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Polar transport of the phytohormone auxin through PIN-FORMED (PIN) auxin efflux carriers is essential for the spatiotemporal control of plant development. The Arabidopsis thaliana serine/threonine kinase D6 PROTEIN KINASE (D6PK) is polarly localized at the plasma membrane of many cells where it colocalizes with PINs and activates PIN-mediated auxin efflux. Here, we show that the association of D6PK with the basal plasma membrane and PINs is dependent on the phospholipid composition of the plasma membrane as well as on the phosphatidylinositol phosphate 5-kinases PIP5K1 and PIP5K2 in epidermis cells of the primary root. We further show that D6PK directly binds polyacidic phospholipids through a polybasic lysine-rich motif in the middle domain of the kinase. The lysine-rich motif is required for proper PIN3 phosphorylation and for auxin transport-dependent tropic growth. Polybasic motifs are also present at a conserved position in other D6PK-related kinases and required for membrane and phospholipid binding. Thus, phospholipid-dependent recruitment to membranes through polybasic motifs might not only be required for D6PK-mediated auxin transport but also other processes regulated by these, as yet, functionally uncharacterized kinases.
The phytohormone auxin is essential for plant differentiation and growth (Leyser, 2010; Teale et al., 2006). Seemingly all organ development as well as all dynamic tropic growth responses, e.g. phototropism and gravitropism, rely on differential auxin distribution patterns and auxin-induced cellular responses (Adamowski and Friml, 2015; Wang and Estelle, 2014). Auxin levels within the plant are controlled by local auxin metabolism and directional auxin transport, which is achieved by the PIN-FORMED (PIN) efflux and the AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) influx carriers (Adamowski and Friml, 2015). PINs are polarly distributed in the plasma membranes of many cells and their differential distribution results in the formation of local auxin maxima and minima that orchestrate the development of the growing plant body (Adamowski and Friml, 2015; Grunewald and Friml, 2010; Habets and Offringa, 2014).
Studies on directional auxin transport and PIN polar distribution have become attractive systems to study cell polarity in plants (Pan et al., 2015; Sachs, 1991; Yang, 2008). Although it is still unclear whether the targeting of de novo synthesized PINs is polar or apolar, it is well established that the maintenance of PIN polarity requires constitutive recycling in combination with a reduced lateral diffusion in the plasma membrane (Feraru et al., 2011; Kleine-Vehn et al., 2011). PIN polarity is seemingly also critically dependent on the activity of the AGCVIII protein kinase PINOID (PID) and PIN phosphorylation (Friml et al., 2004; Kleine-Vehn et al., 2009; Michniewicz et al., 2007; Offringa and Huang, 2013). Tissue-specific PIN polar distribution patterns can sufficiently explain auxin distribution gradients across plant organs like roots and shoots (de Reuille et al., 2006; Grieneisen et al., 2007; Heisler et al., 2010; Mironova et al., 2010; van Berkel et al., 2013; Wabnik et al., 2011). Additionally, gravity as well as light-dependent changes in PIN polarity correlate with observed changes in auxin distribution required for tropic growth processes (Ding et al., 2011; Kleine-Vehn et al., 2010; Rakusová et al., 2011). Thus, understanding PIN polarity and activity control at the plasma membrane can substantially contribute to the understanding of plant growth.
PIN-mediated auxin transport is critically dependent on phosphorylation by protein kinases (Barbosa and Schwechheimer, 2014). The AGCVIII serine/threonine kinase D6 PROTEIN KINASE (D6PK) but also the proposed PIN polarity regulator PINOID (PID) activate PIN-mediated auxin transport through PIN phosphorylation when tested in Xenopus laevis oocytes (Friml et al., 2004; Zourelidou et al., 2014). Arabidopsis mutants of D6PK and the orthologous D6PK-LIKE genes D6PKL1-D6PKL3 are compromised in directional auxin transport and PIN-dependent growth (Willige et al., 2013; Zourelidou et al., 2009). Conversely, pharmacological interference with D6PK abundance at the plasma membrane impairs auxin transport-dependent tropic responses (Barbosa et al., 2014). Although both D6PK and PINs are polarly localized at the plasma membranes of cells of different plant tissues, their recycling kinetics are dramatically different and obviously mediated by different trafficking pathways (Barbosa et al., 2014; Geldner et al., 2003; Zourelidou et al., 2009). Understanding how D6PK associates with the plasma membrane is thus important for understanding D6PK-mediated auxin transport and plant growth regulation.
Peripheral membrane proteins can be anchored to membranes by diverse mechanisms that include protein-protein and protein-lipid interactions (Cho and Stahelin, 2005). The very low abundant phosphoinositides (PIs) are important players for the recruitment of peripheral proteins to membranes (Balla, 2013; Heilmann, 2016). PIs are mono-, bis- and tris-phosphorylated derivatives of phosphatidylinositol (PtdIns) and PI compositions differ between the plasma membrane and different organellar membranes (Balla, 2013). Membrane-specific PI distribution, a result of the differential distribution and tight regulation of specific PI kinases or phosphatases, allows recruiting specific cognate binding proteins that determine organelle function (Behnia and Munro, 2005). In particular, asymmetric PI distribution in the plasma membrane contributes to the organization of polar domains in many organisms and biological processes (Comer and Parent, 2007; McCaffrey and Macara, 2009; Orlando and Guo, 2009; Shewan et al., 2011). In plants, the polar distribution of PIs, PI kinases, PI binding partners and associated processes are extensively studied in tip growing cells such as pollen tubes and root hairs (Kusano et al., 2008; Stenzel et al., 2008; Ischebeck et al., 2010). Analysis of PtdIns(4,5)P2 biosensors and respective PIP5Ks (PtdIns4P 5-KINASES) revealed that PtdIns(4,5)P2 and PIP5Ks are polarly localized in many cell types and required for PIN polarity control (Ischebeck et al., 2013; Tejos et al., 2014). More recently, PtdIns4P was identified as a major PI, conferring negative charges to the plant plasma membrane (Simon et al., 2016).
Peripheral membrane proteins can bind PIs through globular PI species-specific binding domains, e.g. the PtdIns3P-specific FYVE domain or the PtdIns(4,5)P2-specific PH domain (Hammond and Balla, 2015). Alternatively, peripheral membrane proteins can bind membranes through ionic interactions between stretches of positively charged basic amino acids [lysine (K) or arginine (R)] and the negatively charged lipids, such as PIs or PtdOH (phosphatidic acid) (Brzeska et al., 2010; Heo et al., 2006; Jaillais et al., 2011; McLaughlin and Murray, 2005).
D6PK associates also with root hair tips following root hair initiation (Stanislas et al., 2015). PINs, the established D6PK substrates, are reportedly absent from root hair tips and D6PK might have other phosphorylation substrates and PIN-independent roles at this plasma membrane region (Stanislas et al., 2015). The association of D6PK with the root hair initiation site is dependent on PIP5K3 and, reciprocally, D6PK can bind phospholipids in vitro (Stanislas et al., 2015). A basic sequence stretch noted in several AGCVIII kinases is a good candidate region for phospholipid interactions (Zegzouti et al., 2006).
Here, we show that D6PK is able to bind phospholipids with polyacidic headgroups and that phospholipid composition critically determines D6PK plasma membrane recruitment and polarity. We further identify a polybasic K-rich motif within the D6PK middle domain that is required for D6PK phospholipid binding and membrane association as well as for proper PIN phosphorylation and PIN-dependent tropisms. Additionally, we find that K/R-rich motifs are essential for membrane and phospholipid-binding in other, as yet, uncharacterized AGCVIII kinases. Thus, phospholipid-dependent recruitment of AGCVIII kinases through ionic interactions between the polyacidic phospholipid headgroups and polybasic motifs emerge as general control mechanisms of protein-membrane binding, and in the case of D6PK for auxin transport control.
D6PK plasma membrane association is phospholipid dependent
The molecular determinants of the membrane association and polar distribution of D6PK at the basal plasma membrane of root cells are unknown. In lipid overlay assays, D6PK bound to phospholipids with polyacidic headgroups, namely phosphatidic acid (PtdOH) as well as the PIs PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 (Fig. 1A). D6PK binding was strongest to PtdIns3P when using TRIS-buffered saline and to PtdOH when using PBS (phosphate-buffered saline) of comparable ionic strengths (188 mM and 181 mM) (Fig. 1A) (Stanislas et al., 2015). D6PK did not bind to phospholipids with neutral headgroups, namely PtdCho (phosphatidylcholine) and PtdEtn (phosphatidylethanolamine), or to phospholipids with monoacidic headgroups such as PtdIns and phosphatidylserine (PtdSer; Fig. 1A). In a lipid bilayer context using liposomes of mixed phospholipid compositions, D6PK bound to liposomes containing the polyacidic phospholipids PtdIns4P, PtdIns(4,5)P2 or PtdOH, but not to liposomes containing only PtdEtn and PtdCho, phospholipids with neutral headgroups (Fig. 1B).
PI biosynthesis begins with PtdIns and involves several phosphorylation and dephosphorylation steps by PI kinases and phosphatases (Meijer and Munnik, 2003). PtdOH, conversely, is produced either from PIs by phosphoinositide phospholipase C (PI-PLC) and subsequent action of diacylglycerol kinase (DGK) or from PtdCho or PtdEtn by phospholipase D (PLD; Fig. 2A) (Meijer and Munnik, 2003). To address whether the membrane phospholipid composition determined the basal localization of YFP-tagged D6PK (YFP:D6PK) in root cells, we examined YFP:D6PK localization and distribution after 30 min treatments with established PI and PtdOH biosynthesis inhibitors (Fig. 2). For comparisons and to establish the effectiveness of the inhibitor treatments, we also analysed the localization of phosphoinositide-specific biosensors, PIN2:GFP trafficking and the uptake of the lipophylic dye and endocytosis tracer FM4-64 (Fig. S1). Interestingly, the PI3-KINASE inhibitors LY294002 and Wortmannin (WM) led to a significant depletion of YFP:D6PK from the plasma membrane (Baggiolini et al., 1987; Simon et al., 2016; Vlahos et al., 1994). Our WM and LY294002 treatments efficiently altered the localization of the PdtIns3P sensor (P18Y) but not of the PdtIns4P (FAPP1) nor PdtIns(4,5)P2 (P24Y) sensors. With regard to PIN2:GFP, they led to PIN2:GFP accumulation in endosomes, as previously reported for WM treatments, but had no visible effects in overall FM4-64 uptake (Fig. S1) (Jaillais et al., 2006). Conversely, the PI4-KINASE inhibitor PAO caused a strong plasma membrane dissociation of YFP:D6PK (Fig. 2B,C) (Hammond et al., 2012; Simon et al., 2016). As expected, PAO treatment specifically led to the internalization of the PdtIns4P sensor (FAPP1) but, interestingly, also blocked FM4-64 uptake as well as the accumulation of PIN2:GFP in BFA compartments, suggesting that PAO treatment might block endocytosis (Fig. S1). Strikingly, short-term treatments with U73122 (+), an inhibitor of the PI-PLC-mediated hydrolysis of PIs, but not treatments with its inactive analogue U73343 (−), led to a depolarization of YFP:D6PK at the plasma membrane, concomitant with the appearance of the protein in endosomes. Effects on the seemingly polar PdtIns(4,5)P2 biosensor or on polar PIN2:GFP could not be detected (Fig. 2B,C) (Bleasdale et al., 1990). It is likely that the increased plasma membrane dissociation by the U73122 (+) treatment resulted from the apolar redistribution and consequent reduction of YFP:D6PK signals at the basal plasma membrane where we performed our measurements (Fig. 2B,C). Surprisingly, the subcellular fractionation of protein extracts prepared from roots expressing YFP:D6PK from the 35S CaMV promoter indicated that the observed plasma membrane dissociation after WM, LY294002, PAO, and U73122 treatments was accompanied by an obvious increased solubilization of YFP:D6PK only in the case of the PAO treatment (Fig. 2B-E). For all treatments, we observed similar effects on YFP:D6PK localization, regardless of whether the fusion was expressed from the D6PK or the 35S CaMV promoter (Fig. 2; Fig. S1).
As YFP:D6PK strongly bound to PtdOH, we also tested the effects of the modulation of PtdOH abundance with R59022, an inhibitor of DGK- and PI-PLC-dependent PtdOH synthesis (de Chaffoy de Courcelles et al., 1985; Potocký et al., 2014). R59022 led to the occasional accumulation of YFP:D6PK on endosomes, detectable only when YFP:D6PK was expressed from the D6PK promoter (Fig. 2B; Fig. S1B). Interestingly, PIN2:GFP and FM4-64 also accumulated in R59022-induced compartments (Fig. S1). At the same time, inhibition of PLD-dependent PtdOH production by 1-butanol resulted in the partial internalization of YFP:D6PK, an apparent reduction in FM4-64 uptake as previously reported, but no detectable effects on PIN2:GFP trafficking (Fig. 2B,C; Fig. S1) (Li and Xue, 2007). Conversely, anticipated increases in PtdOH abundance as a result of propanolol treatments had neither an obvious effect on YFP:D6PK localization nor on PIN2:GFP trafficking or FM4-64 uptake (Fig. 2B,C; Fig. S1). Thus, the intracellular levels of PtdOH seemingly regulate YFP:D6PK localization more subtly than PI levels, and likely in a differential manner, depending on whether the PtdOH originates from the PI-PLC or the PLD pathway. Fractionation experiments indicated that none of the PtdOH inhibitor treatments resulted in the dissociation of YFP:D6PK from cellular membranes (Fig. 2D,E). Taken together, these findings suggested that PtdIns3P, PtdIns(4,5)P2, and PtdOH, but most importantly PtdIns4P, might be essential for YFP:D6PK plasma membrane localization and also membrane association. By contrast, PtdIns(4,5)P2 or one of its derivatives might critically determine YFP:D6PK polar distribution.
PIP5K1 and PIP5K2 are required for proper targeting of D6PK
PIP5K1 and PIP5K2, two Arabidopsis PIP5Ks isoforms that convert PtdIns4P to PtdIns(4,5)P2, preferentially localize to apical and basal domains in root epidermal cells (Ischebeck et al., 2013). As U73122 treatments indicated that PtdIns(4,5)P2 might control the D6PK membrane association and polarity in root epidermal cells, we introduced the YFP:D6PK transgene into the pip5k1 pip5k2 double mutant. Similarly to the reported internalization of YFP:D6PK in root hair cells of pip5k3 mutants, we noted reduced YFP:D6PK plasma membrane association in the root epidermal cells of the pip5k1 pip5k2 double mutant (Fig. 2F,G) (Stanislas et al., 2015). Thus, the reported alterations of PtdIns(4,5)P2 levels in pip5k1 pip5k2 mutants might exert an influence on the plasma membrane targeting of D6PK (Ischebeck et al., 2013; Tejos et al., 2014).
The D6PK MID domain is required and sufficient for D6PK plasma membrane anchoring
AGCVIII kinases have an insertion between kinase subdomains VII and VIII that has been implicated in the targeting of different AGCVIII kinases (Rademacher and Offringa, 2012). In the case of D6PK and the orthologous D6PK-LIKE 1 to 3 (D6PKL1-D6PKL3), this insertion is between 101 and 104 amino acids long and designated here as the middle (MID) domain (Fig. 3A). A role of the insertion domain for the specific localization of different AGCVIII kinases has previously been proposed (Ek-Ramos et al., 2010; Zegzouti et al., 2006). In addition, the kinase domains are flanked by N- and C-terminal domains of 117 to 199 amino acids and 45 to 57 amino acids, respectively (Fig. 3A) (Rademacher and Offringa, 2012). To examine the importance of the three domains for D6PK localization, we analysed YFP:D6PK variants lacking these three domains in YFP:D6PKΔN, YFP:D6PKΔMID and YFP:D6PKΔC lines. Conversely, we analysed fusions between YFP and the three isolated domains in YFP:D6PK_N; YFP:D6PK_MID and YFP:D6PK_C lines. The deletion of any of the three domains resulted in the loss of the plasma membrane association of D6PK (Fig. 3B; Fig. S2). Regardless, each of these deletion variants remained associated with membranes, possibly endomembranes (Fig. 3C). Specifically in the case of YFP:D6PKΔMID, an endosomal localization was apparent as judged by its co-localization with the membrane lipid dye FM4-64 (Fig. 3B; Fig. S2). In turn, YFP:D6PK_N and YFP:D6PK_C were neither associated with the plasma membrane nor with membrane fractions after subcellular fractionation (Fig. 3B,C). The MID domain, however, was sufficient to recruit YFP:D6PK_MID to the plasma membrane in a polar manner, albeit with a reduced polarity when compared with YFP:D6PK, and the majority of the protein was identified in the membrane fraction after subcellular fractionation (Fig. 3B-D). Thus, the MID domain is sufficient to mediate the plasma membrane association of D6PK, and to some extent also its polar distribution, but all three domains are required for D6PK plasma membrane association.
The rapid recycling of D6PK from the plasma membrane can be blocked by Brefeldin A (BFA), an inhibitor of the ARF-GEF GNOM (Barbosa et al., 2014; Geldner et al., 2003; Zourelidou et al., 2009). Whereas wild-type YFP:D6PK was observed in intracellular compartments after BFA treatments, no obvious accumulation in BFA-compartments was observed with any of the deletion variants, indicating that their intracellular trafficking might be distinct from that of the BFA-sensitive YFP:D6PK (Fig. S2). Although YFP:D6PK_MID localized to the plasma membrane in a strongly polar manner, the protein was partially insensitive to the effects of BFA (Fig. 3B; Fig. S2). Thus, although the MID domain is sufficient for polar plasma membrane targeting, it displays different recycling and polarity properties from YFP:D6PK.
D6PK is an auto-phosphorylating kinase that trans-phosphorylates PINs (Zourelidou et al., 2014). In vitro kinase assays with the PIN1 cytosolic loop showed that D6PKΔN or D6PKΔMID retained the ability for auto-phosphorylation but lost the ability for PIN1 trans-phosphorylation (Fig. 3E). In turn, D6PKΔC had no detectable auto- or trans-phosphorylation activity (Fig. 3E). This suggested that the N-terminus and the MID domain might be required for PIN substrate recognition or trans-phosphorylation, whereas the C-terminus might be required for D6PK kinase activity. Interestingly, D6PK auto-phosphorylation was increased in D6PKΔN and D6PKΔMID when compared with D6PK, suggesting that the N-terminus and the MID domain might have an inhibitory effect on D6PK kinase activity.
A K-rich motif is required for phospholipid binding and D6PK plasma membrane association
In a search for possible motifs in the MID domain that could confer binding to the plasma membrane, our attention was drawn to a positively charged motif including multiple K and R residues in each of the four D6PK and D6PKL proteins (Fig. 4A) (Hammond and Balla, 2015; Zegzouti et al., 2006). We generated alanine (A) replacement variants of the three most highly conserved Ks (D6PK-3KA) or all six Ks (D6PK-6KA) in D6PK and found that phospholipid binding was partially (D6PK-3KA) or strongly (D6PK-6KA) impaired in phospholipid binding assays or in liposome binding assays with PtdIns4P or PtdIns(4,5)P2 (Fig. 4B,C). In turn, these mutations did not affect D6PK auto- or PIN1 trans-phosphorylation (Fig. 4D).
In transgenic plants, the YFP:D6PK-3KA and YFP:D6PK-6KA mutations strongly impaired the plasma membrane association of the protein when expressed from a 35S CaMV promoter in a wild-type Col-0 background (Fig. 4E) or from a D6PK promoter fragment in the d6pk d6pkl1 (d6pk01) double mutant (Fig. 4F). The observed intracellular distribution patterns ranged from a partial accumulation in the case of YFP:D6PK-3KA to an almost complete intracellular accumulation of YFP:D6PK-6KA (Fig. 4E-G). In quantitative terms, the effects of the K-to-A mutations on YFP:D6PK plasma membrane association differed depending on the promoter used, where the stronger 35S CaMV promoter led to stronger dissociation indices than the weaker D6PK promoter (Fig. 4G). This variation could arise from limiting plasma membrane recruiting factor(s), such as phospholipids or protein, that might become limiting when YFP:D6PK is abundant and presents lower affinity to membranes. In contrast, the individual KA mutants revealed surprisingly conserved dissociation indices between independent transgenic lines with different protein levels, regardless of the promoter driving their expression. For instance, the four D6PK promoter lines of YFP:D6PK-6KA with variable absolute signal intensities at the plasma membrane displayed strikingly conserved plasma membrane dissociation indices (Fig. 4F,G).
To test whether absolute protein abundance contributed to the variable plasma membrane abundance of the D6PK promoter YFP:D6PK-6KA lines, we analysed total YFP:D6PK abundance from these lines after immunoprecipitations of YFP:D6PK with anti-GFP. We indeed detected a strong accumulation of the YFP:D6PK-6KA protein in lines #13 and #10 with stronger plasma membrane signals, in comparison with the weaker lines #9 and #11 and to YFP:D6PK and YFP:D6PK-3KA lines (Fig. S3A). These differences could not be explained by differences in transcript abundance between these lines (Fig. S3B). We concluded that K residue mutations combined with the resulting localization defects might lead to a disturbance of YFP:D6PK trafficking, protein turnover and ultimately homeostasis.
Owing to the low protein expression levels, we were unable to perform fractionation experiments with lines expressing YFP:D6PK from the D6PK promoter. However, when we performed subcellular fractionation experiments with the 35S CaMV promoter-driven YFP:D6PK lines, we detected a shift in the distribution of the protein between the soluble and the membrane fraction in the case of YFP:D6PK-6KA (Fig. 4H,I). Whereas ∼80% of YFP:D6PK was retained in the membrane fraction, only ∼40% of YFP:D6PK-6KA of the protein resided in the membrane fraction (Fig. 4H,I). In summary, these findings suggest that the K-rich motif of the D6PK MID domain confers the ability to bind to polyacidic phopholipids in vitro and to the plasma membrane and endomembranes in vivo.
The K-rich motif is required for D6PK function
YFP:D6PK abundance at the plasma membrane is crucial for PIN phosphorylation and auxin transport activation (Barbosa et al., 2014). d6pk d6pkl1 (d6pk01) or d6pk d6pkl1 d6pkl2 (d6pk012) mutants are impaired in auxin transport-dependent growth responses, including negative gravitropism and phototropism of the hypocotyl. Expression of YFP:D6PK from a D6PK promoter fragment is sufficient to suppress these phenotypes in d6pk012 (Willige et al., 2013). Likewise, YFP:D6PK-3KA, when expressed from the D6PK promoter, suppressed the tropism defects of the d6pk01 mutant efficiently (Fig. 5A,B). However, YFP:D6PK-6KA suppressed d6pk01 defects less efficiently and the suppression efficiency varied between the individual YFP:D6PK-6KA lines in correlation with their differential tendency to associate with the plasma membrane (Fig. 4, Fig. 5A,B). We therefore suggest that their differential ability to associate with the plasma membrane is the cause for their differential effects in the suppression of the tropism defects of d6pk01 mutants.
A further indication for an impaired functionality of the proteins came from our analyses of seedlings overexpressing wild-type and mutated YFP:D6PK. Dark-grown seedlings overexpressing YFP:D6PK from the 35S CaMV promoter have strong tropism defects that can be suppressed by treatments with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA), suggesting that these phenotypes are caused by auxin transport defects (Barbosa et al., 2014). Similarly, the overexpression of YFP:D6PK-3KA, but not that of YFP:D6PK-6KA, caused defects in hypocotyl tropism and apical hook formation that were comparable with, but milder than, those observed after overexpression of wild-type YFP:D6PK (Fig. 5C,D). As in root epidermal cells, we observed a reduction in the plasma membrane signal in YFP:D6PK-3KA and YFP:D6PK-6KA lines in epidermal, endodermal and vascular cells in the hypocotyl when compared with the lines expressing wild-type YFP:D6PK (Fig. 4D, Fig. 5E). Thus, the severity of the overexpression phenotypes in the hypocotyl decreased after mutation of the K-rich motif in line with their differential defects in plasma membrane association and their defects in auxin transport regulation at the plasma membrane.
Proper PIN3 phosphorylation depends on an intact K-rich motif in YFP:D6PK
PIN3 has a role in the control of hypocotyl tropisms and is a direct target of D6PK phosphorylation (Willige et al., 2013; Zourelidou et al., 2014). PIN3 phosphorylation is reduced in d6pk01 and d6pk012 (Willige et al., 2013) and BFA treatments, which lead to a dissociation of D6PK from the plasma membrane and impair PIN3 phosphorylation in the wild type (Barbosa et al., 2014). We accordingly expected that the differential accumulation of YFP:D6PK and its mutant variants at the plasma membrane should result in a differential PIN3 phosphorylation. In immunoblots with anti-PIN3, PIN3 can be separated into a slow- and a fast-migrating form, which are better distinguished in intensity profiles (Fig. 6A,B). It was shown that the more slowly migrating form corresponds to a phosphorylated form of PIN3 according to several criteria (Willige et al., 2013). Densitometric analysis revealed that phosphorylated PIN3 was reduced by about 40% in d6pk01 when compared with the wild type or the d6pk012 mutant complemented with the wild-type YFP:D6PK transgene. Whereas the complementation of the phosphorylation defect was still efficient with YFP:D6PK-3KA, the degree of complementation was variable between the different YFP:D6PK-6KA lines. Here, the PIN3 phosphorylation defects were partially suppressed in the YFP:D6PK-6KA lines (#13 and #10, Fig. 6), in line with suppression of d6pk01 tropism defects by YFP:D6PK-6KA in these lines and the fact that they accumulated YFP:D6PK-6KA at the plasma membrane. In turn, PIN3 phosphorylation was not normalized in the YFP:D6PK-6KA lines that could not complement the d6pk01 phenotype and that had reduced YFP:D6PK-6KA plasma membrane abundance (#9 and #11, Fig. 6 and Fig. 5). Thus, the differential complementation efficiency of the different YFP:D6PK-6KA lines on PIN3 phosphorylation correlated with their efficiency to complement d6pk01 tropism defects and also with their abundance at the plasma membrane (Fig. 4F,G, Fig. 5).
Polybasic motifs are present in many members of the AGCVIII kinase family
D6PK and the three D6PKLs belong to the AGC1 subgroup of the 23-member AGCVIII kinase family of Arabidopsis (Rademacher and Offringa, 2012). Besides a number of previously uncharacterized kinases, this family also includes several plasma membrane-resident kinases: the proposed PIN polarity regulators PID, WAG1 and WAG2 (AGC3 subgroup); the blue light receptors PHOTOTROPIN1 (phot1) and phot2 (AGC4 subgroup); and UNICORN (UCN; AGC2 subgroup) (Dhonukshe et al., 2010; Enugutti et al., 2012; Kong et al., 2006; Sakamoto and Briggs, 2002) (Fig. 7). Although all kinases have an insertion corresponding to the D6PK MID domain, the amino acid sequence conservation in this insertion domain and over the K-rich motif of D6PK is rather limited (Fig. 7A). Recently, studies on a biochemically diverse group of proteins from different organisms demonstrated that protein domains required for membrane association could be reliably predicted based on their basic and hydrophobic score (BH score) above a critical threshold value of 0.6 (Bailey and Prehoda, 2015; Brzeska et al., 2010). Accordingly, the biophysical properties, determined by, for example, the BH score rather than amino acid conservation, could control protein-membrane associations. Indeed, when we applied the BH score prediction to D6PK, we identified the K-rich motif with a BH score (0.79) above the critical threshold value (Fig. 7; Fig. S4). Importantly, the 3KA and 6KA mutations reduced the BH score (<0.6), thus supporting the predictive power of this value (Fig. 7; Fig. S4).
We subsequently surveyed the 23 members of the Arabidopsis AGCVIII kinase family for their BH profile and found a peak with BH scores ≥0.6 in 18 Arabidopsis proteins at the end of the MID domain that coincided, in its relative position, with the D6PK K-rich motif (Fig. 7; Fig. S4). We next tested whether the K/R-enriched motifs were indeed required for plasma membrane association and phospholipid binding in hitherto uncharacterized protein kinases. To this end, we examined the localization of representative members of individual AGC1 family subclades, namely AGC1.3 (BH=0.78), AGC1.7 (0.68), AGC1.8 (0.94) and AGC1.12 (0.8), in their wild-type form and after mutation of the K/R-enriched motifs in protoplasts (Fig. 7B). Conversely, we also tested the binding of wild-type and mutant proteins in lipid overlay assays. In each but one case, the protein kinases associated with the plasma membrane in a polybasic motif-dependent manner (Fig. 7C). A noteworthy exception was AGC1.7, which had the lowest BH score of all proteins tested (0.68) and did not associate with the plasma membrane. All four kinases bound to polyacidic phospholipids with comparable binding specificities in their wild-type form but with visibly reduced affinity in their mutant form, for most of the phospholipids, with the exception of AGC1.8 (Fig. 7C). Thus, the K/R-rich motifs with high BH scores are, in most cases, required for plasma membrane binding and good candidate motifs for phospholipid binding.
We have shown that proper phospholipid composition is required for the polar recruitment of D6PK to the plasma membrane and that a K-rich motif in the D6PK MID domain mediates protein-phospholipid interactions. We propose that phospholipid metabolism determines the plasma membrane composition and polar distribution of negatively charged phospholipids that recruit D6PK via its positively charged K-rich motif (Fig. 8). In line with this, we find that mutations of the K-rich motif impair D6PK membrane association and auxin transport-dependent tropic responses that correlate with defects in the phosphorylation of the auxin efflux carrier PIN3.
In in vitro binding assays, D6PK could bind to polyacidic PIs and PtdOH in a K-rich motif-dependent manner. In vivo manipulation of PI or PtdOH metabolism led to the intracellular accumulation of YFP:D6PK and impaired its polar plasma membrane distribution but only manipulation of PtdIns4P synthesis affected YFP:D6PK solubility. At the same time, K-rich motif mutations in D6PK led to increased solubilization of the protein. This suggests that the intact K-rich domain is required for interactions with polyacidic phospholipids in different membranes and that the specific phospholipid composition in a given membrane might determine D6PK recruitment to different membranes. The D6PK interaction with multiple polyacidic phospholipids through ionic interactions resembles previously reported mechanisms for protein-phospholipid ionic interactions and is distinct from interactions mediated between phospholipids and globular protein domains (Hammond and Balla, 2015; Li et al., 2014).
PtdOH biosensors have indicated that PtdOH is enriched at the apical and sub-apical plasma membranes in pollen tubes (Potocký et al., 2014; Simon et al., 2014). Interestingly, the same manipulations that affected the behaviour of a PtdOH biosensor in pollen tubes also affected the intracellular distribution of D6PK in root cells, i.e. the inhibition of PLD by 1-butanol resulted in the internalization of both proteins and inhibition of DGK by R59022 resulted in the accumulation of both proteins in endosomal compartments. PtdOH is thus a good candidate phospholipid for anchoring D6PK at the plasma membrane.
PI-kinases and phosphatases determine the abundance of different PI species in different membranes; PI biosensors indicate that PtdIns4P and PtdIns(4,5)P2 reside in the plasma membrane whereas PtdIns3P resides in the late endosomes and the tonoplast. In turn, genetic evidence indicates that PtdIns(3,5)P2 might be produced at the tonoplast and late endosomes (Heilmann and Heilmann, 2015; Hirano et al., 2015; Novakova et al., 2014; Simon et al., 2014, 2016; Vermeer et al., 2006, 2009). The cellular distribution of PtdIns5P in plants is unknown and neither PtdIns(3,4)P2 nor PtdIns(3,4,5)P3 have as yet been detected in plants (Heilmann, 2009; Munnik and Nielsen, 2011). In our experiments, treatments with WM and LY294002, which are expected to result in the depletion of PtdIns3P (WM and LY294002) and PtdIns4P (only WM), caused D6PK internalization but did not affect D6PK membrane association. As PtdIns3P has not yet been detected at the plasma membrane, the effects at least of WM but not of LY294002 on D6PK plasma membrane localization could be caused by the plasma membrane depletion of PtdIns4P or other indirect effects on membrane or protein trafficking. A recent study supports this hypothesis by demonstrating that PtdIns4P is the predominant phospholipid in plant plasma membranes and that the AGCVIII kinase PID localizes to the plasma membrane in a PtdIns4P-dependent manner (Simon et al., 2016).
The U73122-mediated inhibition of PtdIns(4,5)P2 hydrolysis led to a quick depolarization of D6PK. This resembled the U73122-induced depolarization of a tip-localized PtdIns(4,5)P2 biosensor in pollen tubes, which was observed after inhibition of PI-PLCs with subapical lateral localization (Helling et al., 2006; Zhao et al., 2010). The observed depolarization of YFP:D6PK suggests that PtdIns(4,5)P2 is also polarized in root epidermal cells. There is, however, still some controversy as to whether the PtdIns(4,5)P2 biosensor has an apicobasal localization in these cells (Ischebeck et al., 2013; Simon et al., 2016; Tejos et al., 2014). Regardless, it is tempting to speculate that U73122 treatment blocks a lateral PI-PLC function in the root epidermis, which could then lead to a depolarization of PtdIns(4,5)P2 and D6PK, and that the current PtdIns(4,5)P2 biosensor fails to recapitulate the PI dynamics. Future dissection of the polarity functions of PI4P 5-kinases and PI-PLCs in the root epidermis will be required to examine this hypothesis.
As almost all of our (short-term) manipulations of PI and PtdOH biosynthesis had an impact on D6PK localization, with the strongest effects following the PAO-induced depletion of PtdIns4P, we suggest that not only one particular phospholipid but rather a combination of PtdOH and PIs might be responsible for D6PK plasma membrane recruitment. The retention of a weak but detectable plasma membrane association in the D6PK-3KA and -6KA variants or with a deletion of the MID domain suggests that there are other determinants of membrane association within D6PK. Alternatively, regions other than the D6PK MID domain might mediate additional interactions with membrane proteins or phospholipids, or yield lipid modifications required for D6PK affinity and specificity to membranes. Such a coincident detection has been often described for the recruitment of peripheral membrane proteins (Carlton and Cullen, 2005; Hammond and Balla, 2015; Stahelin et al., 2014). Importantly, we can exclude that kinase activity might control D6PK localization, as we have previously shown that inactive YFP:D6PK maintains similar polarity and recycling kinetics to the wild-type protein (Barbosa et al., 2014).
Despite limited sequence similarity in the insertion domain, many AGCVIII protein kinases have a high BH score, specifically in the region corresponding to the D6PK K-rich motif. We thus propose, and have in part tested, that these other kinases are recruited to the plasma membrane (and possibly internal membranes) through similar mechanisms to those studied in detail for D6PK. While this study was in progress, an analysis of the mechanisms targeting the AGCVIII kinase PID to the plasma membrane identified the corresponding motif in PID to be required for PID plasma membrane association (Simon et al., 2016). Although the BH score might allow prediction of membrane associations, slight differences in motif length and features of nearby residues, e.g. hydrophobicity or phosphorylation, might affect their properties with regard to phospholipid specificity, membrane localization or trafficking as reported for other proteins (Schmick et al., 2014; Yeung et al., 2008). For example, the K/R-rich motif in the middle domain of the D6PK-related ADI3 kinase from tomato determines its dual targeting between nucleus and endomembranes (Ek-Ramos et al., 2010, 2014; Heo et al., 2006; Maures et al., 2011). Similar mechanisms might control the distribution of D6PK between the plasma membrane and endomembranes, or even the nucleus, and the existence of such mechanisms might also explain the differential localization of AGC1.7 in our protoplast studies.
In summary, we propose a model for D6PK plasma membrane anchoring and polarity maintenance where both the phospholipid composition of the plasma membrane and the D6PK surface charge at the K-rich motif of the MID domain critically determine D6PK plasma membrane steady-state levels (Fig. 8). This model provides a basis for the involvement of phospholipids in the control of auxin transport activity, and possibly directionality, by D6PK plasma membrane abundance and distribution. The findings reported here for D6PK might also be relevant for the other, as yet uncharacterized, AGC family kinases or other membrane-associated proteins with a high BH score.
Biological material
The following previously published Arabidopsis thaliana mutants and transgenic lines in the Columbia-0 background were used in this study: d6pk d6pkl1 double mutants (d6pk01) (Zourelidou et al., 2009), pip5k1 pip5k2 double mutants (Ischebeck et al., 2013), 35S:YFP:D6PK in the wild-type background and D6PK:YFP:D6PK in the d6pk d6pkl1 d6pkl2 background (D6PK d6pk012) (Willige et al., 2013; Zourelidou et al., 2009), PIN2:PIN2:GFP (PIN2:GFP) (Abas et al., 2006), as well as the phosphoinositide marker lines P18Y (2xFYVEHRS) for PtdIns3P, YFP-PHFAPP1 for PtdIns4P, and P24Y (2xPHPLC) for PtdIns(4,5)P2 (Simon et al., 2014; Vermeer et al., 2009).
Cloning procedures
Clones of D6PK protein domain variants were obtained by PCR-amplification of Gateway-compatible PCR fragments from pDONR201 (Life Technologies) containing the D6PK coding sequence (Zourelidou et al., 2009) with the following primers: IB2 and IB3 for YFP:D6PKΔN (amino acids 109 to 499); IB1 and IB4 for YFP:D6PK_N (amino acids 1 to 108); IB1 and IB5 for YFP:D6PKΔC (amino acids 1 to 446); IB2 and IB6 for YFP:D6PK_C (amino acids 447 to 499); IB7 and IB8 for YFP:D6PK_MID (amino acids 255 to 335). The PCR fragments were then inserted into the entry vector pDONR201. YFP:D6PKΔMID, with a deletion of amino acids 255 to 355, was obtained by SacI digestion and re-ligation of a pDONR-D6PK clone, which had been mutagenized with the primers IB9 and IB10 to introduce SacI sites flanking the MID domain. To generate the D6PK-3KA variant, lysines K315, K317 and K318 were replaced by alanines in pDONR-D6PK using site-directed mutagenesis with the primer IB70. For the D6PK-6KA mutant variant, lysines K312, K314 and K320 were mutated in D6PK-3KA with IB62. All primer sequences are listed in Table S1.
Inserts of the pDONR201 entry clones with the above-described deletions or mutations were introduced into the destination vectors pDEST15 (Life Technologies) to obtain glutathione-S-transferase (GST) N-terminal fusion constructs for recombinant expression in bacteria or into pEXTAG-YFP-GW to generate constructs for the expression of N-terminal YFP-fusions under control of the 35S CaMV promoter in plants. D6PK mutant variants were also excised as XhoI/NotI fragments from the pEXTAG-YFP-GW vectors and inserted into a pGREEN0229 vector derivative containing a D6PK promoter sequence and a 35S CaMV terminator sequence (Zourelidou et al., 2014). All pEXTAG-YFP-GW overexpression constructs were transformed by floral dip into the wild type (Col-0) and all pGREEN0229 constructs were transformed into d6pk01 for complementation analysis (Clough and Bent, 1998).
The open-reading frames of AGC1.3, AGC1.7, AGC1.8 and AGC1.12 were amplified by PCR and inserted via pDONR201 into the destination vectors pDEST15 (Life Technologies) to obtain GST N-terminal fusion constructs for recombinant expression in bacteria or into pEXTAG-YFP-GW to generate constructs for the expression of N-terminal YFP fusions under control of the 35S CaMV promoter in plants. Polybasic motifs were mutated using overlap extension PCRs with complementary mutated primers (Hansson et al., 2008). In the case of AGC1.8, this PCR was performed in two rounds due to the extended length of the polybasic motif. Protoplast transformation was performed as previously described (Katsiarimpa et al., 2011). Confocal analysis was carried out 14 h after transformation. All primer sequences are listed in Table S1.
Inhibitor treatments and confocal microscopy
Unless otherwise stated, seedlings for microscopic analyses were grown in continuous white light (110 μmol/m–2 s–1) at 21°C for 5-7 days on ½ MS medium (2.15 g/l Murashige and Skoog salts, 0.5 g/l 2-N-morpholino ethane-sulfonic acid, 8 g/l agar, pH 5.8). The following inhibitors were dissolved in DMSO and used as previously described: Wortmaninn (Applichem) (Jaillais et al., 2006); LY294002 (Sigma) (Simon et al., 2016); PAO (Sigma) (Simon et al., 2016); U73343 and U73122 (Sigma) (Andreeva et al., 2010; Zhao et al., 2010); R59022 (Sigma) (Li and Xue, 2007; Potocký et al., 2014); propanolol (Sigma) (Li and Xue, 2007; Potocký et al., 2014); 1- and 2-butanol (Sigma) (Li and Xue, 2007; Potocký et al., 2014); Brefeldin A (Fischer Scientific) (Barbosa et al., 2014). All inhibitor treatments were controlled by solvent control treatments performed in parallel. FM4-64 (2 µM) staining was performed as described in the figure legends. All confocal images were acquired with an Olympus FV1000 confocal microscope with high-sensitivity GaAsP detectors using identical acquisition settings between samples (Olympus). Image analyses and quantification were performed using Fiji and as further specified in the results sections and figure legends (Schindelin et al., 2012).
Phospholipid binding assays
For lipid binding assays, GST fusion proteins were recombinantly expressed in Escherichia coli BL21(DE3) and purified using Glutathione Sepharose 4B (GE Healthcare). Free GST protein was expressed from pGEX-6P-1 (GE Healthcare) as a negative control. Lipid overlay assays using PIP-strips were performed following manufacturer's instructions (Echelon). In brief, membranes were blocked overnight at 4°C in a blocking buffer with 4% BSA either in TBS-T (0.1% Tween) or PBS-T (0.1% Tween). Purified proteins (0.5 μg/ml blocking buffer) were incubated with PIP-strip membranes for 1 h at room temperature, washed three times for 10 min with TBS-T or PBS-T, then incubated for 1 h with anti-GST (1:2000 in blocking buffer; GE Healthcare), washed three times and incubated for 1 h at room temperature with anti-goat peroxidase conjugate antibodies (1:8000 in blocking buffer; Sigma, A5420). After three final washes, detection of bound proteins was performed using Pierce ECL Plus Western Blotting Substrate (Thermo Scientific) in a Fujifilm LAS 4000 Mini (Fuji).
Liposome binding assays were performed as previously described (Julkowska et al., 2013). Liposomes were prepared from 400 nmol of total lipids at the following molar ratios: PtdCho:PtdEtn, 1:1; PtdCho:PtdEtn:PtdIns4P, 2:2:1; PtdCho:PtdEtn:PtdIns(4,5)P2, 2:2:1; PtdCho:PtdEtn:PtdOH, 2:2:1. All lipids were obtained from Avanti Polar Lipids. Proteins were separated by 10% SDS-PAGE and blotted to Protran nitrocellulose membranes (GE Healthcare). GST-tagged D6PK and D6PK-6KA were detected with goat anti-GST (GE Healthcare, 27457701V) and visualized using a rabbit anti-goat antiserum conjugated to an alkaline phosphatase reporter (Sigma).
In vitro kinase assays were performed with purified recombinant kinases using GST:PIN1 CL (cytosolic loop) as a phosphorylation substrate (Zourelidou et al., 2009). Equal protein amounts (∼0.5 µg) were incubated for 60 min at 28°C in a buffer [25 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.2 mM EDTA, 50 μM ATP, 1× Complete Protease Inhibitor Cocktail (Roche)] supplemented with 10 μCi [γ-32P]-ATP [370 MBq, specific activity 185 TBq (Hartmann Analytic)]. Reactions were stopped by adding 5× Laemmli buffer, samples were then boiled and separated in two SDS-PAGE gels, one for autoradiography and one for staining with Coomassie Brilliant Blue as a loading control.
Negative gravitropism and phototropism responses
Seedlings were grown for 2.5 days on ½ MS in the dark. Two hours before the experiment, agravitropically growing seedlings were straightened under green safe light. For phototropism response experiments, etiolated seedlings were transferred to a FloraLED chamber (CLF Plant Climatics) and illuminated for 4 h with 5 μmol m–2 s–1 unilateral blue light. For the hypocotyl negative gravitropism response experiments, plates were reoriented by 90° and kept in the dark for an additional 20 h. Plates were scanned and bending angles were quantified using Fiji (Schindelin et al., 2012).
For the detection of YFP-tagged D6PK and its variants, roots of seven-day-old seedlings were homogenized with protein extraction buffer (PEB; 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1 mM MG132, 0.1 mM PMSF, 1% (v/v) Protease Inhibitor Cocktail (Sigma), and PhosStop phosphatase inhibitor cocktail (Roche)]. For fractionation experiments, extracts were pre-cleared by centrifugation at 10,000 g and the supernatant (T) was subsequently ultra-centrifuged at 100,000 g to yield the soluble (S) and membrane (M) fractions. Membrane fractions were resuspended in the same volume as soluble fractions with PEB. Samples were denatured with 1× Laemmli buffer and heated at 42°C for 15 min. For immunoblotting, the equivalent of 10 µg total protein was separated on a 10% SDS-PAGE and transferred to nitrocellulose membrane (GE Healthcare) by semi-dry blotting. Immunoblots were detected with rabbit anti-GFP (1:3000; Fischer Scientific, A11122) and rabbit anti-UGP (1:2000; Agrisera, AS05086) and secondary antibody goat anti-rabbit HRP (1:2000; Sigma, A9169). Chemiluminescence detection was performed with a Fujifilm LAS 4000 Mini, total band and band profile intensities were quantified using the Fujifilm Multi Gauge v3.0 software.
For immunoprecipitation, protein was extracted in PEB with 0.5% Triton X-100 from seven-day-old seedlings and subsequently diluted to reduce the Triton X-100 concentration to 0.01%. YFP:D6PK was then immunoprecipitated from 3 mg protein extract with 15 µl GFP-Trap_MA slurry (Chromotek) for 1 h at 4°C. Subsequent wash steps followed the manufacturer's protocol. Beads were resuspended in 40 µl 2× Laemmli buffer and 10 µl were used for SDS-PAGE and immunoblotting as described above. A 5 µg aliquot of total protein extract (input) was used for SDS-PAGE and immunoblotting with anti-UGP as a loading control.
For PIN3 immunoblots, protein extraction, quantification and profile analysis was performed using four-day-old dark-grown seedlings as previously described (Barbosa et al., 2014; Willige et al., 2013).
qRT-PCR analyses
RNA extraction and qRT-PCR analysis were performed as previously described (Lutz et al., 2015). Primer sequences are provided in Table S1.
The authors would like to thank Benjamin Weller and Lanassa Bassukas (Technische Universität München) for helpful discussions throughout this study. Jutta Elgner is thanked for her excellent technical assistance. Lanassa Bassukas, Lena Frank and Andreas Scaia (Technische Universität München) are thanked for generating primary cDNA constructs for several AGC1 kinases.
Author contributions
I.C.R.B. performed all experiments and all data analyses; M.Z. performed protein kinase assays; H.S. provided the AGC1.7 clone and performed initial lipid overlay assays; I.H. and M.H. provided mutant material and performed liposome binding assays; I.C.R.B., I.H. and C.S. discussed and interpreted the data and wrote the manuscript.
This work was supported by a fellowship from the Fundação para a Ciência e a Tecnologia (FCT) to I.C.R.B. (SFRH/BD/73187/2010); and grants from the Deutsche Forschungsgemeinschaft [SCHW751/12-1 to C.S. and HE3424/6-1 to I.H.].
Competing interests
The authors declare no competing or financial interests.


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Turkey - Q&A - Excerpts from the daily press briefing (06.02.19)
Q : Did Turkey respond to France after President Macron's decision to establish a day to commemorate the Armenian genocide?
A : In establishing this day of commemoration, the President is fulfilling his well-known pledge to honor French citizens of Armenian descent.
We have had several opportunities to exchange views with the Turkish authorities on this issue. They have been informed of our positions just as we have been informed of theirs.

subset: commoncrawl


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Scream Factory has released, the under seen Lauren Bacall-starring The Fan to Blu-Ray. The stalker-thriller comes to homevideo filled with extras features.
The Film
Not to be mistaken with the 1996 Tony Scott Directed film, though they are both about stalkers, this The Fan is not as flashy as Scott's film. Rather The Fan is a middling film that toes the line but never fully commits to being the gory overwrought slasher you want it to be.
Sally Ross (Bacall) is a Star on what could be the possible downslope. She's in a new musical that seems to be imploding on itself. Do not tell this or any other disparaging remarks to Douglas (Michael Biehn). A record store employee by day, a stalker by night; Douglas sends daily letters to his Star. Armed with his typewriter (this is the 80s after all) and a cache of knives, Douglas invades into Sally's personal life. As he ups the ante in various ways, Sally and those around her are in danger. Will Sally be able to survive Douglas's homicidal obsession.
The biggest issue with The Fan is its lack of commitment to being a straight thriller or gore-filled horror film. Studio interference caused most of these issues but even either side of the coin is only slightly effective. The set pieces though complex, in execution, lacks the sort of Brian De Palma-esque flair needed to truly be great. Though that is not to take away from those moments as it's in those pieces where Biehn is literally stalking prey is where the film comes alive.
Unfortunately, the rest of the film does not equal to what Biehn is doing until the twenty-minute finale that is inspired but without the weight of an entire film around it. Stapleton is the best performer besides Biehn. Both Bacall and Gardener sleepwalk through the film. Though it should be noted that this reviewer wasn't sure if Bacall's performance was informed by the fact she was angry for having been hoodwinked into this film or was just that fed up with acting.
The Fan is well worth your time if you're a Michael Biehn fan or need to see everything that Lauren Bacall ever did. If not a "fan" of either one will only be frustrated at far it does or doesn't go depending on your tastes.
The Transfer
Unfortunately, the bad news keeps coming for fans of The Fan. The transfer sourced from an older transfer does not have Scream Factory's usual luster. One is not sure if it was the film stock of the era (during that time film stock was cheap and not all that great) or the transfer they used but everything is a bit soft and almost too grainy.
The Extras
They include the Following:
- Audio Commentary with David DeCoteau, David Del Valle, Moderated By Scream Factory Marketing Director Jeff Nelson
- Number One Fan – An Interview With Actor Michael Biehn
- Fan Service – An Interview With Director Edward Bianchi
- Fanning The Flames – An Interview With Editor Alan Heim
- Theatrical Trailer
- TV Spots
- Still Gallery
The commentary by David DeCoteau and David Del Valle is hosted by Jeff Nelson (VP of Marketing). Beginning with a discussion of their love of the film, and also how John Lennon's Assassination led to Paramount picking up the film. The men do a great job of deep-diving into the film, the history behind the production/release, the thriller-horror hybrid, it's cult status, and so much more. Definitely worth a listen.
Number One Fan – Interview with Michael Biehn is an all-new 25-minute interview. If anyone has watched or listened to Biehn recently will know how honest and unfiltered the actor is. He discusses how he was cast, his excitement over the role because of the lack of work. Meeting Lauren Bacall, working on the film, the changes including the change in director is discussed, Maureen Stapleton anecdotes that's crazy, his less-than-great relationship with Bacall, and some truly candid comments about this part of his career.
Fan Service – Interview with Edward Bianchi is an all-new 38-minute interview with the director. The director's interview has some overlap with Biehn's interview with the Stapleton story (but not the extent as Biehn did). Bianchi discusses a huge accident that happened during production. Also some tidbits about composer Pino Donaggio, how Lennon's assassination affected the box office and the film's reception and a gallows humor comment from Joel Silver. It's an interesting interview.
Fanning the Flames – Interview with Editor Alan Heim is an all-new 18-minute interview with the editor. Heim discusses how he moved from All The Jazz to The Fan and how that came about and his need to work. How he tried to get out of the project but could not once the film changed from a traditional thriller to a thriller-horror hybrid. A discussion of how Bacall's attitude (which is confirmed by both Biehn and Bianchi. Heim is great about the technical discussion about editing, editing with coverage, and the less than great footage of the song and dance scene. Heim more than anyone approaches the subject of the film being a cult item for the gay community.
Rounding out the special features is a Theatrical Trailer, TV Spots and a Still Gallery. The 2-minute trailer is effective, the 3 30-second TV spots less so, and the still gallery runs automatically and can be paused but not navigated.
The Final Thought
The Fan is a mixed bag that may not please either fans of psychological thrillers or fans of horror films. Scream Factory's special features do make this an instant-purchase for any fan of the film.


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"He said, 'I don't know if I should go, I'm from the U of S, there's going to be people from Harvard there, I won't know anything,''' recounted Szafron, an assistant professor in the School of Public Health and director of its collaborative biostatistics program.
"I said, 'I guarantee if you go, you will be fine,'" Szafron said. "Thankfully, he took my advice. He came back and said, 'I'm so glad I went! Not only did I know as much as they knew, I ended up helping people who were in Harvard, Johns Hopkins, or Yale. They were just shocked that they had never heard of where I was from, yet I knew more than them.'"
"That's the quality of our students."
As Master of Public Health (MPH) practicum co-ordinator, Szafron was one of the faculty presiding over and judging the School of Public Health Annual Poster Day held in the D-Wing Atrium of the Health Sciences complex on October 23. The event featured 59 posters from MPH practicum students as well as two dozen posters from the thesis-based graduate programs.
Thesis-based graduate programs (epidemiology, vaccinology and immunotherapeutics, and collaborative biostatistics) projects covered a wide range of public health issues. A few examples were lifestyle factors contributing to heart disease, risks of petroleum contamination of soils, economic burden of dental care on families, and the challenges of delivering cervical cancer screening and prevention programs in rural Colombia.
"I am happy to see the dedication and the hard work our graduate students have put in conducting and communicating their research findings," said Dr. Suresh Tikoo, professor in the School of Public Health and interim assistant executive director (research) at the school.
Zhubing Li, a PhD student in vaccinology and immunotherapeutics, is looking at how the body tries to defend itself against the hepatitis C virus (HCV), under the supervision of VIDO-InterVac research scientist Qiang Liu. HCV is a chronic liver disease that afflicts about 250,000 Canadians. Specifically, Li looked at a protein called PCSK9 which is naturally produced in the body.
The good news: PCSK9 helps protect cells by deactivating the receptors the virus uses to grab a hold of the cell before it invades. Plus, the body can be induced to produce more PCSK9. The bad news: this is probably not a good idea.
"Actually the upregulation of this protein is kind of harmful to your body, because if you upregulate this protein, it will downregulate the low-density lipoprotein receptors, and that will increase the plasma cholesterol level," Li said, explaining hers is basic research to find out what is happening and how.
"So far we have shown that PCSK9 can inhibit HCV replication and now we want to identify the mechanism involved," she said. "It's still a long way to go to an actual anti-HCV therapeutic."
Master's student Xueying (Elsie) Zhang, also in the vaccine and immunotherapeutics program, is working on another high-profile disease, HIV, the virus that causes AIDS. One of the challenges in research to develop vaccines against the disease is to come up with a safe, economical, non-human model to study the virus over time.
"HIV-1 is a chronic infection in AIDS patients, so we want to build a chronic infection model in mice," Zhang said. She explained initial results testing a vaccine on these mice are promising, and should help contribute to efforts to eventually develop a human vaccine.
Other Research Day projects focused on people's behavior, such as how they get around. Epidemiology graduate student Dana Ramsay is taking advantage of a lucky accident in the Saskatoon Household Travel Survey, commissioned by the City of Saskatoon, Warman and Martensville.
"They were looking at different travel behaviours, how different people take the bus, what routes, how long, but what they did is also collect information on health," Ramsay said.
This made the survey a rich source of data for studying active transport—that is, walking, bicycling and transit— and its effect on people's body mass index (BMI) as a measure of health. Ramsay uncovered that, as one might expect, active travel was favourably associated with BMI.
However, a different picture emerged once she looked, not only at income, but also education, employment levels and social deprivation (e.g. single parents and people living alone). Once these factors were considered, Ramsay found active travel had a beneficial association with BMI—but only for higher income groups.
"For the three highest income categories, we see lower BMIs, so active travel appears to confer a benefit," she said. "The lowest income category—that's below $25,000 in household income—the data show they are not benefitting from using active travel."
Ramsay explained the findings could guide policy makers to ensure programs to encourage active travel do not leave anyone behind.
Another behavioral question, people's reluctance to get vaccinations, is something Vaccinology and Immunotherapeutics PhD student Stephanie Ortynsky hopes to explore. This reluctance, formally referred to as vaccine hesitancy, is an increasing global health concern, with diseases such as whooping cough and measles making a comeback due to insufficiently vaccinated populations.
Ortynsky explains the WHO (World Health Organization) and UNICEF have recognized the problem and added two questions to their biennial survey on vaccines, supplied to all 195 WHO member countries. Of the 180 that responded, 131 reported vaccine hesitancy and identified people's reasons.
"Things like religion, culture, gender, socioeconomic factors—many countries reported that's what they were coming across when they were looking at recommendations and why people were hesitant," Ortynsky said.
The narrative also differs greatly by country. For example, in some African countries some people think if they get vaccinated they won't be able to have kids, while in Canada, it is often a question of people demanding vaccination be an individual choice. Ortynsky said initial findings show vaccine hesitancy is on the rise globally, and more study is needed to develop strategies to combat the trend.
"It's still in its infancy that they're actually monitoring it at a global level," she said.
Dr. George Mutwiri, interim executive director of the School of Public Health, emphasized the "very high quality" of the student research presented.
"This is a reflection of the ability and commitment of our students and the leadership of our faculty," he said.
School of Public Health Poster Day
Michael Szafron likes to share an anecdote from one of his students who was intimidated by attending a course at Johns Hopkins, one of the top medical schools in North America.
"He said, 'I don't know if I should go, I'm from the U of S, there's going to be people from Harvard there, I won't know anything,''' recounted Szafron, an assistant professor in the School of Public Health and director of its collaborative biostatistics program.


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Gabe Zichermann's new book rocks.
Game-Based Marketing: Inspire Customer Loyalty Through Rewards, Challenges, and Contests
What i like is his book is full of real world suggestions that are easy to implement instead of esoteric ideas. You know how when you read some books you think ok...but how does this apply to me - not this book, by the end of every chapter you realise hmm thats interesting i could use this example here etc.
According to this Venture Beat article - http://venturebeat.com/2011/04/14/by-2015-50-percent-of-companies-will-embrace-gamification-gartner-says of companies will use it in some form or another by 2015 to encourage interaction between companies and their users/customer and even vendors.
We used some of his suggestions on http://www.LiveBaseballChat.com so will be interesting to see how well we can execute on Gabe Zichermanns ideas
Thursday, April 14, 2011
Gamification ...it's not a toy - Gabe Zichermann
Posted by Dean Collins at 7:23 AM
2 comments:
Dean, your comment above notes implementing certain suggestions into your site; any results that you care to share; did the game ideas work for you?ReplyDelete
What i really liked about the book (and others check out the comments on the amazon reviews) is that the book is full of really straight forward ideas you can implement right now eg not "esoteric" pie in the sky guidlines.ReplyDelete
We did fan walls (eg http://www.LiveFootballChat.com/fan/Jets ) and Top 10 rankings http://www.livefootballchat.com/lfbc/top10page.do based on direct examples i read about in this book .
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text: DuPz0r 5361 Report post Posted August 4, 2016 I've been watching this series for the last couple of weeks and i find it pretty cool so far. There's 4 seasons out, a 5th on it's way. I'm only just starting season 3 though. Has anyone else seen it? Quote Share this post Link to post Share on other sites


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"8/10", Beautiful vintage piece, one of the most wanted shirts of the club, all the Badges are stitched. It has some tiny bobbles or pulls (front and back) but Generally nice feel material.
This is a player issue shirt probably prepared or used by the player during that season. The sponsor is printed within material. The number on the back has wear and minor details. Please see the photos and do not hesitate to ask. Measurements: length: 28,75/30,70 inches (73/78 back cm), width (armpit to armpit) 24 inches (61 cm).
Vintage Jerseys Collectors DIFFICULT TO GET in these conditions and size. ANY OTHER QUESTIONS OR INFORMATION, you can request it.Normative: We do not sell to buyers with less than 5 qualifications. THE FEEDBACK IS VERY IMPORTANT TO US. WE WILL SOLVE YOUR ISSUE FASTER.
International Buyers - Please Note. This item is in the category "Sports Mem, Cards & Fan Shop\Fan Apparel & Souvenirs\Soccer-International Clubs". The seller is "trikotsjerseys" and is located in this country: ES. This item can be shipped worldwide.


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One picture is worth a thousand words but every picture of winters in Zermatt, Switzerland taken by Mikolaj Gospodarek, a Polish photographer is not less than a fairy tale in itself. Based out of Germany, Mikolaj has been on a photography tour of winter in the Alps for past few years and this year, he landed in the stunning alpine village of Zermatt in Switzerland.
Top 18 Places To Visit In Switzerland In Winter 2021 For A Memorable Vacay
Enjoying an awesome weather in Zermatt, Mikolaj takes the world through incredible winters in Zermatt via his incredible pictures that he shot everyday from 7am to 6pm for one full week by riding in the gondolas. Capturing the biggest peak of magnetic Matterhorn, Mikolaj portrays the best of winters in Zermatt in his pictures.
Out of all these, Gornergrat is his favorite spot as being at 3136 above sea level enabled him to witness not one, not two but a total of 29 incredible Swiss peaks. Treating your eyes with a bit of sunshine, frost and breathtaking panoramas, here's Zermatt for you through the lens of Mikolaj Gospodarek.
Take a look!
1. High On Adrenaline At Breithorn
2. Happiness Is… Watching Sunrise At Weisshorn
14 Spectacular Switzerland Tourist Attractions That Are Worth A Visit In 2022!
3. An Alpine Ibex Chills In The Hills Of Gornergrat
4. A Piece Of Heaven At Weisshorn
5. This Is What Paradise Must Feel Like At Matterhorn
10 Best Ski Resorts In Switzerland
6. Mountains Are Calling & I Must Go!
7. Gornergrat Railway Joyride
8. There Is Beauty Everywhere, You Just Have To Look Around!
A Fantastical Honeymoon Trip In A Fairytale World Of Switzerland
9. This Is Where You Find Solace
10. A Place Straight Out Of Dreams!
11. Heard Of A Glacier Palace Ever?
A Fantastical Honeymoon Trip In A Fairytale World Of Switzerland
12. Survival Of The Fittest In Alps
13. Who Wouldn't Want To Wake Up To This?
14. Mighty. Majestic. Matterhorn!
15. This Is How Switzerland Fills Your Soul!
Wanderlusting already? Customize your own thrilling Switzerland tour package with TravelTriangle and make some room for that adrenaline rush!


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The Coaching Program
"They guide, they provide, and they never leave your side". Rebecca Miller - AFLW Richmond Football Club player and past MDCC student
Sports coaches assist athletes by helping them to perform at their best. They are responsible for analysing performances, instructing in relevant skills and providing encouragement. This assistance is not only provided in relation to sporting pursuits, but also to the athletes' lives in general.
These principles are aligned with the Coaching Program at Mater Dei Catholic College, however, instead of working with athletes, the coaches work with students to assist them to feel good and function effectively during their time at the College.
"Not everything should be done on your own... You need someone to bounce ideas off, you need someone who can support and guide you, and you also need someone to help keep you grounded and give you perspective". Harry Cunningham - AFL Sydney Swans Football Club player and past MDCC student
Students have the opportunity to book collaborative planning conversations with their chosen coach to map their way forward with a focus on academic achievement, future direction and life balance. The coaches act as a wellbeing support resource which caters to the individual circumstances of each of the members of our Mater Dei community.
Bookings can be made through the Coaching Tile in Canvas.


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This page has been archived and is being provided for reference purposes only. The page is no longer being updated, and therefore, links on the page may be invalid.
|Read the magazine story to find out more.
See our information kit to find out more.
New Poultry Litter Applicator Holds Promise for Chesapeake Bay AreaBy Don Comis
August 2 2010
A machine that can inject dry poultry litter and composted cattle manure below the soil surface in pastures and no-till fields is on order from a research coalition across five Chesapeake Bay states: Delaware, Maryland, Virginia, Pennsylvania and New York. There are currently no machines on the market that can do this.
The coalition is led by U.S. Department of Agriculture (USDA) scientist Peter Kleinman. He works at the Pasture Systems and Watershed Management Research Unit operated by USDA's Agricultural Research Service (ARS) in University Park, Pa. ARS is USDA's principal intramural scientific research agency.
Kleinman and his research partners at Pennsylvania State University at University Park and Virginia Polytechnic Institute and State University at Blacksburg received a $786,000 grant to test four prototypes of the Poultry Litter Subsurfer.
Soil scientist Dan Pote, at the ARS Dale Bumpers Small Farms Research Center in Booneville, Ark., invented the Subsurfer, which injects litter with the minimal soil disturbance required by no-till farmers who do not clear their fields of crop residue before planting a new crop.
In tests on Arkansas pastures, Pote found that the Subsurfer lowers nutrient runoff and ammonia emissions by at least 90 percent, while increasing forage yields. Kleinman and colleagues documented lower phosphorus runoff and ammonia loss and greater corn yields in Maryland and Pennsylvania. Jack Meisinger, an ARS soil scientist in Beltsville, Md., also reported lower ammonia losses.
As a collaborative project, Pote led development of the Subsurfer, while agricultural engineer Tom Way's team at the ARS National Soil Dynamics Laboratory in Auburn, Ala., focused on developing a different prototype with adjustable row spacing for litter application in row-crop systems and pastures.
The two machines have such different delivery systems that Pote and Way sought different patents. Pote's Subsurfer uses a unique auger system that crushes litter and distributes it to soil trenches, allowing precise control, including very low rates not previously feasible. His tractor-drawn Subsurfer carries up to five tons of litter and simultaneously opens eight trenches (two inches wide and three inches deep), with one foot between each trench.
ARS is applying for U.S. and international patents on Pote's Subsurfer. One company has applied for a license to commercialize it. Way's invention has been patented.
Read about this and other research to help protect the Chesapeake Bay in the August 2010 issue of Agricultural Research magazine.


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The GatorWell Health Hut is a peer-based outreach program committed to encouraging and empowering University of Florida students to achieve overall health, happiness and success. Staffed by our trained Wellness Educators, we work to help UF students' development of skills related to their 8 Dimensions of Wellness to provide balance and support optimal wellness and ultimately student success.
When visiting the Health Hut, peer Wellness Educators will provide:
- Thought provoking information about relevant health and wellness topics through rotating bi-weekly health messages and accompanying skill-based activities.
- Distribution of related Health Behavior Tools related to the bi-weekly messages in order to support your behavior change journey.
- Campus resources and referrals to best connect students to other health and wellness campus partners.
The Health Hut travels across areas of campus to highly trafficked areas in order to best reach students. Please check out our calendar to find the Health Hut's current location.
For more information please contact the Health Hut Program Coordinator, Samantha Evans.
Interested in collaborating with GatorWell or having the Health Hut at your next event? Please check out our Collaborate with GatorWell page for more information.


text: Branding season commences. We have most of our baby calves on the ground. We have to pick the right days for branding–after the calves are big enough to not be too stressed, but not so big that they will cause the branding crew too much stress. These calves have reached that "Goldilocks Moment". In the last few days, we have branded one set of calves on the Home Ranch, and one set of calves in the desert at the Powder Flat Headquarters. We even had a photographer from the Library of Congress, Carol Highsmith, to document the great American branding. As usual, we had child labor on hand.


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Swimming pool builder, Jim Chandler, completes a beautiful swimming pool and spa in Roseville. When I first saw the beautiful landscaped yard I was impressed by the established mature peaceful park like setting. Features included in the landscape setting were a stone bridge which the homeowner wanted to keep and an existing curved stone and […]Read More
Jim Chandler PoolsCustom Swimming Pool Construction From A Trusted Pool Builder
Lic. #585004 Call Now: (916) 624-5296


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AttractionsBrou Monastery
Located just next to Bourg-en-Bresse in the town of Brou is the Brou Monastery. Its location, it is on the way to the Alps, make it a nice place to stretch your legs and discover a 16th monastery built to commemorate the life Philibert le Beau.
The Brou Monastery was the inspiration of Margaret of Austria who built it to as tribute to her husband, Philibert le Beau who died just three years after marrying Margaret. The monastery was designed to house the tombs of Philibert, Margaret and Philibert's mother, Margaret of Bourbon.
It was actually Margaret of Bourbon who had the idea of building a monastery at Brou. But she died before construction could be started.
Construction of the Brou Monastery, as we know it today started in the early 16th century and took about 25 years. Sadly, Margaret did not live to see her dream completed as she died in 1530.
The monastery includes a gothic church, three sets of cloisters and a museum. It all can be visited in about two hours and has few stairs (except for the museum, which is on two levels). There are free wheelchairs for anyone with limited mobility.
The museum is in, what were the monks' rooms and cells. It has a selection of paintings from the 15th to the 19th century along with sculptures. The church is the centerpiece of the Brou Monastery. It has good indoor natural lighting. This aluminates it's many features. Which include, sculptures, stained glass windows and of course the three tombs. The tombs are located near the altar and can't be missed.
If you are in the area or just passing through the Brou Monastery makes a nice stop. Behind the monastery is a park where you can picnic if you like.
The monastery has documentation in English.
The Brou Monastery is accessible from the A40. You can use either exits 5 or 6 and then follow the signs marked 'Brou'. The monastery is only about a 20 minute once you exit the A40.
Related Links:


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SOUTH PADRE ISLAND, Texas (ValleyCentral) — The first official lighting of the Port Isabel Lighthouse in 117 years will take place on Dec. 9.
Funded and coordinated by The Texas Historical Commission, the reproduction of a 3rd Order Fresnel Lens will be fitted into the lantern room at the top of the lighthouse, said the city of Port Isabel.
The $200,000 lens will offer visitors a glimpse into the past of the lighthouse and the service it provided to the Gulf Coast.
"The Port Isabel Lighthouse is a major heritage attraction for visitors, offering a glimpse into the past of the community and its maritime history," said Mark Wolfe, executive director of the Texas Historical Commission. "Thanks to the support of the Texas Legislature and our statewide leaders, visitors will now see the lighthouse much as it would have appeared more than a century ago."
While working at the lighthouse, Dan Spinella and the team at ArtWorks Florida have replicated a functional Fresnel lens, pedestal, and lamp, based on a 19th-century plan showing the design of the lens-specific to the site, said the city's news release.
The lighting will take place from 6:30- 8 p.m. at 421 East Queen Isabella Blvd.


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Between work, shuttling the kids to and from school and activities, meeting up with friends, and walking the dog, finding healthy dinner ideas that are easy to whip up on the weeknights isn't always your top priority.
Yet fast food is notoriously unhealthy, and even getting takeout from your favorite restaurant can be filled with nutrition pitfalls. Not to mention the fact that eating "out," even if it's on the go, can really take a toll on your pocketbook.
Choosing healthy dinner ideas is one of the best ways you can nourish your body — and your soul. With a little planning, some pantry staples, and maybe a bit of weekend prep work, you can come up with inexpensive, easy to prepare healthy dinner ideas to last you all week long.
What are you waiting for? Grab your shopping list and let's plan some delicious meals!
Make Your Own Grain Bowls
Grain bowls, sometimes called "Buddha Bowls," are all the rage these days — and for good reason. They are easy to prepare, they are infinitely customizable, and they're generally inexpensive to make.
Not surprisingly, a grain bowl starts with whole grains. Skip the Minute Rice and choose brown rice, barley, quinoa, farro, or even wheatberries.
Next, load up your bowl with veggies. This is the perfect time to clean out your crisper and use up some odds and ends: a few bites of grilled zucchini from last weekend's cookout, half a bell pepper, some shelled edamame.
Now add some protein. The leftover approach works well here, too, so dig out the last of the rotisserie chicken, sauteed shrimp, or baked tofu. A poached or soft-boiled egg is perfect in a pinch.
Top your grain bowl with your go-to dressing, or drizzle with pesto, harissa, hummus, tahini sauce, hoisin…the options are endless.
Other additions that can elevate a bowl from blah to extraordinary include sliced avocado, fruit, nuts, pickled vegetables, nori, and fresh herbs.
Choose Noodles
Grains not your style? No worries. You can assemble a delicious noodle bowl that incorporates all the fun healthy dinner ideas of a grain bowl. Steer clear of white pasta and choose nutty soba noodles, light, and refreshing rice noodles, or even "noodles" made from spiralized vegetables.
Noodle bowls can be tossed with a vinaigrette or light sauce, or warm the whole thing in some chicken or vegetable broth for a cozy, slurpable soup that's far better than anything you'd get out of a can.
Beans, Beautiful Beans
Millions of people around the world regularly make a meal of beans and rice in some form.
Try red beans (with just a touch of sausage for flavor), black beans, pintos, black-eyed peas or pigeon peas, to name just a handful. Dry beans are generally a better choice because they're much cheaper; canned varieties can also be high in sodium. Cook up a bag of beans in your pressure cooker or crockpot and freeze them in 1- or 2-cup quantities to make meal prep easy.
Brown rice is the classic partner to any cooked bean, but one of the best healthy dinner ideas is to make a soupier preparation, like frijoles borrachos, and serve it with cornbread or corn muffins.
Simple Stir Fry
Don't be intimidated by stir fries. While they may seem complicated, there are many simple recipes that incorporate only a few ingredients. Try shrimp with broccoli or snap peas, for instance. You can also find plenty of stir fry shortcuts — bagged vegetable mixes, for example, eliminate much of the prep work that gives stir fry dishes a bad rap.
While they may seem complicated, there are many simple recipes that incorporate only a few ingredients. Try shrimp with broccoli or snap peas, for instance. You can also find plenty of stir fry shortcuts — bagged vegetable mixes, for example, eliminate much of the prep work that gives stir fry dishes a bad rap.
Another quick stir fry fix is to use a jarred sauce. While using a stir-fry sauce (or an Indian or Thai simmer sauce) is certainly convenient, and won't require you to buy a lot of exotic ingredients that you might never use again (hello, tamarind paste and Szechuan pepper), read the ingredients list carefully. Some of these sauces can be high in sugar and sodium content.
One way to achieve the ease of a prepared sauce while still keeping things healthy is to make your own in advance. Devote a few weekend hours to whipping up a batch of peanut sauce, teriyaki sauce, or green curry sauce. Most of these will freeze well, or refrigerate them in a tightly sealed mason jar for a week or two.
Breakfast for Dinner
Who doesn't love to mix it up and make breakfast for dinner? For healthy dinner ideas, however, you'll have to ditch the greasy sausage and the cinnamon rolls.
Multigrain pancakes with fresh fruit are fun for kids and adults. Or whip up a quick batch of breakfast burritos using whole-wheat tortillas, low-fat cheese, scrambled eggs and whatever vegetables suit your fancy (we recommend sauteed poblano chiles, mushrooms, or spinach).
Super short on time? Avocado toast to the rescue. It's filling, provides healthy fats, and tastes great to boot. Use a whole-grain bread, and add extra veggies if you have them on hand. A poached egg perched on the smashed avo is one of the trendiest healthy dinner ideas around!
Build A Better Salad
Let's face it — eating the same old salad can get boring. Worse, if you buy a head of lettuce and don't eat it up quickly enough, it'll go bad. That's a big waste of money.
The solution? Build a better salad with vegetables that can hang out in your fridge without being the worse for wear. Cabbage, whether white, red or a Chinese variety like Napa, makes coming up with healthy dinner ideas a snap. Shred it finely, add other veggies like bell peppers, carrots, and celery, and then toss with dressing. A cabbage-based salad stands up well to a heartier dressing, like peanut sauce.
Cabbage, whether white, red or a Chinese variety like Napa, makes coming up with healthy dinner ideas a snap. Shred it finely, add other veggies like bell peppers, carrots, and celery, and then toss with dressing. A cabbage-based salad stands up well to a heartier dressing, like peanut sauce.
Prewashed kale and other greens also tend to last longer than lettuce and pack a nutritional punch. Make a delicious kale or collard greens Caesar, and you may never return to romaine again!
Finding healthy dinner ideas doesn't have to mean scouring cookbooks for new recipes or spending tons of money on prepared foods from your local organic market. A little imagination and creativity, in addition to a fridge stocked with vegetables and a few healthy staples, will help you plan better meals, save money, and even lose weight.


text: SIX Newsroom Media Releases The SMI Celebrates 30th Anniversary with Exhibition One of the predecessor organizations of SIX launched the SMI 30 years ago. On 1 July 1988, the then Association of Swiss Securities Exchanges published the Swiss Market Index® (SMI) for the first time. To mark this anniversary, the Swiss Finance Museum in Zurich-West is holding a year-long special exhibition from 1 June 2018 onward: "30 years SMI: stock market indices explained". Published at 31 May 2018 Medium Media A great deal has happened since the SMI was launched. Over the course of its eventful history, the index has risen almost sixfold. The benchmark now covers around 80% of the Swiss equity market's entire capitalization. As the Swiss stock market's best-known reference index, it is still used by providers of financial products from across the world. The SMI is also used as a reference value and underlying for index-based investments such as ETFs. The SMI is as important as ever: the SMI stocks of Nestlé and Novartis have the two highest market capitalizations in Europe and along with Roche are among Europe's top five. As you would expect, events over the past 30 years have left their mark on the SMI's performance over time, which provides a portrayal of defining events and illustrates the context in which corporate mergers, changes to the index rules and product launches took place. You can find a concise selection of historical highlights here: www.six-group.com/smi-30years The Swiss Finance Museum is marking the anniversary by re-telling the history of the SMI while explaining the origin and significance of stock market indices in general. For example, a share certificate of General Electric Company takes us back to the era in which the Dow Jones indices and the Wall Street Journal were founded. The new special exhibition also uses touchscreen installations, 3D elements and an extended free smartphone audio guide to outline the trend in the Swiss economy over the last 30 years. In addition, historical securities from eight selected Swiss companies that have played a key role in the country's commercial life give visitors an insight into the recent past of the Swiss economy. The exhibits are part of the world's largest and most important collection of historical securities. Pictures of the exhibition can be found here: www.finanzmuseum.ch/medien Any questions? Please do not hesitate to contact Julian Chan. Julian Chan Deputy Head Media Relations +41 58 399 2508 Send Inquiry Generate PDF SIX SIX operates and develops infrastructure services in the areas of securities, payment transactions and financial information with the aim of raising efficiency, quality and innovative capacity across the entire value chain of the Swiss financial center. The company is owned by its users (127 banks). With a workforce of some 4,000 employees and a presence in 23 countries, it generated operating income in excess of CHF 1.9 billion and group net profit of CHF 207.2 million in 2017. www.six-group.com Swiss Finance Museum The Swiss Finance Museum is the first of its kind in the banking nation of Switzerland. It houses a multimedia exhibition on the fundamental significance of the financial market and its infrastructure for our everyday life, and illustrates the origins of our economic system. It focuses on stock exchange trading, the capital market, financial instruments and payment transactions. The museum is run by the foundation Collection of Historical Securities, a charitable foundation established by SIX in 2001. The foundation owns one of the most significant collections of historical securities worldwide with some 10,000 securities from more than 150 countries. The highlights of this collection are exhibited in the museum. www.finanzmuseum.ch


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Arsenal are 5/11 with GunnersGaming to win their London derby against QPR at Loftus Road on Saturday afternoon.
The Gunners have never won at Loftus Road in the Premier League era, but come into the game in top form having lost just one of their last 13 matches in the top flight.
The home side were relegated last weekend and have gone 275 minutes without scoring in the Premier League. Additionally, they are without a win in any of their last six matches.
However history suggest that this may well be a low scoring affair as Arsenal have scored exactly one goal in seven of their last eight league meetings with Harry Redknapp's side.
That makes under 2.5 goals well worth considering at odds of 23/20, as well as a 1-0 win for Arsenal at 17/3 in the correct score market.
Theo Walcott, who got back on the goals trail when netting in last week's 1-1 draw with Manchester United, looks a worthy favourite at 4/1 in the first goalscorer betting.
However a bit of value could be Aaron Ramsey at 12/1 as he has had more shots without scoring than any other player in the Premier League.
Follow GunnersGaming on Twitter - @GunnersGamingBB
Podolski to score first @ 9/2
Copyright 2023 The Arsenal Football Club Limited. Permission to use quotations from this article is granted subject to appropriate credit being given to www.arsenal.com as the source.


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Market News
It is a tale told by an idiot. Full of sound and fury, signifying nothing.
A famous lamentation about life from a medieval Scottish king seems to have captured investor sentiment about the possible impact of anti-trust congressional hearings held last Wednesday. As the CEOs of Amazon, Apple, Google parent Alphabet, and Facebook took a bipartisan bombardment about their potentially anti-competitive and monopolistic business practices for over five hours, the stocks of those companies advanced 1% to 2% in share price. The following day all four companies released quarterly financial reports that smashed Wall Street expectations, leading to further impressive stock gains.
Back in 2013, the acronym FANG (Facebook, Amazon, Netflix, and Google) gained currency in investing circles as a pithy way to refer to the technology companies that seemed to dominate market news. By 2017, Apple was added to the group to form the less-grammatical FAANG. This year, Microsoft's resurgence has seen the ill-formed FAANGM acronym gain popularity. No matter how you abbreviate them, a small group of big tech companies have only grown more prominent during the coronavirus lockdown and their outperformance has lead to an outsized dominance in the marketplace. The societal and political implications of their stature is best left to other publications, but we think it is important to look at how unusual this market concentration is from a financial perspective.
As of July 31, 2020, Apple accounted for about 5.4% of the US stock market by market capitalization ( the total dollar value of the company's public shares). The top 5 largest companies (Apple, Microsoft, Amazon, Facebook, and Google) made up about 19% of the US stock market. Is such concentration unusual? Well, historically no. The graph below from Dimensional Investing and the University of Chicago shows the overall market capitalization (or market cap) % of the largest 10, the largest 5, and the overall largest stocks.
At the end of 1967, the technology titan of the time, IBM, represented a larger portion (5.8%) of the market than Apple does today. In that year the top 5 stocks accounted for over 20% of the total market capitalization. In fact, the graph below shows that over the last 100 years, there has usually been a more concentrated market then there is now, and the hi-tech heavyweights of the time (whether AT&T, General Motors, General Electric, or IBM) were the main drivers of stock market growth.
So the current situation is not atypical historically. However, will the current top companies be able to leverage their dominance on the internet to continue to increase their market concentration? Well, the answer to that question deserves another quote from the play Macbeth:
If you can look into the seeds of time, and say which grain will grow and which will not, speak then to me.
Read More >>


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New Heffy Doodle Release – Coming Soon!
Hello everyone!
It's Lesley here – and I am here to share some wonderful news! That's right – we have a NEW RELEASE right around the corner! YAY!
Grab a pen and mark your calendars, because we have 6 new stamp sets and 4 new sets of dies that will be released on 1 November – just little over a week away! We are so excited!
Be sure to visit us here on the blog to see some sneak peeks on 29th, 30th and 31st October, and then the full reveal will be shown on 1st November. We will also have the new release available to buy from 1st November via our Heffy Doodle shop and Amazon store.
What's more, we are working hard to ensure that our retailers will have the stock ready for the 1st November launch as well. YAY! You can check out all our retail partners here.
To celebrate our new release, we will also be having a blog hop on Saturday 4th November- with lots of inspiration – and I can't wait to show you all the amazing projects that our Heffy Doodlers have been making!
Why not sign up to our newsletter so you won't miss out on any important updates – as there will be LOADS of exciting things happening and you won't want to be out of the loop!
HAPPY CRAFTING my friends,
2 Comments on "New Heffy Doodle Release – Coming Soon!"
I'm excited to see the next release of Heffy Doodle stamps! Your stamps are so much fun, Lesley! I love that you think of all of the little details and that the first release included so many small items to help convey the theme with or without words. I think those little stamps help add personality to each of the animals.


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$88.40 $97.68
2.4G full-scale four-wheel drive off-road climbing car, 1:14 has both climbing and high-speed off-road functions.
Equipped with built-in shock absorbers for smoother off-road performance.
The product uses 2.4GHz technology to support multiple high-speed cars at the same time.
The product is a full-scale remote control four-wheel drive system.
The product has shock absorption and anti-collision design.
Item type:toy
Servo:2G 5line Package:44*20*20.5cm Color:Yellow green Brand:SUBOTECH Battery:NI-cd 6v 500Mah Charge:USB Frequency: 2.4G ESC: Brush Using time:20min Max speed:15km/h Control way:Radio control Control distance:50m Charging time:150min Model:BG1522 Transmitter battery:3x1.5AA(not included) Channel:2CH Size:33*16*17cm
Configure the full-scale steering servo, the remote control configuration steering fine-tuning, steering fine-tuning. Configure the front and rear bridge structure of the three differential gearbox. Configure 2 sets of LED headlights and configure a hidden battery compartment. Configure removable tool hammer, tool axe, fuel tank, spare tire and other decorative items. Configure anti-collision collision protection frame. Configure the bridge steel frame girder vehicle structure.
Packing list:
1x RC Car 1x Transmitter 1x Charger 1x Battery 1x English manual
The colors deviation might differ due to different monitor settings.


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The Princesa Resort was built in 1999 in Puerto Penasco, Rocky Point, Mexico located on Sandy Beach and the Sea of Cortes. With lush, well maintained grounds, 5 buildings and a clubhouse, the Princesa resort and been constructed to maximize the direct views of the ocean and beach from all points in the Resort, giving owners and renters a feeling of luxury and privacy.
Featuring three swimming pools, fitness center, children's pool, clubhouse and three Jacuzzis, the Princesa has amenities for every member of the family. Volleyball court, resort bar and restaurant ,and palapas for relaxing in the shade right on the white sandy beach
Condominiums as the Princesa are available in 1, 2, and 3 bedrooms. Ground floor condominiums have direct access to the resort grounds from their walkout patio. The upper floor units offer incredible views of the ocean, old port, sunset and resort grounds. Each condos has a fully equipped kitchen, has it own laundry room, walkout patio and includes TV/VCR/DVD and cable.
Contact Kyle Wood at Playa Sonora Realty for additional information on properties at The Princesa Resort. If you are interested in selling your unit, please contact Kyle for your private listing consultation.
Contact Kyle Wood at 480-297-8079
Email Kyle Wood at email@example.com
Request to be updated on Rocky Point Condos for Sale
Need a Floor Plan or Building Location at Princesa - See our Information Section
Site customization by The Real Estate Web Guy


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Last month, Foursquare announced a partnership with Voice Media Group, publishers of regional magazines like Miami New Times, L.A. Weekly, and Village Voice. The partnership brings Voice Media Groups's products like "Voice Places" and "Best of" to Foursquare's location services.
Today, that partnership goes live as VMG content should now be integrated into Foursquare.
"A couple weeks ago, we announced our partnering with Voice Media Group to bring you a ton of great tips, lists, and tens of thousands of events related to arts, culture, and food in cities across the U.S. Today, you can see those events when you open up the app. To find great goings-on near you, just open up Explore and tap through to your favorite places. You'll see things like gallery openings and author appearances, along with the movies, concerts, and sporting events we already show you," says Foursquare in a blog post.
The new data from "Voices Places" and "Best Of" will be seen in over 50 different venue categories spread across 11 different markets. Those markets include New York, Los Angeles, Denver, Phoenix, Houston, Dallas, St. Louis, Miami, Minneapolis, Palm Beach, and Orange County.
So, when you're browsing Foursquare and come across a venue in these major cities, you may just see some additional info like reviews, upcoming events, and other cool tips.