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Masaru Fujimoto1,6, Yasuyuki Suda4,7, Samantha Vernhettes2,3, Akihiko Nakano1,4 and Takashi Ueda1,5,* 1 Laboratory of Developmental Cell Biology, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan 2 INRA, UMR1318, Institut Jean-Pierre Bourgin, Saclay Plant Sciences, F-78000 Versailles, France 3 AgroParisTech, Institut Jean-Pierre Bourgin, RD10, F-78000 Versailles, France 4 RIKEN Center for Advanced Photonics, Live Cell Molecular Imaging Research Team, Extreme Photonics Research Group, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan 5 Japan Science and Technology Agency (JST), PRESTO, 4-1-8 Honcho Kawaguchi, Saitama, 332-0012 Japan 6 Present address: Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyoku, Tokyo, 113-8657 Japan. 7 Present address: Department of Molecular Cell Biology, Graduate School of Comprehensive Human Sciences and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan. *Corresponding author: E-mail, [email protected]; Fax, +81-3-5841-7613. (Received September 14, 2014; Accepted December 1, 2014) Keywords: Cellulose synthase CESA Endocytosis Membrane trafficking Phosphoinositides. Abbreviations: CESA, cellulose synthase; CGA, CGA 3250 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; mRFP, monomeric red fluorescent protein; MS, Murashige and Skoog; PAO, phenylarsine oxide; PI, phosphoinositide; PI3K, phosphatidylinositol 3-kinase; PI4K, phosphatidylinositol 4-kinase; PM, plasma membrane; PtdIns, phosphatidylinositol; PVC, pre-vacuolar compartment; SCLIM, super-resolution 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. 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 (Green 1962, Baskin 2005). 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 family, which is classified into two subgroups (Richmond and Somerville 2000). CESA1, CESA3 and a member of the CESA6like clade are responsible for primary cell wall synthesis (Persson et al. 2005, Desprez et al. 2007, Persson et al. 2007, Wang et al. 2008), and three other isoforms, CESA4, CESA7 and CESA8, are Plant Cell Physiol. 56(2): 287–298 (2015) doi:10.1093/pcp/pcu195, Advance Access publication on 15 December 2014, available FREE online at www.pcp.oxfordjournals.org ! The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected] Editor-in-Chief’s Choice 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 (PM), which comprise the cellulose synthases (CESAs). The abundance and localization of CSCs at the PM should be strictly controlled for precise regulation of cellulose deposition, which strongly depends on the membrane trafficking system. However, the mechanism of the intracellular 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 proteintagged 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. Special Focus Issue – Regular Paper Phosphatidylinositol 3-Kinase and 4-Kinase Have Distinct Roles in Intracellular Trafficking of Cellulose Synthase Complexes in Arabidopsis thaliana M. Fujimoto et al. | Phosphoinositide requirements in CESA trafficking implicated in secondary cell wall synthesis (Taylor et al. 1999, Taylor et al. 2003, Brown et al. 2005). Cellulose synthesis by CSCs is assumed to occur solely at the plasma membrane (PM) (Reiter 2002, Scheible and Pauly 2004, Cosgrove 2005, Oikawa et al. 2013). 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 CESAcontaining compartments (SmaCCs) (Haigler and Brown 1986, Paredez et al. 2006, Desprez et al. 2007, Crowell et al. 2009, Gutierrez et al. 2009, Gu et al. 2010, Li et al. 2012). 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 m2 subunit of the adaptor protein complex 2 (AP2), which is involved in clathrin-mediated endocytosis (CME) (Bashline et al. 2013, Di Rubbo et al. 2013, Kim et al. 2013, Yamaoka et al. 2013, Gadeyne et al. 2014), 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). Green fluorescent protein (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-b-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 2014, Vain et al. 2014). However, the molecular mechanisms mediating CSC trafficking remain largely unknown. Although membrane lipids are known to play pivotal roles in membrane trafficking (Sprong et al. 2001, Hanzal-Bayer and Hancock 2007), possible roles of lipids in CSC trafficking have 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 trisphosphates (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, pre-vacuolar compartments (PVCs) and vacuoles; PtdIns4P in the Golgi apparatus, TGN and PM; and PtdIns(4,5)P2 is abundant in the PM (Vermeer et al. 288 2006, van Leeuwen et al. 2007, Vermeer et al. 2009, Simon et al. 2014). 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 (Thole and Nielsen 2008, Krishnamoorthy et al. 2014). 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, PI4Kb1 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 (Preuss et al. 2006, Kang et al. 2011). PI4Kb1 is also reported to be involved in the late step of CME at the tip of the pollen tube (Zhao et al. 2010). PIP5K3 and PIP5K4/ 5, members of PtdIns4P 5-kinases (PIP5Ks) that mediate the synthesis of PtdIns(4,5)P2 from PtdIns4P, have also been shown to co-ordinate 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 (Mei et al. 2012, Ischebeck et al. 2014) and growing pollen tubes (Zhao et al. 2010). Moreover, A. thaliana FAB1A and FAB1B, close homologs of the yeast PtdIns3P 5-kinase (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 (Wm; a PI3K and PI4K inhibitor) (Jung et al. 2002, Aggarwal et al. 2013), LY294002 (a PI3K inhibitor) (Jung et al. 2002, Lee et al. 2008, Aggarwal et al. 2013) 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. 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 sucrosefree medium. Using confocal laser scanning microscopy (CLSM), we observed temporal changes of CESA3 localization exposed to sucrose-free conditions in root epidermal cells of Plant Cell Physiol. 56(2): 287–298 (2015) doi:10.1093/pcp/pcu195 A. thaliana. Immediately after the transfer from a normal sucrose medium (30 mM) to a sucrose-free medium (0 mM), 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; Fig. 1B, arrowheads). Conversely, under the continuous normal sucrose condition, the rapid increase of GFP–CESA3 in intracellular compartments was not observed (Supplementary Fig. S1). Pearson’s correlation coefficients between fluorescence from GFP–CESA3 and CLC–mOrange, which were calculated from six 100 mm2 CLSM images for each time point, 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 affect CESA trafficking from the PM differently PIs are known as regulatory components of membrane trafficking pathways including endocytic trafficking. To examine the involvement of PIs in CESA trafficking, we first analyzed the effect of 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 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 mm. (B) Magnified images of the region surrounded by the white lines in (A). Arrowheads indicate ring-shaped localization of GFP–CESA3. Scale bar = 5 mm. (C) Average of Pearson’s correlation coefficients between fluorescence from GFP–CESA3 and CLC–mOrange calculated from six 100 mm2 CLSM images taken with samples prepared in the same way as samples presented in (A) and Supplementary Fig. S1. The results are indicated as the means ± SD. Different letters indicate statistically significant differences (Tukey’s honestly significant difference; a = 0.05, n = 6). 289 M. Fujimoto et al. | Phosphoinositide requirements in CESA trafficking compartments, and PM localization was also evident (Fig. 2). In contrast, treatment with 80 mM Wm severely inhibited the internalization of GFP–CESA3 into the cytoplasm. Intriguingly, lower concentrations of Wm (4 and 20 mM) resulted in the removal of GFP–CESA3 from the PM and its accumulation in dot-shaped 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 (Jung et al. 2002, Vermeer et al. 2006, Vermeer et al. 2009, Aggarwal et al. 2013), 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 mM PAO for 60 min, most of the GFP–CESA3 signals were retained on the PM, similarly to the 80 mM Wm treatment (Fig. 2). Conversely, after the 60 min 40 mM LY294002 treatment, the GFP–CESA3 signals entirely disappeared from the PM and accumulated at punctate intracellular compartments, similarly to the 4 and 20 mM Wm treatment. Weaker but similar effects were observed when cells were treated with 10 mM PAO or LY294002, respectively, whereas 4 mM 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. Fig. 2 Effect of PI4K and PI3K inhibitors on the localization of GFP–CESA3 triggered by hypotonic stimulus. Arabidopsis thaliana root epidermal cells expressing GFP–CESA3 (green) treated with 0.1% DMSO or the indicated concentrations of chemicals in sucrose-free medium for 60 min were observed using CLSM. Scale bars = 10 mm. 290 Plant Cell Physiol. 56(2): 287–298 (2015) doi:10.1093/pcp/pcu195 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 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 of root epidermal cells treated with PAO for 15 min, GFP–CESA3 foci at the PM were barely 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 (Fig. 3E, F). A similar effect on CLC–mOrange was also observed for a 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 mM 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 ringshaped 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 quantitative analysis also demonstrated that the co-localization 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 illumination microscopy (SIM) (Langhorst et al. 2009). We observed GFP–CESA3labeled 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 monomeric red fluorescent protein (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. 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. Inhibited PI3K reduced the size of the Golgi apparatus 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 (Figs. 5A, B, 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 postGolgi organelles visualized using RFP-tagged organelle makers in ten to fifteen 100 mm2 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 with 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. 291 M. Fujimoto et al. | Phosphoinositide requirements in CESA trafficking 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 mM 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 mM PAO (C). Scale bars = 10 mm (A, C) or 5 mm (B, D). (E) Average of Pearson’s correlation coefficients between fluorescence from GFP–CESA3 and CLC–mOrange calculated from six 100 mm2 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; a = 0.05, n = 6). (F) Average number of CLC–mOrange foci calculated from 121 mm2 VIAFM images of A. thaliana root epidermal cells treated with 0.1% DMSO or 40 mM PAO (n = 6). The results are indicated as the means ± SD. ***P < 0.001, Student’s t-test. 292 Plant Cell Physiol. 56(2): 287–298 (2015) doi:10.1093/pcp/pcu195 Fig. 4 Effects of LY294002 on the hypotonic stimulus-induced re-localization of GFP–CESA3. (A) Arabidopsis 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 mM LY294002 (lower panels). Scale bars = 10 mm. (B) Average of Pearson’s correlation coefficients between fluorescence from GFP–CESA3 and CLC–mOrange calculated from six 100 mm2 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; a = 0.05, n = 6). PI4K is also required for CGA-induced relocation of CESA3 from the PM Treatment with a cellulose synthesis inhibitor, CGA 3250 615 (CGA), has been reported to stimulate the relocation of CESA from the PM to intracellular compartments, the MASCs/ SmaCCs and the Golgi 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 pre-treatment with PAO for 15 min inhibited the relocation of GFP–CESA3 to the cytoplasm (Fig. 7, right panels). These results indicated that the re-localization of CESA3 induced by the cellulose synthesis inhibitor also requires PI4K. Fig. 5 Fine structures of cytoplasmic compartments with GFP– CESA3 observed by super-resolution microscopy. (A, B) Arabidosis 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 mM LY294002 (B). Arrowheads indicate ringshaped structures. Scale bars = 3 mm. 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 293 M. Fujimoto et al. | Phosphoinositide requirements in CESA trafficking Fig. 6 Accumulation of GFP–CESA3 at the Golgi apparatus after treatment with LY294002. (A) Arabidopsis thaliana root epidermal cells expressing GFP–CESA3 (green) and mRFP-tagged post-Golgi organelle markers (red) were observed after 60 min incubation in sucrose-free medium in the presence of 0.1% DMSO (upper panels) or 40 mM LY294002 (lower panels) using CLSM. Scale bars = 10 mm. (B) Average of Pearson’s correlation coefficients between fluorescence from GFP–CESA3 and mRFP-tagged organelle markers calculated from four 100 mm2 CLSM images taken with samples prepared in the same way as the samples presented in (A). Different letters indicate statistically significant differences (Tukey’s honestly significant difference; a = 0.05, n = 4). The results are indicated as the means ± SE. (C) Average planar areas of organelles in root epidermal cells incubated with 0.1% DMSO (blue bar) or 40 mM LY294002 (red bar) for 60 min and calculated from 100 mm2 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 3250 615 (CGA)-induced accumulation of GFP–CESA3 in intracellular compartments. Arabidopsis 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 mM PAO (right panels) with CGA for 60 min after pre-treatment with DMSO or PAO for 15 min were observed using CLSM. Scale bars = 10 mm. 294 hypotonic treatment in A. thaliana root epidermal cells. Although the physiological significance of re-localization of CESA upon hypotonic treatment remains unknown, hypertonic treatment is also shown to promote endocytosis of CESA proteins (Gutierrez et al. 2009, Crowell et al. 2010), which is associated with modification of the cell wall. The response of the cell wall to the hypotonic condition should also be examined in 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 (Figs. 3A, C, 7). Considered together with a recent report on the involvement of the adaptor complex 2 (AP2) in CESA endocytosis (Bashline 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, Zhao et al. 2010, Ischebeck Plant Cell Physiol. 56(2): 287–298 (2015) doi:10.1093/pcp/pcu195 a lower concentration of Wm should mainly inhibit PI3K and a 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 the 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 PM. Different concentrations of Wm, in combination with other inhibitors such as LY294002 and PAO, would be convenient tools to analyze the requirements for PI3K and PI4K in plant functions. 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 the secretory and/ or recycling pathway from the Golgi apparatus. CSC, cellulose synthase complex comprising CESA; MASC/SmaCC, microtubule-associated cellulose synthase compartments/small CESA-containing compartments. et al. 2013), 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). In contrast to the effect of PAO, the treatment with LY294002 did not perturb the 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 (Hall et al. 2006, Low et al. 2010, Dominguez et al. 2011). 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 (Robatzek et al. 2006, Beck et al. 2012, Ito et al. 2012a, Di Rubbo et al. 2013). In this study, we demonstrated that a lower concentration of Wm had a different effect from a 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 (Matsuoka et al. 1995, Jung et al. 2002, Vermeer et al. 2006, Vermeer et al. 2009, Aggarwal et al. 2013), 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 the morphology of the Golgi and TGN in addition to its known effect on multivesicular endosomes. In particular, a significant reduction in the size of the Golgi apparatus was evident (Fig. 6A, C). 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 (Vermeer et al. 2006, Simon et al. 2014), 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 (Burda et al. 2002, Arighi et al. 2004, 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 localized at the TGN as well as in the endosomes/PVC (Oliviusson et al. 2006, Niemes et al. 2010). 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 proteins (Robatzek et al. 2006, Zhao et al. 2010, Beck et al. 2012, Di Rubbo et al. 2013) and lipids labeled with FM dye from the PM (Van Damme et al. 2011, Bandmann et al. 2012), 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 295 M. Fujimoto et al. | Phosphoinositide requirements in CESA trafficking cells, which should also be effective to elucidate a possible coordination between cell wall synthesis and PI metabolism. Statistical analysis The results are expressed as the means ± SD or ± SE 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. Materials and Methods Plant materials and growth conditions Arabidopsis thaliana plants (accession Columbia-0) were grown on Murashige and Skoog (MS) medium plates at 23 C under continuous light. The transgenic plant expressing GFP–CESA3 in the je5cesa3 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 the markers; F1 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. Supplementary data Supplementary data are available at PCP online. Funding This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan [Grants-in-Aid for Scientific Research (to A.N. and T. U.)]; Japan Science and Tecnology Agency (JST) [PRESTO grant (to T.U.)]; Japan Society for the Promotion of Science (JSPS) [Grant-in-Aid for JSPS Fellows (to M.F.)]. Hypotonic and drug treatments Ten-day-old A. thaliana seedlings cultured on MS plates containing 30 mM sucrose and 0.3% phytagel were transferred to liquid MS medium containing 0 mM or 30 mM sucrose and incubated at 23 C before microscopic observation. DMSO (0.1%, v/v), Wm (4 or 80 mM), PAO (4 or 40 mM), LY294002 (4 or 40 mM) 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 mM 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 0.12–0.17 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 (NA) objective, the Nikon TIRF2 system for VIAFM with a CFI Apo TIRF 100 1.49 NA objective, and the N-SIM system for SIM with a CFI Apo TIRF 100 1.49 NA objective. For SCLIM, we used a custom-made system that we developed, which consists of an IX-70 microscope equipped with UPlanSApo 100 1.40 NA 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) (Matsuura-Tokita et al. 2006, Nakano and Luini 2010, Ito et al. 2012b, Okamoto et al. 2012, Kurokawa et al. 2013, Nakano 2013, Suda et al. 2013, Kurokawa et al. 2014). GFP and mOrange or mRFP were excited with 488 and 561 nm lasers, respectively. For 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 excited with different illumination patterns. Each frame in VIAFM and SIM was acquired by 200 ms and 1.8 s 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 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 40 images taken at 0.1 mm vertical intervals using a theoretical point-spread function optimized for CSU10 (Yokogawa Electric). The acquired images were processed with Photoshop CS6 Extended (Adobe Systems). 296 Acknowledgments We thank K. Kurokawa and M. Ishii (RIKEN) for kind support in the SCLIM experiment. We also thank K. Schumacher (University of Heidelberg) and K. Shoda (RIKEN, Japan) for sharing materials. Disclosures The authors have no conflicts of interest to declare. References Aggarwal, C., Labuz, J. and Gabrys, H. 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