modulate a wide range of cellular processes including membrane trafficking (Wenk and De Camilli 2004). PtdIns consists of diacylglycerol connected to a myo-inositol head with five free hydroxyl groups, of which positions 3, 4, and 5 are targets for phosphorylation by lipid kinases, generating mono-, bis, and tri-phosphates (Munnik and Nielsen 2011). Among the derivatives, five types of PIs, PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,5)P2, and PtdIns(4,5)P2, have been detected in plants (Mueller-Roeber and Pical 2002). Specific PI-binding domains tagged with fluorescent proteins have been developed as biosensors for PIs, which allowed the elucidation of the intracellular distribution and dynamics for some of these PIs; for example, PtdIns3P is enriched in membranes of endosomes, prevacuolar compartments (PVC), and vacuoles; PtdIns4P in the Golgi apparatus, TGN, and PM; and PtdIns(4,5)P2 is abundant in the PM (Simon et al. 2014; van Leeuwen et al. 2007; Vermeer et al. 2009; Vermeer et al. 2006). To identify the requirements for PIs in each pathway of the membrane trafficking system in plants, investigation of lipid kinases that mediate PI metabolism (PI kinases) has been important

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2014; Vain et al. 2014). However, the molecular mechanisms mediating CSC trafficking remain largely

(Krishnamoorthy et al. 2014; Thole and Nielsen 2008). For example, the activity of PtdIns 3-kinase (PI3K), which generates PtdIns3P from PtdIns, is required for vacuolar trafficking and the late step of endocytosis in Arabidopsis thaliana (Kim et al. 2001; Lee et al. 2008). Regarding PtdIns 4-kinase that synthesizes PtdIns4P from PtdIns, PI4Kβ1 has been identified as an effector of a small GTPase (RabA4b) and shown to act in the polarized exocytosis of cell wall materials such as pectin and xyloglucan in root hairs (Kang et al. 2011; Preuss et al. 2006). PI4Kβ1 is also reported to be involved in

PtdIns4P 5-kinases (PIP5K) that mediate the synthesis of PtdIns(4,5)P2 from PtdIns4P, have also been shown to coordinate apical secretion of pectin during root hair formation (Kusano et al. 2008; Stenzel et al. 2008) and pollen tube growth (Ischebeck et al. 2008; Ischebeck et al. 2010). Three other isoforms of PIP5K, PIP5K1/2 and PIP5K6 have also been reported to regulate CME in root cells (Ischebeck et al. 2014; Mei et al. 2012) and growing pollen tubes (Zhao et al. 2010). Moreover, A. thaliana FAB1A and FAB1B, close homologs of yeast PtdIns3P 5-kinases (PI3P5K) Fab1p that catalyze the conversion of PtdIns3P to PtdIns(3,5)P2, have been implicated in endosomal trafficking associated with maintenance of vacuolar morphology and homeostasis (Hirano et al. 2011; Serrazina et al. 2014). These lines of evidence also suggest a potential contribution of PIs in cellulose synthesis, which is strongly dependent on the membrane trafficking system. In the present study, to elucidate the requirements for PIs in CESA trafficking, we conducted pharmacological analyses using several inhibitors of PI synthesis: wortmanninn (a PI3K and PI4K inhibitor) (Aggarwal et al. 2013; Jung et al. 2002), LY294002 (a PI3K inhibitor) (Aggarwal et al. 2013; Jung et al. 2002; Lee et al. 2008), and phenylarsine oxide (PAO, a PI4K inhibitor) (Vermeer et al. 2009). These inhibitors affected different steps of transport of fluorescently tagged CESA3, which indicated that PI3K and PI4K are specifically required for CESA trafficking in A. thaliana.

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the late step of CME at the tip of the pollen tube (Zhao et al. 2010). PIP5K3 and PIP5K4/5, members of

Results Hypotonic stimulus accelerates re-localization of CESA3 from the PM into the intracellular compartments Hypertonic stress has been demonstrated to induce internalization of PM-localized CESA in the hypocotyl epidermal cells of A. thaliana (Crowell et al. 2009; Gutierrez et al. 2009). In this study, we investigated the effect of hypotonic conditions on CESA internalization by transplanting seedlings into a

changes of CESA3 localization exposed to sucrose-free conditions in root epidermal cells of A. thaliana. Immediately after the transfer from a normal sucrose medium (30 mM) to a sucrose-free medium (0 mM), green fluorescent protein (GFP)-tagged CESA3 was predominantly localized in the PM (Fig. 1A). As time passed, intracellular signals of GFP-CESA3 gradually increased, a large part of which was co-localized with monomeric Orange-tagged clathrin light chain (CLC-mOrange) (Fig. 1A, 15 min), which is on the TGN (Ito et al. 2012a). After 30 min incubation, a major part of the signals accumulated on ring-shaped structures (Fig. 1A, 30 min and Fig. 1B, arrowheads). Conversely, under the continuous normal sucrose condition, the rapid increase of GFP-CESA3 in intracellular compartments was not observed (Fig. S1). Pearson’s correlation coefficients between fluorescence from GFP-CESA3 and 2

CLC-mOrange, which were calculated from six 100 µm CLSM images for each timepoints, also supported internalization of GFP-CESA3 into CLC-positive intracellular compartments in root epidermal cells exposed to the sucrose-free condition (Fig. 1C). These results indicated that the hypotonic stimulus also triggers accelerated re-localization of PM-localized CESA3 into intracellular compartments via the TGN.

Inhibitors of PI4K and PI3K differently affect CESA trafficking from the PM Phosphoinositides (PIs) are known as regulatory components of membrane trafficking pathways

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sucrose-free medium. Using confocal laser scanning microscopy (CLSM), we observed temporal

including endocytic trafficking. To examine the involvement of PIs in CESA trafficking, we first analyzed the effect of wortmannin (Wm) on CESA3 re-localization from the PM in A. thaliana root epidermal cells. At 60 min after exposure to a sucrose-free medium containing 0.1% dimethylsulfoxide (DMSO), the fluorescent signals of GFP-CESA3 were mainly distributed at the obvious ring-shaped compartments, and PM localization was also evident (Fig. 2). In contrast, treatment with 80 µM Wm severely inhibited the internalization of GFP-CESA3 into the cytoplasm. Intriguingly, lower concentrations of Wm (4 µM

structures in the cytoplasm (Fig. 2). Given that a high concentration of Wm inhibits both PI3K and PI4K and a low concentration of Wm more specifically inhibits PI3K (Aggarwal et al. 2013; Jung et al. 2002; Vermeer et al. 2009; Vermeer et al. 2006), these distinct effects could result from different requirements for PI4K and PI3K activities in CESA3 trafficking. Therefore, we examined the effects of more specific inhibitors of PI4K and PI3K (PAO and LY294002, respectively) on the re-localization of CESA3 caused by the hypotonic treatment in root epidermal cells. After treatment with 40 µM PAO for 60 min, most of GFP-CESA3 signals were retained on the PM, similarly to the 80 µM Wm treatment (Fig. 2). Conversely, after the 60 min 40 µM LY294002 treatment, the GFP-CESA3 signals entirely disappeared from the PM and accumulated at punctate intracellular compartments, similarly to the 4 µM and 20 µM Wm treatment. Weaker but similar effects were observed when cells were treated with 10 µM PAO or LY294002, respectively, whereas 4 µM PAO and LY294002 did not markedly affect the endocytic trafficking of GFP-CESA3. These results indicated that PI4K and PI3K play distinct roles in CESA3 trafficking after the hypotonic treatment.

PI4K is required for clathrin-mediated CESA3 internalization from the PM To identify the process requiring PI4K in CESA3 trafficking, we monitored hypotonicity-stimulated re-localization of GFP-CESA3 in the presence or absence of PAO by CLSM and variable incidence

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and 20 µM) resulted in the removal of GFP-CESA3 from the PM and its accumulation in dot-shaped

angle fluorescence microscopy (VIAFM). In the absence of PAO, the cytoplasmic fluorescent signals of GFP-CESA3 rapidly increased and were detected in the punctate compartments labeled with CLC-mOrange and on the PM (Fig. 3A). On the PM as observed by VIAFM, GFP-CESA3 signals assembled into discrete foci after 15 min exposure to the sucrose-free medium without PAO and partially co-localized with CLC-mOrange (Fig. 3B). Conversely, a rapid increase in cytoplasmic GFP-CESA3 signals was not observed in the presence of PAO (Fig. 3C). Moreover, in VIAFM images

co-localized with CLC-mOrange, which formed large aggregates instead of the punctate foci on the PM (Fig. 3D). These observations were further supported by quantitative analyses (Figure 3E and F). A similar effect on CLC-mOrange was also observed for the similar concentration of Wm in our previous study (Ito et al. 2012a). These results suggest that PI4K inhibitors affect the internalization of CESA3 at the early step of endocytosis, which should be due to an inhibitory effect on a clathrin-mediated process of endocytosis.

Inhibition of PI3K results in CESA3 accumulation at the periphery of the Golgi apparatus To distinguish the role of PI3K in CESA3 trafficking, we investigated a spatio-temporal change in endocytic transport of GFP-CESA3 triggered by the hypotonic treatment in the presence or absence of 40 µM LY294002 in root epidermal cells by CSLM, which was also followed by the quantitative analysis. Under the control conditions (0.1% DMSO), GFP-CESA3 was time-dependently internalized into the cytoplasm after hypotonic stimulus and predominantly localized in the PM and ring-shaped compartments closely associated with the CLC-mOrange-labeled TGN after 60 min (Fig. 4A, upper panels). Conversely, in the presence of LY294002, GFP-CESA3 was internalized into the cytoplasm to accumulate in the punctate compartments with a gradual removal of the PM-localized GFP-CESA3, and GFP-CESA3 totally disappeared from the PM after 30-60 min (Fig. 4A, lower panels). The

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of root epidermal cells treated with PAO for 15 min, GFP-CESA3 foci at the PM were barely

quantitative analysis also demonstrated that the colocalization rate between GFP-CESA3 and CLC-mOrange was significantly reduced at 30 min after the hypotonic treatment (Fig. 4B), further indicating the effect of LY294002 on subcellular trafficking of GFP-CESA3. To define the fine structures of the LY294002-induced punctate compartments containing GFP-CESA3, we applied a combination of different super-resolution microscopic techniques, specifically, super-resolution confocal live imaging microscopy (SCLIM) (Nakano 2013) and structured

compartments in root epidermal cells after incubation with or without LY294002 for 60 min. In both SCLIM and SIM images of the control plants, the ring-shaped localization of GFP-CESA3, which was also frequently associated with CLC-mOrange, was clearly observed (Fig. 5A). Also, in plants treated with LY294002, a similar ring-shaped localization of GFP-CESA3 was evident via SCLIM and SIM, but the diameters of the ring-shaped structures seemed to be smaller than those observed in the control plants (Fig. 5B). We then characterized the ring-shaped structures observed in LY294002-treated cells in more detail. We co-expressed GFP-CESA3 with markers of several post-Golgi organelles and compared their localization in cells treated with or without LY294002 for 60 min. In CLSM images of the control and LY294002-treated cells (Fig. 6A, upper and lower panels, respectively), large parts of the GFP-CESA3 signals were localized in the periphery of the trans-Golgi cisternae as labeled by sialyltransferase fused to mRFP (ST-mRFP) (Boevink et al. 1998). Conversely, GFP-CESA3 was rarely co-localized with TGN markers, vacuolar ATPase a1 subunit (VHAa1) or Syntaxin of plants 43 (SYP43) fused to mRFP, but they were observed in close association as observed for the Golgi and TGN markers (Uemura et al. 2004; Viotti et al. 2010) (Crowell et al. 2009). We hardly observed co-localization between GFP-CESA3 and the multivesicular endosome marker mRFP-ARA7, which exhibited dilated morphology upon LY294002 treatment as observed in Wm-treated cells (Ebine et al.

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illumination microscopy (SIM) (Langhorst et al. 2009). We observed GFP-CESA3-labelled

2011) (Fig. 6A). Quantitative analysis using Pearson’s correlation coefficient between GFP and mRFP fluorescence also supported the predominant Golgi localization of GFP-CESA3 in both the control and LY294002-treated cells (Fig. 6B). Treatment with LY294002 also reduced co-localization/association between GFP-CESA3 and mRFP-ARA7 (Fig. 6B). These results indicated that the inhibition of PI3K causes accumulation of GFP-CESA3 on the Golgi apparatus, leading to the removal of CESA3 from the PM.

After treatment with LY294002, the sizes of the ring-shaped structures bearing GFP-CESA3 seemed to be smaller than those in control cells in images taken with SCLIM, SIM, and CLSM (Fig. 5A, 5B, and 6A), which raised the possibility that LY294002 affects the size of the Golgi apparatus. To verify this possibility, we compared the planar sizes of the post-Golgi organelles visualized using RFP-tagged 2

organelle makers in ten to fifteen 100 µm CLSM images of root epidermal cells treated with or without LY294002 for 60 min. As we expected, the size of the Golgi apparatus was significantly reduced by the LY294002 treatment, which suggested that PI3K activity is required for maintaining the size of the Golgi (Fig. 6C). Conversely, a slight but significant increase in the planar size of the TGN (VHAa1-mRFP and mRFP-SYP43) was detected, and the size of the multivesicular endosomes (mRFP-ARA7) was obviously enlarged compared to the size in control plants (Fig. 6C). Thus, inhibition of PI3K had distinct effects on post-Golgi organelles, indicating specific requirements for PI3K in homeostasis of post-Golgi organelles.

PI4K is also required for CGA-induced relocation of CESA3 from the PM Treatment with a cellulose synthesis inhibitor, CGA 325'615 (CGA), has been reported to stimulate the relocation of CESA from the PM to intracellular compartments, the MASCs/SmaCCs and the Golgi

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Inhibited PI3K reduced the size of the Golgi apparatus

apparatus in hypocotyl epidermal cells in A. thaliana (Crowell et al. 2009). We investigated whether PI4K is also required for CGA-induced re-localization of CESA3. In A. thaliana root epidermal cells treated with CGA in the presence of DMSO for 60 min, GFP-CESA3 accumulated in ring-shaped and dot-shaped compartments, which were the Golgi apparatus and presumably MASCs/SmaCCs, respectively, and was hardly observed on the PM as previously reported (Crowell et al. 2009; Gutierrez et al. 2009) (Fig. 7, left panels). In contrast, co-incubation with CGA and PAO for 60 min after

panels). These results indicated that the re-localization of CESA3 induced by the cellulose synthesis inhibitor also requires PI4K.

Discussion Plant PI4K and PI3K are required for distinct steps in the endocytic and/or secretory trafficking of CESA In this study, we demonstrated that CESA3 was internalized into the intracellular compartments from the PM upon the hypotonic treatment in A. thaliana root epidermal cells. Although the physiological significance of re-localization of CESA upon the hypotonic treatment remains unknown, the hypertonic treatment is also shown to promote endocytosis of CESA proteins (Crowell et al. 2010; Gutierrez et al. 2009), which is associated with modification of the cell wall. The response of the cell wall to the hypotonic condition should be also examined in the future studies. We demonstrated that the inhibitors of PI4K (PAO) and PI3K (LY294002) affect different steps of CESA trafficking. The treatment with PAO severely impaired sucrose depletion or cellulose synthesis inhibitor-stimulated internalization of fluorescently labeled CESA3 from the PM, causing abnormal clathrin aggregation in A. thaliana root epidermal cells (Fig. 3A, 3C, and Fig. 7). Considered together with a recent report on the involvement of the adaptor complex 2 (AP2) in CESA endocytosis (Bashline

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pretreatment with PAO for 15 min inhibited the relocation of GFP-CESA3 to the cytoplasm (Fig. 7, right

et al. 2013), PI4K is thought to participate in clathrin-dependent endocytic vesicle formation. PM-enriched PtdIns4P and PtdIns(4,5)P2, whose metabolism involves PI4K, were consistently demonstrated to play a critical role in the regulation of plant CME (Ischebeck et al. 2010; Ischebeck et al. 2013; Zhao et al. 2010), but the specific processes requiring PI4K activity in plant CME has not been identified thus far. Further studies are needed to elucidate the specific function of PI4K in the endocytosis of CESA from the PM (Fig. 8).

internalization of CESA3 from the PM into the intracellular compartments. However, LY294002 resulted in the accumulation of CESA3 in the periphery of the Golgi apparatus and in dot-shaped organelles, leading to disappearance of CESA3 from the PM. This finding raises the possibility that reduced activity of PI3K results in the impairment of secretion of CESA from the Golgi apparatus, but it is also possible that PI3K is specifically required for recycling of endocytosed CESA to the PM. To our knowledge, a function for PI3K in secretory trafficking has not been elucidated in plants; however, PI3K is implicated in the secretion of insulin and cytokines in animal cells and in the exocytosis of a surface glycoprotein in trypanosomes (Dominguez et al. 2011; Hall et al. 2006; Low et al. 2010). Our results could indicate that there is a common step critical for secretory trafficking mediated by PI3K in animal and plant cells. It would be an interesting future project to examine the function of PI3K and its product PtdIns3P in the secretory/exocytic pathway, in which CESA is a promising cargo molecule. It was suggested that Wm blocks the initial step of CME in plant cells (Beck et al. 2012; Di Rubbo et al. 2013; Ito et al. 2012a; Robatzek et al. 2006). In this study, we demonstrated that a lower concentration of Wm had a different effect from higher concentration of Wm on the trafficking of CESA3; the lower concentration of Wm exerted a similar effect to that of LY294002, whereas the higher concentration of Wm inhibited internalization of CESA3 in a manner similar to that of PAO. Given the reported dose-dependent specific inhibitory effects of Wm on PI3K and PI4K (Aggarwal et al. 2013;

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In contrast to the effect of PAO, the treatment with LY294002 did not perturb the

Jung et al. 2002; Matsuoka et al. 1995; Vermeer et al. 2009; Vermeer et al. 2006), a lower concentration of Wm should mainly inhibit PI3K and the higher concentration of Wm should inhibit both PI3K and PI4K. This notion is more consistent with a requirement for PI4K in an upstream process than a PI3K-dependent step in endocytic pathway rather than a PI4K blockage of a downstream event and a secondary effect on the initial step of endocytosis because of impaired recycling of machinery components in the plasma membrane. Different concentrations of Wm, in combination with other

PI4K in plant functions.

PI3K is involved in maintenance of morphology of post-Golgi organelles Another important finding in this study is that the inhibition of PI3K also results in the alteration of morphology of the Golgi and TGN in addition to its known effect on multivesicular endosomes. Especially, a significant reduction in the size of the Golgi apparatus was evident (Fig. 6A and 6C). It was recently reported that treatment with LY294002 promotes the homotypic fusion of vacuoles in A. thaliana root epidermal cells (Zheng et al. 2014). Despite the preferential localization of PtdIns3P in the vacuolar and late endosomal membranes (Simon et al. 2014; Vermeer et al. 2006), our results suggest that PI3K also plays some role in the maintenance of structures in the Golgi and TGN. Although it is not clear whether this effect is direct or indirect, the reduced size of the Golgi after LY294002 treatment may reflect the existence of an unknown mechanism of membrane supply from PtdIns3P-enriched endosomal/vacuolar compartments to the Golgi apparatus. The retrograde membrane flow from endosomal compartments mediated by the retromer complexes, whose function was shown to be required for PI3K activity (Arighi et al. 2004; Burda et al. 2002; Oliviusson et al. 2006; Zelazny et al. 2013), could be a candidate for the source of membranes for the Golgi. Subunits of the retromer complex in A. thaliana, VPS29, sorting nexin 1 (SNX1), and sorting nexin 2a (SNX2a) consistently

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inhibitors such as LY294002 and PAO, would be convenient tools to analyze requirements of PI3K and

localized at the TGN as well as in the endosomes/PVC (Niemes et al. 2010; Oliviusson et al. 2006). Further studies on retromer functions and the effect of mutations of retromer complexes on the Golgi apparatus are needed to elucidate the role of PI3K in trafficking between the Golgi and endosomal/vacuolar compartments. In this study, we found that transport of CESA specifically requires PI3K and PI4K at certain trafficking steps. PI4K activity has been shown to be possibly required for the endocytosis of several

labeled with FM dye from the PM (Bandmann et al. 2012; Van Damme et al. 2011), which indicates that PI4K is an essential component for general endocytosis. Conversely, it is currently unclear whether the effects of LY294002 and a low concentration of Wm are specific to CESA transport or whether the secretory/recycling pathway generally requires PI3K activity. Future studies on functions of PIs in the secretory/recycling pathway are needed to elucidate mechanisms of PI-dependent membrane trafficking in plant cells, which should also be effective to elucidate a possible coordination between cell wall synthesis and PI metabolism.

Materials and Methods Plant materials and growth conditions A. thaliana plants (accession Columbia-0) were grown on Murashige-Skoog (MS) medium plates at cesa3

23 °C under continuous light. The transgenic plant expressing GFP-CESA3 in the je5

background

and the plant co-expressing GFP-CESA3 and CLC-mOrange were previously described (Crowell et al. 2009; Miart et al. 2014). Transgenic plants that expressed combinations of GFP-CESA3 and mRFP-tagged markers of post-Golgi organelles, ST-mRFP (Ito et al. 2012b), VHAa1-mRFP (Viotti et al. 2010), mRFP-SYP43 (Uemura et al. 2012), and mRFP-ARA7 (Ebine et al. 2011) were generated by crosses with transgenic plants expressing GFP-CESA3 and plants expressing each of markers; F1

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proteins (Beck et al. 2012; Di Rubbo et al. 2013; Robatzek et al. 2006; Zhao et al. 2010) and lipids

plants were used for microscopic observation. Plants expressing VHAa1-mRFP and ST-mRFP were kindly provided by Karin Schumacher (University of Heidelberg) and Keiko Shoda (RIKEN), respectively.

Hypotonic and drug treatments Ten-day-old A. thaliana seedlings cultured on MS plates containing 30 mM sucrose and 0.3% phytagel

before microscopic observation. DMSO (0.1%, v/v), Wm (4 µM or 80 µM), PAO (4 µM or 40 µM), LY294002 (4 µM or 40 µM), or CGA (5 nM) was added to the liquid MS medium for drug treatments. Before CGA treatment, seedlings were pre-incubated in liquid MS medium containing 30 mM sucrose and 0.1% DMSO or 40 µM PAO for 15 min.

CLSM, VIAFM, SCLIM, and SIM Transgenic A. thaliana roots were placed on a glass slide (76 × 26 mm, Matsunami) and covered with a 0.12- to 0.17-mm-thick cover glass (24 × 60 mm, Matsunami). Root epidermal cells were observed with the Carl Zeiss LSM780 system for CLSM using a 63× 1.40 numerical aperture (N.A.) objective, the Nikon TIRF2 system for VIAFM with a CFI Apo TIRF 100× 1.49 N.A. objective, and the N-SIM system for SIM with a CFI Apo TIRF 100× 1.49 N.A. objective. For SCLIM, we used a custom-made system that we developed, which consists of IX-70 microscope equipped with UPlanSApo 100× 1.40 N.A. objective (Olympus), a custom-made low-noise and high-speed confocal scanner (Yokogawa electric), a custom-made spectroscopic filter system (Hamamatsu Photonics), and custom-made cooled image intensifiers (Hamamatsu Photonics) (Ito et al. 2012b; Kurokawa et al. 2013; Kurokawa et al. 2014; Matsuura-Tokita et al. 2006; Nakano 2013; Nakano and Luini 2010; Okamoto et al. 2012; Suda et al. 2013). GFP and mOrange or mRFP were excited with 488-nm and 561-nm lasers, respectively. For

17


were transferred to liquid MS medium containing 0 mM or 30 mM sucrose and incubated at 23 °C

CLSM observation, acquired images were processed with Photoshop CS6 Extended (Adobe Systems). Pearson’s correlation coefficient was calculated with Image Pro Plus 4.0 (Media Cybernetics). In VIAFM and SIM observations, fluorescence emission spectra were separated with a 565 LP dichroic mirror and filtered through 515/30 (GFP) and 580 LP (mOrange) filters for VIAFM and 515/30 (GFP) and 610/40 (mOrange) filters for SIM. Images were acquired with an iXonEM EMCCD camera (Andor Technology). Each high-resolution SIM image was constructed from nine original structured images

milliseconds and 1.8 seconds exposure, respectively, and the acquired images were analyzed with Photoshop CS6 Extended (Adobe Systems) and NIS-Elements (Nikon). In SCLIM observation, GFP and mOrange were excited with 491-nm and 542-nm lasers, respectively. The fluorescence emission spectra were separated with a 545 LP dichroic mirror and filtered through either a 515/30 (GFP) or 570/40 (mOrange) filter. Images were acquired with ImagEM EM-CCD cameras (Hamamatsu Photonics). High-resolution images were constructed via the deconvolution analysis performed with Volocity (PerkinElmer). A SCLIM image was constructed from forty images taken at 0.1 µm vertical intervals using a theoretical point-spread function optimized for CSU10 (Yokogawa Electric). The acquired images were processed with Photoshop CS6 Extended (Adobe Systems).

Statistical analysis The results are expressed as the means ± s.d. or ± s.e. calculated from appropriate numbers of experiments as indicated in the text. Student’s t-test or Tukey’s honestly significant difference were employed to analyze statistical significance.

Funding This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education,

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excited with different illumination patterns. Each frame in VIAFM and SIM was acquired by 200

Culture, Sports, Science, and Technology of Japan (A.N. and T. U.), JST, PRESTO (T.U.), and a Grant-in-Aid for JSPS Fellows (M.F.).

Acknowledgements We thank K. Kurokawa and M. Ishii (RIKEN) for kind support in the SCLIM experiment. We also thank Karin Schumacher (University of Heidelberg) and Keiko Shoda (RIKEN, Japan) for sharing materials.

The authors have no conflicts of interest to declare.

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Disclosures

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Legends to figures Fig. 1 Temporal changes of GFP-CESA3 localization in response to the hypotonic stimulus. (A) Time-course CLSM images of A. thaliana root epidermal cells expressing GFP-CESA3 (green) and CLC-mOrange (red) taken at 0, 15, or 30 min after the transfer from normal medium (30 mM sucrose) to sucrose-free medium (0 mM sucrose). Scale bars = 10 µm. (B) Magnified images of the region surrounded by the white lines in (A). Arrowheads indicate ring-shaped localization of GFP-CESA3.

2

GFP-CESA3 and CLC-mOrange calculated from six 100 µm CLSM images taken with samples prepared in the same way as samples presented in (A) and Fig. S1. The results are indicated as the means ± standard deviation (SD). Different letters indicate statistically significant differences (Tukey’s honestly significant difference; α = 0.05, n = 6).

Fig. 2 Effect of PI4K and PI3K inhibitors on the localization of GFP-CESA3 triggered by hypotonic stimulus. A. thaliana root epidermal cells expressing GFP-CESA3 (green) treated with 0.1 % DMSO or indicated concentrations of chemicals in sucrose-free medium for 60 min were observed using CLSM. Scale bars = 10 µm.

Fig. 3 Effects of PAO on the re-localization of GFP-CESA3 induced by hypotonic stimulus. (A, C) Time-course CLSM images of A. thaliana root epidermal cells expressing GFP-CESA3 (green) and CLC-mOrange (red) taken at 0, 15, or 30 min after transfer to hypotonic conditions in the presence of 0.1 % DMSO (A) or 40 µM PAO (C). (B, D) VIAFM images of A. thaliana root epidermal cells expressing GFP-CESA3 (green) and CLC-mOrange (red) incubated in sucrose-free medium for 15 min in the presence of 0.1 % DMSO (A) or 40 µM PAO (C). Scale bars = 10 µm (A, C) or 5 µm (B, D). (E) Average of Pearson’s correlation coefficients between fluorescence from GFP-CESA3 and CLC-mOrange

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Scale bar = 5 µm. (C) Average of Pearson’s correlation coefficients between fluorescence from

calculated from six 100 µm2 CLSM images taken with samples prepared in the same way as samples presented in (A) and (C). The results are indicated as the means ± SD. Different letters indicate statistically significant differences (Tukey’s honestly significant difference; α = 0.05, n = 6). (F) Average 2

number of CLC-mOrange foci calculated from 121 µm VIAFM images of A. thaliana root epidermal cells treated with 0.1 % DMSO or 40 µM PAO (n = 6). The results are indicated as the means ± SD. ***P < 0.001, Student’s t-test.

thaliana root epidermal cells expressing GFP-CESA3 (green) and CLC-mOrange (red) were observed after incubation in sucrose-free medium for 0, 15, 30, or 60 min in the presence of 0.1 % DMSO (upper panels) or 40 µM LY294002 (lower panels). Scale bars = 10 µm. (B) Average of Pearson’s correlation coefficients between fluorescence from GFP-CESA3 and CLC-mOrange calculated from six 100 µm2 CLSM images taken with samples prepared in the same way as samples presented in (A). The results are indicated as the means ± SD. Different letters indicate statistically significant differences (Tukey’s honestly significant difference; α = 0.05, n = 6).

Fig. 5 Fine structures of cytoplasmic compartments with GFP-CESA3 observed using super-resolution microscopy. (A, B) A. thaliana root epidermal cells expressing GFP-CESA3 (green) and CLC-mOrange (red) were observed using SCLIM and SIM after incubation in sucrose-free medium for 60 min in the presence of 0.1 % DMSO (A) or 40 µM LY294002 (B). Arrowheads indicate ring-shaped structures. Scale bars = 3 µm.

Fig. 6 Accumulation of GFP-CESA3 at the Golgi apparatus after treatment with LY294002. (A) A. thaliana root epidermal cells expressing GFP-CESA3 (green) and mRFP-tagged post-Golgi organelle

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Fig. 4 Effects of LY294002 on the hypotonic stimulus-induced re-localization of GFP-CESA3. (A) A.

markers (red) were observed after 60 min incubation in sucrose-free medium in the presence of 0.1 % DMSO (upper panels) or 40 µM LY294002 (lower panels) using CLSM. Scale bars = 10 µm. (B) Average of Pearson’s correlation coefficients between fluorescence from GFP-CESA3 and 2

mRFP-tagged organelle markers calculated from four 100 µm CLSM images taken with samples prepared in the same way as samples presented in (A). Different letters indicate statistically significant differences (Tukey’s honestly significant difference; α = 0.05, n = 4). The results are indicated as the

with 0.1 % DMSO (blue bar) or 40 µM LY294002 (red bar) for 60 min and calculated from 100 µm

2

CLSM images (n = 10~16). The results are indicated as the means ± SE. *P < 0.05. **P < 0.01. ***P < 0.001, Student’s t-test.

Fig. 7 Effect of PAO on CGA 325'615 (CGA)-induced accumulation of GFP-CESA3 in intracellular compartments. A. thaliana root epidermal cells expressing GFP-CESA3 (green) and CLC-mOrange (red) incubated in the medium containing 30 mM sucrose in the presence of 0.1 % DMSO (left panels) or 40 µM PAO (right panels) with CGA for 60 min after pretreatment with DMSO or PAO for 15 min were observed using CLSM. Scale bars = 10 µm.

Fig. 8 Schematic illustration of trafficking steps requiring PI4K and PI3K in CESA trafficking. PI4K is required for the initial step of internalization of CESA, and PI3K plays a pivotal role(s) in secretory and/or recycling pathway from the Golgi apparatus. CSC, cellulose synthase complex comprising CESA;

MASC/SmaCC,

microtubule-associated

CESA-containing compartments.

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cellulose

synthase

compartments/small


means ± standard errors (SE). (C) Average planar areas of organelles in root epidermal cells incubated


119x136mm (300 x 300 DPI)


160x213mm (300 x 300 DPI)


119x255mm (300 x 300 DPI)


209x164mm (300 x 300 DPI)


119x140mm (300 x 300 DPI)


165x131mm (300 x 300 DPI)


92x70mm (300 x 300 DPI)


77x50mm (300 x 300 DPI)

Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis.

CGR2 and CGR3 have critical overlapping roles in pectin methylesterification and plant growth in Arabidopsis thaliana.

The trafficking of the cellulose synthase complex in higher plants.

The roles of cross-talk epigenetic patterns in Arabidopsis thaliana.

The condensin complexes play distinct roles to ensure normal chromosome morphogenesis during meiotic division in Arabidopsis.

Intracellular trafficking of cationic liposome-DNA complexes in living cells.

Allyl Isothiocyanate Inhibits Actin-Dependent Intracellular Transport in Arabidopsis thaliana.

Unidirectional movement of cellulose synthase complexes in Arabidopsis seed coat epidermal cells deposit cellulose involved in mucilage extrusion, adherence, and ray formation.

Involvement of Phosphatidylinositol 3-kinase in the regulation of proline catabolism in Arabidopsis thaliana.

Distinct sensitivities to phosphate deprivation suggest that RGF peptides play disparate roles in Arabidopsis thaliana root development.

Distinct Roles of Meiosis-Specific Cohesin Complexes in Mammalian Spermatogenesis.

The Roles of β-Oxidation and Cofactor Homeostasis in Peroxisome Distribution and Function in Arabidopsis thaliana.

Arabidopsis cytosolic acyl-CoA-binding proteins ACBP4, ACBP5 and ACBP6 have overlapping but distinct roles in seed development.

Phosphatidylinositol phosphate 5-kinase genes respond to phosphate deficiency for root hair elongation in Arabidopsis thaliana.

The roles of autophagy in development and stress responses in Arabidopsis thaliana.

Trafficking of endoplasmic reticulum-retained recombinant proteins is unpredictable in Arabidopsis thaliana.

Different replication protein A complexes of Arabidopsis thaliana have different DNA-binding properties as a function of heterotrimer composition.

Corrigendum: Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders.

Characterization of Arabidopsis thaliana GCN2 kinase roles in seed germination and plant development.

Mechanisms and Physiological Roles of the CBL-CIPK Networking System in Arabidopsis thaliana.

AINTEGUMENTA-LIKE genes have partly overlapping functions with AINTEGUMENTA but make distinct contributions to Arabidopsis thaliana flower development.

Cellulose synthase complexes act in a concerted fashion to synthesize highly aggregated cellulose in secondary cell walls of plants.

Automatic Quantification of the Number of Intracellular Compartments in Arabidopsis thaliana Root Cells.

CELLULOSE SYNTHASE-LIKE A2, a glucomannan synthase, is involved in maintaining adherent mucilage structure in Arabidopsis seed.