Author response: The Arabidopsis SAC9 enzyme is enriched in a cortical population of early endosomes and restricts PI(4,5)P2 at the plasma membrane
2022-05-19

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Abstract Membrane lipids, and especially phosphoinositides, are differentially enriched within the eukaryotic endomembrane system. This generates a landmark code by modulating the properties of each membrane. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] specifically accumulates at the plasma membrane in yeast, animal, and plant cells, where it regulates a wide range of cellular processes including endocytic trafficking. However, the functional consequences of mispatterning PI(4,5)P2 in plants are unknown. Here, we functionally characterized the putative phosphoinositide phosphatase SUPPRESSOR OF ACTIN9 (SAC9) in Arabidopsis thaliana (Arabidopsis). We found that SAC9 depletion led to the ectopic localization of PI(4,5)P2 on cortical intracellular compartments, which depends on PI4P and PI(4,5)P2 production at the plasma membrane. SAC9 localizes to a subpopulation of trans-Golgi Network/early endosomes that are enriched in a region close to the cell cortex and that are coated with clathrin. Furthermore, it interacts and colocalizes with Src Homology 3 Domain Protein 2 (SH3P2), a protein involved in endocytic trafficking. In the absence of SAC9, SH3P2 localization is altered and the clathrin-mediated endocytosis rate is reduced. Together, our results highlight the importance of restricting PI(4,5)P2 at the plasma membrane and illustrate that one of the consequences of PI(4,5)P2 misspatterning in plants is to impact the endocytic trafficking. Editor's evaluation Phosphoinositide phosphates (PIPs) are lipids that can convey distinct identities to different cellular membranes via different phosphorylation patterns. Here, Lebecq, Doumane, and co-authors document the effects of the previously-characterized sac9 mutant, affecting a putative PIP-5-phosphatase in Arabidopsis, on PIP localization and endocytic trafficking. This work confirms that disrupting PI(4,5)P2 localization or abundance can affect endocytic trafficking in plants and will be of interest to the plant and cell biology research fields. https://doi.org/10.7554/eLife.73837.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Phosphoinositides constitute a family of low abundance lipids differentially enriched in the membranes of eukaryotic cells (Platre and Jaillais, 2016; Balla, 2013; Noack and Jaillais, 2020a). These versatile lipids can be interconverted into one another. For example, phosphatidylinositol 4,5-biphosphate [PI(4,5)P2] is synthetized from phosphatidylinositol 4-phosphate (PI4P) by PI4P-5 kinases and is dephosphorylated into PI4P by PI(4,5)P2 5-phosphatase (Noack and Jaillais, 2017). Furthermore, PI(4,5)P2 and PI4P are hydrolyzed by phospholipases C (PLC) into diacylglycerol and soluble phosphorylated inositol (Balla, 2013). PI(4,5)P2 strictly localizes at the plasma membrane in plants and animal cells (Van Leeuwen et al., 2007; Carim et al., 2019; Del Signore et al., 2017; Simon et al., 2014; Ben El Kadhi et al., 2011) despite the plasma membrane being constantly turned-over by endocytosis and exocytosis. Throughout this paper, we define endocytosis in its broader sense, including the internalization step sensus stricto (i.e. recruitment of cargo and coat components of the ‘clathrin-mediated endocytosis’ machinery, formation of clathrin-coated pits, scission of the clathrin-coated vesicles and uncoating) followed by the subsequent transport of lipid and proteins through the endosomal system. As such, the endocytosis, or here after the endocytic trafficking, is the process that allows (1) cells to transport particles and molecules across the plasma membrane and (2) the termination of signaling through transport toward the vacuole for degradation. At the plasma membrane, PI(4,5)P2 interacts with a variety of extrinsic membrane proteins such as endocytic protein adaptors (Zhang et al., 2015) and actin-regulatory proteins (Paez Valencia et al., 2016), which are recruited and/or activated by the binding to PI(4,5)P2. Therefore, PI(4,5)P2 subcellular patterning is likely critical to regulate the recruitment of proteins that act at the plasma membrane, and the cellular processes they mediate, including clathrin-mediated endocytosis. Consistent with a critical role of PI(4,5)P2 in the recruitment of early clathrin-mediated endocytosis factors, a pip5k1 pip5k2 double mutant in Arabidopsis thaliana (Arabidopsis) lacking two ubiquitously expressed PI4P-5 kinases, has abnormal auxin distribution and defective endocytic trafficking of the transmembrane auxin efflux carriers PIN-FORMED 1 (PIN1) and PIN2 (Tejos et al., 2014; Ischebeck et al., 2013; Mei et al., 2012). Furthermore, the pip5k1 pip5k2 double mutant has an altered dynamic of CLATRHIN LIGHT CHAIN2 (CLC2), with the density of CLC2 foci at the plasma membrane being reduced in the mutant (Ischebeck et al., 2013). Overexpression of Arabidopsis PI4P-5 kinase 6 in tip-growing pollen tubes induced massive aggregation of the plasma membrane in pollen tube tips due to excessive clathrin-dependent membrane invagination, supporting a role for PI(4,5)P2 in promoting early stages of clathrin-mediated endocytosis (Zhao et al., 2010). The inducible overexpression of a highly active human PI4P-5 kinase leads to an increased PI(4,5)P2 production, very strong developmental phenotypes, and heightened endocytic trafficking toward the vacuole (Gujas et al., 2017). In addition, we recently showed that inducible depletion of the PI(4,5)P2 from the plasma membrane using the iDePP system leads to a decrease in the fraction of the clathrin adaptor protein AP2-μ and the Src Homology (SH3)-domain containing protein 2 (SH3P2) at the plasma membrane (Doumane et al., 2021). Furthermore, FM4-64 uptake experiments confirmed an impact of PI(4,5)P2 depletion from the plasma membrane on bulk endocytic flow. In animal cells, several PI(4,5)P2 phosphatases are required for the late stages of clathrin-mediated endocytosis (He et al., 2017; Pirruccello et al., 2014). Many PI(4,5)P2 phosphatases belong to the 5-phosphatase enzyme family, including OCRL and synaptojanins (Syn1/2) in animals, and synaptojanin-like proteins (Inp51p/Snjl1p, Inp52p/Sjl2p, and Inp53p/Sjl3p) in Saccharomyces cerevisiae. The Arabidopsis genome contains 15 genes encoding 5-phosphatases, but only a few are characterized. Mutation in the 5-phosphatase nine leads to osmotic stress tolerance, with reduced reactive oxygen species production and Ca2+ influx (Golani et al., 2013). The 5-phosphatase 6/COTYLEDON VASCULAR PATTERN2 (CVP2) and 5-phosphatase 7/CVP2 LIKE 1 (CVL1) are specifically required for vascular differentiation (Rodriguez-Villalon et al., 2015; Carland and Nelson, 2009; Carland and Nelson, 2004). Finally, the 5-phosphatase 15/FRAGILE FIBER 3 (FRA3) is expressed in developing fibers and vascular cells, which is consistent with the defective fiber and vessel phenotypes seen in the loss-of-function fra3 mutant (Zhong et al., 2004). Proteins containing SUPPRESSOR OF ACTIN (or Sac1-like) domains constitute another family of phosphoinositide phosphatases (Zhong and Ye, 2003). In Arabidopsis, there are nine SAC proteins, forming three clades (Zhong and Ye, 2003). The first clade is composed of SAC1, a PI(3,5)P2 5-phosphatase (Zhong et al., 2005), and its relatives SAC2 to 5, putative PI(3,5)P2 5-phosphatases (Nováková et al., 2014). The second clade corresponds to SAC7/RHD4 a PI4P 4-phosphatase (Thole et al., 2008) and its relatives SAC6 and SAC8 putative PI4P 4-phosphatases (Song et al., 2021). The third clade is composed of a single member, a plant-specific protein called SAC9. SAC9 has a unique structure, with a SAC phosphoinositide phosphatase domain at its N-terminus, immediately followed by a putative protein/protein interaction domain (WW domain), and a long C-terminal region of 1104 amino acids where a putative coil-coiled domain is predicted (Figure 1A, Zhong and Ye, 2003). The sac9 mutant is dwarf, it constitutively accumulates anthocyanins and it expresses genes from stress response pathways (Williams et al., 2005). Loss-of-function alleles of SAC9 display a threefold increase in PI(4,5)P2 content, together with a decrease in PI4P level, suggesting that it acts as a PI(4,5)P2 5-phosphatase in planta (Williams et al., 2005). Figure 1 with 1 supplement see all Download asset Open asset Structure-function analysis of SUPPRESSOR OF ACTIN9 (SAC9). (A) Schematic representation of SAC9 protein. The SAC catalytic domain, as well as the WW domain and the coil-coiled domain, are represented. (B) Representative images of the macroscopic phenotype observed in (i) wild-type (Col-0), (ii) sac9-1-/- and sac9-3-/- loss of function mutants, (iii) sac9-3-/- complemented lines expressing full-length genomic DNA encoding SAC9 fused to yellow (mCIT-SAC9, line #1000-9-1) or red (TdTOM-SAC9, line #987-5-4) fluorescent proteins and a mutated version of the putative catalytic cysteine residue within the C-x(5)-R-[TS] catalytic motif in the SAC domain (mCIT-SAC9C459A, line #1354-12-14; right panel). Pictures are taken 12 days post germination (dpg). Note that a second independent transgenic line is presented for each construct in Figure 1—figure supplement 1. (C) Quantification of primary root length in sac9-3-/- homozygous mutants expressing mCIT-SAC9 and mCIT-SAC9C459A under the control of the native promoter (SAC9prom) and TdTOM-SAC9 under the expression of the Ubq10 promoter. Wild-type (Col-0) seedlings and two independent mutant alleles of SAC9, sac9-1-/-, and sac9-3-/-, are used as controls. (D) Same as (C) but for the quantification of the lateral root density (ratio of the number of lateral roots to primary root length). In the plots, middle horizontal bars represent the median, while the bottom and top of each box represent the 25th and 75th percentiles, respectively. At most, the whiskers extend to 1.5 times the interquartile range, excluding data beyond. For range of value under 1,5 IQR, whiskers represent the range of maximum and minimum values. Details for statistical analysis can be found in the Methods section and Supplementary file 1C. N=number of replicates; n=number of roots. Using in vivo confocal microscopy, we found that SAC9 localizes in a subpopulation of trans-Golgi network/early endosomes (TGN/EEs) enriched in a region close to the cell cortex. Loss of SAC9 results into PI(4,5)P2 mis-patterning at the subcellular level, leading to the accumulation of PI(4,5)P2 in subcortical compartments associated with TGN/EEs. Similar cellular and developmental phenotypes were observed when SAC9 was mutated on its putative catalytic cysteine, suggesting that the phosphoinositide phosphatase activity of SAC9 is required for function. SAC9 interacts and colocalizes with the endocytic component SH3P2. In the absence of SAC9, and therefore when the patterning of the PI(4,5)P2 is compromised, SH3P2 localization is affected and the clathrin-mediated endocytosis is significantly reduced. Thus, SAC9 is required to maintain efficient endocytic uptake, highlighting the importance of restricting the PI(4,5)P2 pool to the plasma membrane. Results The cysteine 459 in the catalytic domain of SAC9 is required for SAC9 function We investigated the root phenotype in the already described mutant alleles of SAC9 (Vollmer et al., 2011; Williams et al., 2005). As previously described, sac9-1-/- and sac9-3-/- knock-out mutants are two times shorter compared to the wild-type (WT) Col-0, at 12 days post-germination (dpg; Figure 1A-C). We also observed a three-time decrease in the lateral root density of sac9-1-/- and sac9-3-/- compared to WT plants (Figure 1B-D). To confirm that the phenotypes observed were due to the loss-of-function of SAC9, we generated Arabidopsis lines expressing SAC9 fused to a fluorescent protein (mCIT-SAC9 and TdTOM-SAC9) under SAC9 native promoter (SAC9pro) or the UBIQUITIN10 promoter (UBQ10pro), respectively (Figure 1, Figure 1—figure supplement 1A and B). We found that both SAC9pro:mCIT-SAC9 and UBQ10pro:TdTOM-SAC9 rescued sac9-3-/- mutant phenotypes in two independent homozygous T3 transgenic lines for each construct (Figure 1B and C; Figure 1—figure supplement 1A). These results indicate that the root phenotypes described above are caused by SAC9 loss-of-function, and that N-terminally tagged SAC9 proteins are functional. Next, we mutated the cysteine in the conserved C-x(5)-R-[T/S] catalytic motif found in all SAC domain-containing phosphoinositide phosphatase (Hsu et al., 2015; Hsu and Mao, 2013; Figure 1A and Figure 1—figure supplement 1A). Such cysteine-to-alanine substitution was shown to block the catalytic phosphatase activity of other SAC domain-containing proteins (Manford et al., 2010; Tani and Kuge, 2014), and thus mCIT-SAC9C459A is a putative catalytically dead version of the enzyme. In contrast to wild-type mCIT-SAC9, we could not find any transgenic lines expressing SAC9pro:mCIT-SAC9C459A that were able to rescue the sac9-3 phenotype, out of 24 independent lines analyzed in T1 (Figure 1B, C, Figure 1—figure supplement 1A, B). Further analyses on two independent T3 homozygous lines confirmed these initial results and showed that mCIT-SAC9C459A fusions were stable and accumulated to similar extent as wild-type mCIT-SAC9 (Figure 1B and C, Figure 1—figure supplement 1A, B). Thus, the putative catalytic cysteine, C459, is required for SAC9 function but not for the stability of the protein, suggesting that the phosphatase activity of SAC9 is participating in the observed phenotypes. SAC9 localizes to a population of early endosomes close to the plasma membrane Some phosphoinositide-phosphatases such as Metazoan Sac1 and yeast Sac1p are able to dephosphorylate in vitro several phosphoinositide species, but display a narrower specificity in vivo (Hughes et al., 2000; Guo et al., 1999; Rivas et al., 1999). Also, some enzymes involved in phosphoinositide metabolism, such as the yeast PI 4-kinases Stt4p and Pik1p, specifically impact distinct pools of a given phosphoinositide species depending on their subcellular localization (Roy and Levine, 2004; Yoshida et al., 1994; Flanagan et al., 1993). Using homozygous T3 transgenic lines expressing the functional SAC9pro:mCIT-SAC9 or Ub10pro:TdTOM-SAC9 constructs, we assessed the subcellular distribution of SAC9 in rescued sac9-3-/- homozygous plants using live-cell fluorescence imaging. In meristematic epidermal cells of Arabidopsis roots, mCIT-SAC9 and TdTOM-SAC9 were mainly diffused in the cytosol and excluded from the nucleus (Figure 2A-C, Figure 2—figure supplement 1A). At the cortex of the cell, in close vicinity to the plasma membrane (Zi focal plane, Figure 2A; Figure 2—figure supplement 1B), mCIT-SAC9 localized to a cortical population of mobile dotty structures (Figure 2B, Video 1). To assess whether this discrete subcellular localization was relevant for SAC9 function, we generated transgenic lines expressing a mCIT-SAC9 variant in which the predicted coil-coiled motif was deleted, under the native promoter (Figure 1A, SAC9pro:mCIT-SAC9∆CC). Analyses of 24 independent T1 revealed that mCIT-SAC9∆CC did not complement sac9-3-/- dwarf phenotype (Figure 1—figure supplement 1A). Similar to mCIT-SAC9C459A, these results were confirmed on two independents homozygous T3 lines and western blot analyses showed that mCIT-SAC9∆CC accumulated to similar extent as the wild-type mCit-SAC9 in each of these lines (Figure 1—figure supplement 1B). However, mCIT-SAC9∆CC did not localize to endosome and remained entirely cytosolic (Figure 2B, Figure 2—figure supplement 1C). Thus, even though SAC9 localization in cortical intracellular compartments is discreet, and only slightly enriched compared to its cytosolic localization, it appears critical for function. Furthermore, this analysis excludes a scenario in which the mCit-SAC9-containing compartments are cytosolic densities, because they are not seen with a cytosolic mutant of SAC9. Figure 2 with 1 supplement see all Download asset Open asset SUPPRESSOR OF ACTIN9 (SAC9) localizes in the cytosol and in intracellular compartment enriched at the cell cortex. (A) Schematic representation of two root epidermal cells (cell #1 indicated as C1 and cell #2 indicated as C2) imaged at two different focal planes (cortical plane, close to the plasma membrane and designated as Zi and a median plane, in the middle of the cell designated as Zii). Note that the C1/C2 and Zi/Zii notations are used consistently according throughout the figures. (B) Confocal images of the subcortical part (i.e. Zi) of the Arabidopsis root epidermis expressing mCIT-SAC9, mCIT-SAC9∆CC mutated in the predicted coil-coiled domain, and mCIT-SAC9C459A mutated in its putative catalytic cysteine, under the control of SAC9 native promoter (SAC9prom). (C) Representative images of the fluorescent signal observe in sac9-3-/- mutant expressing mCIT-SAC9, TdTOM-SAC9, and mCIT-SAC9C459A. (D) Comparison of the number of labeled intracellular compartments per cell in sac9-3-/- root epidermis expressing mCIT-SAC9 and TdTOM-SAC9, mCIT-SAC9C459A. (E) Colocalization analysis on Ub10pro:TdTOM-SAC9 (magenta) and SAC9pro:CIT-SAC9C459A (green). (F) Confocal images of the subcortical part of the Arabidopsis root epidermis (upper panels, Zi) or the center of the cell (lower panels, Zii) expressing mCIT-SAC9 together with the PI(4,5)P2 biosensor 2mCH-2xPHPLC (note that the cells shown in Zi and Zii are the same, just on different focal plane). (G), Quantification of the number of endosomes labeled by mCIT-SAC9 observed per cell at two different focal planes (Zi and Zii). (H), Quantification of the number of endosomes labeled by FM4-64 observed per cell at two different focal planes (Zi and Zii). (I), Co-visualization of SAC9pro:mCIT-SAC9C459A (green) together with the plasma membrane marker Lti6b-2xmCH (magenta) in epidermal root cells taken in the Zi focal plane. The inset shows a magnification, with the yellow arrows indicating some of the intracellular compartments decorated by mCIT-SAC9C459A observed at the close vicinity to the labeled plasma membrane in cell C1. Scale bar: 10 μm in A-F; 5 μm in G (left panel) and 2 μm in the close-up of G (right panel). In the plot, middle horizontal bars represent the median, while the bottom and top of each box represent the 25th and 75th percentiles, respectively. At most, the whiskers extend to 1.5 times the interquartile range, excluding data beyond. For range of value under 1,5 IQR, whiskers represent the range of maximum and minimum values. Details of the statistical analysis could be found in Supplementary file 1D. The plane (Zi or Zii) in each image is mentioned, and the image display is representative for the plane used for the analysis. N=number of replicates; n=number of roots. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Time-lapse imaging of mCIT-SAC9 using spinning disk confocal microscope (1 s per frame). The putative catalytically dead version, mCIT-SAC9C459A, accumulated to a lesser extent in the cytosol compared to the native form. In addition, SAC9C459A showed a threefold increase in the number of labeled dotty structures compared with the signal collected for the wild-type fusion proteins mCIT-SAC9 and TdTOM-SAC9 (Figure 2C, D). This result suggests that the putative catalytic cysteine of SAC9 is required for the dynamic interaction of SAC9 with intracellular membranes. However, co-visualization of TdTOM-SAC9 and mCIT-SAC9C459A (Figure 2E) in Col-0 background demonstrated that all TdTOM-SAC9 dotty structures colocalized with mCIT-SAC9C459A at the cortex of the cell. By comparing the signal observed at the cortex of the cell (Zi) with the signal collected at the center of the cell (Zii), we observed and quantified that mCIT-SAC9 labeled more intracellular compartments close to the plasma membrane than at a distal position, while this bias was less pronounced for FM4-64 labeled compartments (Figure 2F-H). Similarly, when imaging cells in their cortical part close to the plasma membrane (Zi), the mCIT-SAC9 and mCIT-SAC9C459A signal was concentrated in intracellular compartments at the close vicinity with the plasma membrane (Figure 2F and I). These results suggest a function for SAC9 in regulating phosphoinositide homeostasis either at or in the close vicinity of the plasma membrane. We next investigated the nature of the intracellular structures labeled by mCIT-SAC9. Both mCIT-SAC9 and mCIT-SAC9C459A colocalized with the amphiphilic endosomal compartments at the just the plasma membrane, while the soluble mCIT-SAC9∆CC did not (Figure and Figure 2—figure supplement 1B, C, Figure supplement 1A). are to while late endosomes are not and We observed that led to the aggregation of mCIT-SAC9 and mCIT-SAC9C459A into (Figure suggesting that both functional and SAC9 fusion proteins may localize to Figure 3 with 2 see all Download asset Open asset SUPPRESSOR OF ACTIN9 (SAC9) localizes to a subpopulation of Confocal images of SAC9pro:mCIT-SAC9 or SAC9C459A in vivo in Arabidopsis root epidermis together with the endocytic FM4-64 Confocal images of Arabidopsis root epidermis SAC9pro:mCIT-SAC9 or SAC9C459A together 2 fused to fluorescent observed in the fluorescent observed after (E) Confocal images of Arabidopsis root epidermis SAC9pro:mCIT-SAC9 (green) and the late compartment marker and the marker (F) of mCIT-SAC9 and a given endosomal compartment marker per cell, at N=number of roots, n=number of In the plots, middle horizontal bars represent the median, while the bottom and top of each box represent the 25th and 75th percentiles, respectively. At most, the whiskers extend to 1.5 times the interquartile range, excluding data beyond. For range of value under 1,5 IQR, whiskers represent the range of maximum and minimum values. The plane (Zi or Zii) in each image is mentioned, and the image display is representative for the plane used for the analysis. Scale 10 To more into the localization at the we Arabidopsis lines expressing fluorescent tagged SAC9 and SAC9C459A proteins with et al., Using live-cell imaging in root meristematic cells at a Zi focal plane, we observed and quantified that mCIT-SAC9 colocalized with Figure and The mCIT-SAC9 showed very with a late endosome marker Figures and and with a marker Figure and Similarly, mCIT-SAC9C459A colocalized with and it did not colocalized with the marker (Figure supplement 2C, Figure This confirmed that SAC9 and SAC9C459A fusion proteins localized to the with the that SAC9 localization in endomembrane compartments was to the cortex of the cell, suggesting a role of SAC9 in PI(4,5)P2 homeostasis the plasma membrane and early endosomal SAC9 is required to maintain the pool of PI(4,5)P2 at the plasma membrane was previously that Arabidopsis loss-of-function mutant a of PI4P and a threefold accumulation of suggesting that SAC9 has a PI(4,5)P2 5-phosphatase catalytic activity (Williams et al., 2005). In to the of the of the phosphoinositide in the absence of SAC9, we two independent PI(4,5)P2 et al., into sac9-3-/- As previously described, both PI(4,5)P2 labeled the plasma membrane and were excluded from intracellular compartments in wild-type cells (Figure and B). In sac9-3-/-, and not only labeled the plasma membrane, but also decorated cortical intracellular dotty structures (Figure and B). In to the absence of observed for sac9-3 mutants expressing the putative SAC9C459A variant (Figure 1B and C, Figure 1—figure supplement the localization for the PI(4,5)P2 biosensor was observed in these lines (Figure These results that is required for the of the pool of PI(4,5)P2 and out toward a role of SAC9 for the of the PI(4,5)P2 at the plasma membrane via its catalytic phosphatase We also observed an increase in the number of intracellular compartments labeled by the PI4P et al., 2014; Simon et al., and but not for (Figure and B). This result is consistent with a of the PI4P pool at the plasma membrane and therefore the of the PI4P biosensor in intracellular compartments, as previously et al., The subcellular localization of and was Col-0 and cells (Figure Figure supplement We a but decrease in the density of intracellular compartments decorated by biosensor in sac9-3-/- (Figure Figure supplement 1). these results indicate that leads to a massive in PI(4,5)P2 subcellular which is on intracellular cortical structures of only being at the plasma membrane. Figure with 2 see all Download asset Open asset SUPPRESSOR OF ACTIN9 (SAC9) PI(4,5)P2 at the plasma membrane. (A) Confocal images of Arabidopsis root epidermis expressing in WT (Col-0) and sac9-3-/- is (B) Quantification of density of labeled intracellular compartments in roots epidermis expressing lipid in wild-type (Col-0) and with In the plots, middle horizontal bars represent the median, while the bottom and top of each box represent the 25th and 75th percentiles, respectively. At most, the whiskers extend to 1.5 times the interquartile range, excluding data beyond. For range of value under 1,5 IQR, whiskers represent the range of maximum and minimum values. Details for statistical analysis can be found in the Methods section and Supplementary file 1. N=number of replicates; n=number of roots. (C) Confocal images of in root epidermal cell of sac9-3 sac9-3-/- (left panel) or sac9-3-/- expressing (right roots. (D) Confocal images of sac9-3-/- Arabidopsis root epidermal cell and C2) expressing (green) and (magenta) at their cortex (upper Zi) and at their center roots. Scale bar: 10 The plane (Zi or Zii) in each image is mentioned, and the image display is representative for the plane used for the analysis. Note that in the cells are shown in the and but at different focal using microscopy, a massive accumulation of containing cell at the close vicinity to the plasma membrane in the sac9 mutant (Vollmer et al., together with in sac9-3-/- (Figure the dotty structures decorated by the PI(4,5)P2 biosensor were observed at the cortex of the cell (Zi), in the close vicinity of the plasma membrane (Figure structures were observed in the part of the cell (Figure panel). dotty structures containing PI(4,5)P2 were associated with compartments but they did not strictly Confocal imaging of sac9-3-/- mutant with endosomal revealed that the dotty structures containing PI(4,5)P2 were found associated but strictly with the FM4-64 endocytic as well as (Figure supplement 1C). Furthermore, induced in sac9-3-/- but did not affect the distribution of compartments in the cells (Figure supplement Therefore, intracellular compartments in sac9-3-/- are of any activated by and are associated with (Figure supplement In vivo imaging of PI(4,5)P2 biosensor and in sac9-3-/- mutant