Supplementary MaterialsSupplementary Data. did not locate to membranes but was present in the cytosol and nucleus. Treatment with short-chain DAG or PMA (phorbol-12-myristate-13-acetate), a phorbol ester that binds the C1a domain name of PKC, caused the recruitment of the biosensor to the plasma membrane. These results indicate that this biosensor works and that the basal DAG concentration in the cytoplasmic leaflet of membranes (i.e. accessible to the biosensor) is usually in general too low, and confirms that this known pools in plastids, the endoplasmic reticulum and mitochondria are located at the luminal face of these compartments (i.e. inaccessible to the biosensor). Nevertheless, detailed further analysis of different cells and tissues discovered four novel DAG pools, namely at: (i) the and six genes. PLCs Vorapaxar ic50 and NPCs have been implicated in diverse functions (Gaude et al. 2008, Nakamura et al. 2009, Dowd and Gilroy 2010, Peters et al. 2010, Munnik 2014, Nakamura 2014, Peters et al. 2014, Pokotylo et al. 2014, Hou et al. 2016). Analysis of the different DAG pools in plants has been challenging. DAG is not a bilayer-forming lipid, so its levels are kept relatively low, which in Arabidopsis is usually approximately 1% of the polar lipids (Kaup et al. 2002, Gaude et al. 2007). Membrane isolation and fractionation procedures have recognized unique DAG pools at chloroplasts, the ER and mitochondria, i.e. all sites where lipid metabolism takes place (Dong et al. 2012, Muthan et al. 2013). A disadvantage of such analyses is that the procedures are relatively long, so DAG levels and pools can easily change due to modifying enzymes or transporters present in the various membrane fractions (Muthan et al. 2013). To map DAG pools in plastids, Bennings lab recently generated transgenic Arabidopsis lines expressing a DAG kinase (DGK) from and plants. DAG binding was validated using a short-chain analog and PMA (phorbol-12-myristate-13-acetate), a phorbol ester that mimics DAG binding to the C1a domain name and in TIAM1 animal cells activates PKC (Oancea et al. 1998). We found that the biosensor was mostly localized in the cytosol, indicating that the concentration of DAG in the cytoplasmic leaflet of membranes is normally too low to be detected by YFPCC1aPKC. Detailed further analysis, however, revealed four novel DAG pools: one at the cytoplasmic leaflet of Vorapaxar ic50 Golgi membranes and three very local and temporal pools at the plasma membrane, i.e. in root epidermal cells of the transition zone, in dividing cells at the growing cell plate and during polarized tip growth in root hairs. Vorapaxar ic50 The results provide new insights into the spatiotemporal dynamics of herb DAG and offers a new tool to monitor this in vivo. Results YFPCC1aPKC localization in tobacco BY-2 cells In mammalian cells, YFPCC1aPKC has been shown to function as a strong DAG biosensor (Oancea and Meyer 1998, Oancea et al. 1998). To investigate its use in herb cells, stable transgenic tobacco BY-2 cells were generated that expressed YFPCC1aPKC under the control of the constitutive 35S promoter. As shown in Fig. 1, most of the YFPCC1aPKC fluorescence was localized in the cytosol and nucleus, like YFP alone (Fig. 1). Nonetheless, some transmission was present as motile, punctate structures (arrowheads in Fig. 1), but no obvious plasma membrane transmission was visible. To test the functionality of the DAG biosensor, the phorbol ester PMA was tested. PMA mimics the binding of DAG to the C1a domain name and is therefore a potent activator of PKC activity in vivo and causes a rapid recruitment of YFPCC1aPKC to the plasma membrane of animal cells (Oancea and Meyer 1998, Oancea et al. 1998). Treatment of our tobacco YFPCC1aPKC cells with 1 ?M PMA also resulted in a strong relocalization of the biosensor to the plasma membrane (Supplementary Fig. S1). As a second control, we tested a short-chain analog of Vorapaxar ic50 DAG, i.e. 1,2-dioctanoyl 0.5 ? EC7) by Student em t /em -test. EYFP is usually shown in green and mRFP or mCherry is usually shown in magenta. Scale bars = 10 ?m. The fungal toxin brefeldin A (BFA) has been shown to inhibit Golgi trafficking and to induce the appearance of large, so-called BFA compartments (Geldner et al. 2003). BFA treatment (50 ?M, 45 min) resulted in a strong accumulation of both YFPCC1aPKC and RabA1g in BFA compartments (Fig. 4), again suggesting that YFPCC1aPKC detects DAG at the TGN. As was reported previously, mRFPCPHFAPP1 labeled structures that were Vorapaxar ic50 much more resistant to BFA treatment and only showed a poor accumulation in BFA compartments (Fig. 4). BFA treatment of UBQ10::EYFPCC1aPKC and UBQ10::EYFPC2 ? C1aPKC seedlings co-incubated with FM4-64 (a fluorescent lipophilic membrane dye) also revealed a clear accumulation of both YFP and FM4-64 transmission in BFA compartments (Supplementary Fig. S7). YFPCC1aPKC.