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IKKε inhibits PKC to promote Fascin-dependent actin bundling
Tetsuhisa Otani, Yosuke Ogura, Kazuyo Misaki, Takuya Maeda, Akiyo Kimpara, Shigenobu Yonemura, Shigeo Hayashi
Development 2016 143: 3806-3816; doi: 10.1242/dev.138495
Tetsuhisa Otani
1Laboratory for Morphogenetic Signaling, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan
ORCID record for Tetsuhisa Otani
For correspondence: otani@nips.ac.jp shayashi@cdb.riken.jp
Yosuke Ogura
Kazuyo Misaki
2Electron Microscope Laboratory, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan
Takuya Maeda
Akiyo Kimpara
Shigenobu Yonemura
Shigeo Hayashi
3Department of Biology, Kobe University Graduate School of Science, Kobe, Hyogo 657-8501, Japan
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Signaling molecules have pleiotropic functions and are activated by various extracellular stimuli. Protein kinase C (PKC) is activated by diverse receptors, and its dysregulation is associated with diseases including cancer. However, how the undesired activation of PKC is prevented during development remains poorly understood. We have previously shown that a protein kinase, IKKε, is active at the growing bristle tip and regulates actin bundle organization during Drosophila bristle morphogenesis. Here, we demonstrate that IKKε regulates the actin bundle localization of a dynamic actin cross-linker, Fascin. IKKε inhibits PKC, thereby protecting Fascin from inhibitory phosphorylation. Excess PKC activation is responsible for the actin bundle defects in IKKε-deficient bristles, whereas PKC is dispensable for bristle morphogenesis in wild-type bristles, indicating that PKC is repressed by IKKε in wild-type bristle cells. These results suggest that IKKε prevents excess activation of PKC during bristle morphogenesis.
Drosophila bristles are external sensory organs involved in mechanosensation and chemosensation that are formed by the elongation of single cells during the pupal stage (Lees and Waddington, 1942; Tilney and DeRosier, 2005). We and others have been investigating the role of a serine threonine kinase, IκB kinase ε (IKKε, also known as Ik2), in bristle cell morphogenesis (Oshima et al., 2006; Shapiro and Anderson, 2006; Koto et al., 2009; Bitan et al., 2010; Otani et al., 2011, 2015). IKKε is locally activated at the tip of growing bristles, and acts as a signaling center for bristle cell elongation by coordinately regulating cytoskeleton organization and vesicle trafficking (Bitan et al., 2010; Otani et al., 2011, 2015). IKKε regulates the shuttling movement of recycling endosomes through phosphorylation of the Rab11 effector molecule Nuclear fallout (Nuf) (Otani et al., 2011). Inhibition of Nuf can restore the Rab11 accumulation phenotype in IKKε-deficient bristles, but does not rescue the actin bundle phenotype, suggesting that IKKε regulates actin bundle organization through a distinct signaling pathway that remains to be characterized (Otani et al., 2011).
In Drosophila bristles, parallel actin bundles run beneath the cellular cortex throughout the bristle shaft (Overton, 1967; Appel et al., 1993) and are assembled through the sequential action of two actin-bundling proteins: Forked and Fascin (also known as Singed) (Tilney et al., 1995, 1996, 1998; Wulfkuhle et al., 1998). Forked initially bundles the newly generated actin filaments in the tip region, whereas Fascin subsequently promotes the hexagonal paracrystalline packing of actin filaments (Tilney et al., 1995, 1996, 1998). The loss of either Forked or Fascin results in disorganized actin bundles accompanied by a gnarled morphology of the bristles, suggesting that the proper cross-linking of actin filaments is essential to maintain the morphology of the bristles (Lees and Waddington, 1942; Lees and Picken, 1944; Overton, 1967; Cant et al., 1994; Petersen et al., 1994; Tilney et al., 1995).
In this study, we sought to understand how IKKε regulates actin bundle organization in bristle morphogenesis. We demonstrate that IKKε regulates actin bundle organization by promoting Fascin-dependent actin bundling. Mechanistically, IKKε inhibits PKC, thereby protecting Fascin from PKC-dependent inhibitory phosphorylation. Interestingly, although excess PKC activation is responsible for actin bundling defects in IKKε-deficient bristles, PKC loss of function in a wild-type background yields no visible phenotype, suggesting that PKC activity is repressed in wild-type bristles by the action of IKKε. These results suggest that IKKε prevents undesired PKC activation during bristle morphogenesis.
IKKε regulates Fascin localization
Phalloidin staining of thorax macrochaetes revealed that actin bundles run in parallel along the long axis of developing bristles (Fig. 1A). The actin bundle organization was severely disorganized when IKKε is inactivated through mutation (IKKε1 or IKKεalice, strong loss-of-function alleles with missense mutations in the kinase domain), RNAi (IKKεRNAi), or the expression of dominant-negative IKKε (IKKεDN; IKKε[K41A], a point mutation in the ATP-binding loop) (Fig. 1B,C, Fig. S1A,B) (Bitan et al., 2010; Otani et al., 2011). Antibody staining revealed that Forked and Fascin, the two actin-bundling proteins involved in bristle morphogenesis, are localized along actin bundles in control bristles (Fig. 1D,G) (Cant et al., 1994; Petersen et al., 1994), but show discontinuous staining, which is most likely due to poor penetration of antibodies into the tightly packed actin bundles (∼8 nm distance between actin filaments when bundled by Fascin; Jansen et al., 2011). Intriguingly, Fascin localization on actin bundles was severely decreased in IKKε-deficient bristles (Fig. 1E,F, Fig. S1C,D). Nevertheless, cytoplasmic Fascin signals were detected in IKKε-deficient bristles, suggesting that IKKε regulates the localization but not the expression of Fascin (Fig. 1E,F, Fig. S1C,D). By contrast, Forked was able to localize along actin bundles in IKKε-deficient bristles (Fig. 1H,I, Fig. S1E,F). These results suggest that IKKε regulates actin bundling by regulating the localization of Fascin.
IKKε regulates Fascin. (A-C) Phalloidin staining of bristles. The parallel organization of actin bundles is disorganized in IKKε-deficient bristles. (D-F) Anti-Fascin antibody staining of bristles. Fascin localization along the actin bundles was severely reduced in IKKε-deficient bristles. (G-I) Anti-Forked antibody staining of bristles. Forked localization along the actin bundles was not perturbed in IKKε-deficient bristles. (J-M) Genetic interactions between IKKε, fascin [singed (sn)] and forked (f). (J-L′) SEM analysis shows that the reduction of forked significantly exaggerates the bristle morphology phenotype of IKKεDN bristles. Representative bristles are shown at higher magnification in J′-L′. (M) Quantification of the bristle morphology phenotype. The percentage of normal scutellar bristles is shown as mean±s.d. n=30 each. ***P<1.0×10−8 by two-tailed t-test. Scale bars: 10 µm in A-I; 100 µm in J-L. See also Figs S1 and S4.
To determine the functional relationship between Forked, Fascin and IKKε, we examined the potential genetic interactions between these proteins. The expression of dominant-negative IKKε resulted in a bent and hooked bristle morphology (Fig. 1J) (Oshima et al., 2006; Shapiro and Anderson, 2006; Koto et al., 2009; Bitan et al., 2010; Otani et al., 2011), whereas control bristles did not show any morphological defects (Fig. 1M). Further reduction of forked in IKKεDN bristles significantly exaggerated the bristle morphology phenotype, resulting in frequent bristle branching (Fig. 1L,M), suggesting that IKKε and Forked regulate bristle morphogenesis in a coordinated manner. By contrast, the reduction of fascin (singed) did not significantly modify the bristle morphology phenotype in IKKεDN bristles (Fig. 1K,M). Heterozygotes of either singed3 or forked36a did not show any visible bristle morphology phenotype (Fig. S4A,B). Taken together, these results suggest that IKKε and Forked coordinately regulate Fascin-dependent actin bundling during bristle morphogenesis.
IKKε regulates the hexagonal packing of actin filaments
The hallmark of Fascin-dependent actin bundling is the hexagonal packing of actin filaments (DeRosier and Tilney, 1982; Tilney et al., 1998). Transmission electron microscopy (TEM) analysis of the microchaetes on the dorsal thorax revealed that actin filaments are hexagonally packed in a paracrystalline manner in control bristles, indicating that Fascin cross-linking is present in control actin bundles (Fig. 2A-C, Fig. S1G,I) (Tilney et al., 1995). In IKKεRNAi microchaetes, the size and the number of actin bundles were not severely altered, although the hexagonal packing of the actin filaments was perturbed and the filaments were irregularly packed (Fig. 2D-F, Fig. S1H,J). These results indicate that Fascin-dependent actin bundling is compromised in IKKεRNAi microchaetes.
IKKε regulates the hexagonal packing of actin filaments. (A-C) TEM analysis of control bristles. Magnified views (B,C) of actin bundles show that the actin filaments are hexagonally packed in a paracrystalline manner. (D-F) TEM analysis of IKKεRNAi bristles. Magnified views (E,F) of actin bundles show that the actin filament packing is irregular, displaying liquid order. (G) Radial distribution function of control bristles shows multiple distinct peaks, indicating that actin filaments are spaced regularly. (H) Radial distribution function of IKKεRNAi bristles does not show clear peaks, indicating that actin filament spacing is irregular. Scale bars: 1 µm. See also Fig. S1.
The packing pattern of actin filaments was quantitatively evaluated. First, the centroids of actin filaments were determined, and the radial distribution function, which represents the probability of finding other actin filament centroids at a distance r from a given reference actin filament centroid, was calculated (Fig. S1K). In control bristles, the radial distribution function showed multiple peaks with 15-16 nm intervals, indicating that actin filaments were regularly spaced (Fig. 2G). The interval size was in agreement with the sum of actin filament diameter and the size of Fascin-dependent cross-links (actin filament diameter of 5-9 nm, Fascin size of ∼8 nm; Alberts et al., 2015; Jansen et al., 2011). By contrast, the radial distribution function of IKKεRNAi bristles did not yield clear peaks, suggesting that the actin filaments were irregularly spaced (Fig. 2H). Voronoi tessellation analysis also suggested that the hexagonal packing of actin filaments was disorganized in IKKεRNAi bristles (Fig. S1L-P). These results demonstrate that IKKε regulates the Fascin-dependent formation of paracrystalline actin bundles during bristle morphogenesis.
Fascin dynamically exchanges in the actin bundles
Fascin has been shown to be a dynamic actin cross-linker that rapidly exchanges within the actin bundles (Vignjevic et al., 2006; Aratyn et al., 2007; Hwang et al., 2015). To examine whether Fascin can dynamically exchange within paracrystalline actin bundles, we examined the dynamics of Fascin in the growing bristles. Fluorescence recovery after photobleaching (FRAP) analysis revealed that Fascin-GFP dynamically exchanges in the actin bundles of growing bristles. (Fig. 3A,C, Movie 1). By contrast, GFP-actin fluorescence recovered little over this timecourse, demonstrating that the actin bundle itself is stably maintained (Fig. 3B,C, Movie 2). These results suggest that Fascin dynamically exchanges within paracrystalline actin bundles during bristle morphogenesis.
Fascin dynamically exchanges in actin bundles. (A) FRAP analysis indicates that Fascin-GFP fluorescence recovered rapidly after photobleaching. (B) By contrast, GFP-actin fluorescence recovery was not observed within this timecourse. Yellow boxes indicate the regions that were photobleached in A and B. (C) Quantification of Fascin-GFP and GFP-actin fluorescence recovery. Fascin-GFP fluorescence recovered rapidly after photobleaching and ∼60% of Fascin-GFP molecules were mobile. The recovery rate was similar for the tip and shaft region. GFP-actin fluorescence did not recover within 90 s. The fluorescence recovery rates are shown as mean±s.d. n=4 for Fascin-GFP, n=3 for GFP-actin. *P<0.001 by two-tailed t-test, Fascin-GFP versus GFP-actin. Scale bars: 10 µm. See also Fig. S3, Movies 1-4.
Rab35 does not play a major role in regulating Fascin localization
To elucidate the molecular mechanisms underlying the IKKε-dependent regulation of Fascin, we examined the relationship of IKKε with known regulators of Fascin. Rab35 has been reported to interact with Fascin in a GTP-dependent manner to promote its membrane recruitment to regulate bristle morphogenesis (Zhang et al., 2009). However, contrary to previous observations, overexpression of Rab35[S22N] (GDP-bound form) did not affect bristle morphology (Fig. S2A) or Fascin localization (Fig. S2F) in our experimental conditions. Moreover, although Rab35[Q67L] (GTP-bound form) overexpression partially suppressed the phenotype of IKKεDN bristles (Fig. S2B-E), it did not restore Fascin localization along the actin bundles (Fig. S2G). These results suggest that IKKε regulates Fascin localization independently of Rab35.
IKKε regulates Fascin through control of serine 52 phosphorylation
PKC is known to phosphorylate Fascin at a conserved serine residue (serine 39 in mammalian cells, serine 52 in Drosophila) to inhibit its actin bundling activity (Yamakita et al., 1996; Ono et al., 1997). A phosphoresistant mutant of Fascin has been shown to possess a PKC-resistant actin bundling activity (Ono et al., 1997), whereas the phosphomimetic mutant of Fascin lacks actin bundling activity (Vignjevic et al., 2006). The phosphomimetic mutant of Fascin, Fascin[S52E], fails to rescue the bristle morphogenesis defects of Drosophila fascin mutants, suggesting that this phosphorylation site might be important in bristle morphogenesis (Zanet et al., 2009).
To determine the relationship between IKKε and PKC-dependent phosphorylation, we expressed wild-type Fascin (Fascin[WT]), phosphoresistant Fascin[S52A] or phosphomimetic Fascin[S52E] in developing bristles. In IKKε-deficient bristles, Fascin[WT]-GFP localization along actin bundles was diminished (Fig. 4A-C). By contrast, Fascin[S52A]-GFP was able to localize along actin bundles in IKKε-deficient bristles (Fig. 4D-F), whereas the actin bundle localization of Fascin[S52E]-GFP was reduced (Fig. 4G-I). Fascin[S52A]-GFP and Fascin[S52E]-GFP showed similar recovery kinetics compared with Fascin[WT]-GFP in FRAP experiments, although the initial recovery rate of Fascin[S52E]-GFP was more rapid compared with that of Fascin[WT]-GFP and Fascin[S52A]-GFP (Fig. S3). These results suggest that IKKε controls Fascin localization by regulating the serine 52 phosphorylation status. Consistent with this idea, expression of Fascin[S52A]-GFP, but not Fascin[WT]-GFP or Fascin[S52E]-GFP, in IKKεDN bristles significantly suppressed the bristle morphology defects (Fig. 4J-M), whereas overexpression of any of the three forms in the wild-type background did not affect bristle morphology (Fig. S4C-E). The incomplete suppression of the bristle morphology defects by Fascin[S52A]-GFP in IKKεDN bristles probably reflects the contribution of other pathways (such as Nuf) operating downstream of IKKε in bristle morphogenesis (Otani et al., 2011). These results indicate that IKKε regulates actin bundling in part by controlling serine 52 phosphorylation of Fascin.
IKKε regulates Fascin by controlling the phosphorylation status of serine 52. (A-I) GFP fluorescence images of live pupae expressing Fascin-GFP in bristles. Fascin[WT]-GFP localization along the actin bundles was decreased in IKKε-deficient bristles (A-C), whereas Fascin[S52A]-GFP was resistant to the loss of IKKε (D-F). The actin bundle localization of Fascin[S52E]-GFP was reduced (G-I). (J-L′) SEM analysis shows that expression of phosphoresistant Fascin[S52A] suppresses the phenotype of IKKεDN bristles. Representative bristles are shown at higher magnification in J′-L′. (M) Quantification of the bristle morphology phenotype. The percentage of normal scutellar bristles is shown as mean±s.d. n=69 for IKKεDN, n=60 for IKKεDN/Fascin[WT], n=40 for IKKεDN/Fascin[S52A] and n=59 for IKKεDN/Fascin[S52E]. *P<0.05 by two-tailed t-test, compared with IKKεDN. Scale bars: 10 µm in A-I; 100 µm in J-L. See also Figs S2 and S4.
IKKε protects Fascin from PKC-dependent inhibitory phosphorylation
To determine whether PKC family kinases are involved in IKKε-Fascin signaling, we examined the genetic interactions between IKKε and PKC family genes. Knockdown of either PKC53E (homologous to PKCα/β) or PKCδ led to a significant suppression of the bristle morphology phenotype of IKKεDN bristles (Fig. 5A,B,D), whereas the knockdown of PKC98E (homologous to PKCε), aPKC or PKN did not modify this phenotype (Fig. 5C,D). Furthermore, knockdown of either PKC53E or PKCδ restored the actin bundle localization of Fascin in IKKεDN bristles (Fig. 5H-J). Knockdown of PKC family genes in a wild-type background did not affect the actin bundle localization of Fascin (Fig. 5E-G) and did not yield any visible phenotype in bristle morphogenesis (Fig. S4F-H), suggesting that PKC is repressed by IKKε in wild-type bristles. These results suggest that IKKε promotes Fascin-dependent actin bundling in bristle morphogenesis by protecting Fascin from PKC-dependent inhibitory phosphorylation.
IKKε regulates Fascin localization by antagonizing PKC. (A-C′) SEM analysis shows that knockdown of PKC53E or PKCδ suppresses the phenotype of IKKεDN bristles. Representative bristles are shown at higher magnification in A′-C′. (D) Quantification of the bristle morphology phenotype. The percentage of normal scutellar bristles is shown as mean±s.d. n=69 for IKKεDN, n=30 for others. IKKεDN is identical to Fig. 4M. **P<0.0005 by two-tailed t-test, compared with IKKεDN. (E-J′) Phalloidin (F-actin) and anti-Fascin antibody staining show that knockdown of PKC53E or PKCδ restores the actin bundle localization of Fascin in IKKεDN bristles. Scale bars: 100 µm in A-C; 10 µm in E-J′. See also Fig. S4.
IKKε regulates PKC membrane translocation
To gain insight to the molecular mechanisms underlying the crosstalk between IKKε and PKC, we expressed the two molecules in cultured Schneider 2 (S2) cells. We focused on PKC53E for further analysis, as its mammalian homolog PKCα is known to phosphorylate Fascin (Yamakita et al., 1996; Ono et al., 1997). IKKε-myc and PKC53E-GFP interacted with each other and were co-immunoprecipitated from S2 cell lysates (Fig. S5). PKC53E-GFP showed a cytoplasmic localization when expressed in S2 cells (Fig. 6A,E), and coexpression of IKKε[WT] did not alter this localization (Fig. 6F). Interestingly, inhibition of IKKε by RNAi or by expression of dominant-negative IKKε[K41A] resulted in membrane translocation of PKC53E-GFP (Fig. 6B,G). Quantitation confirmed that IKKε inhibits the membrane localization of PKC53E-GFP (Fig. 6C,D). In developing bristles, PKC53E-GFP was localized to the cytoplasm, with some punctate signals in wild-type bristles (Fig. 6H), whereas PKC53E-GFP localized to the plasma membrane and vesicle-like structures in IKKεRNAi bristles (Fig. 6I). These results demonstrate that IKKε suppresses the membrane localization of PKC53E-GFP.
IKKε inhibits PKC membrane translocation. (A,B) PKC53E-GFP translocates to the membrane in IKKε-depleted S2 cells. PKC53E is diffusely localized in the cytoplasm in control RNAi (A), but translocates to the membrane in IKKε RNAi (B). (A′,B′) Plot profile along yellow arrows in A,B. (C) Quantitation of plasma membrane localization of PKC53E-GFP in IKKε-depleted S2 cells. **P<0.001 by two-tailed t-test, compared with control RNAi. (D) Quantitation of plasma membrane localization of PKC53E-GFP in IKKε-overexpressing S2 cells. *P<0.01 by two-tailed t-test, compared with mock. (E-G) IKKε kinase activity is required to inhibit PKC53E-GFP membrane translocation. PKC53E-GFP is enriched in the cytoplasm in mock-transfected (E) or IKKε[WT]-myc-transfected (F) cells, but becomes concentrated at the membrane in IKKε[K41A]-myc-transfected cells (G). Cells with moderate expression level of PKC53E-GFP were examined in A-G. (E′-G′) Plot profile along yellow arrows in E-G. (H,I) IKKε inhibits PKC53E-GFP membrane translocation in developing bristles. (H′,I′) Plot profile along yellow arrows in H,I. PM, plasma membrane. Scale bars: 10 µm. See also Fig. S4.
IKKε inhibits PKC
Previous studies have shown that newly synthesized PKC is membrane associated but inactive, whereas fully phosphorylated mature PKC localizes to the cytoplasm and can translocate to membranes in response to upstream signals (Newton, 2003). These results suggest that the membrane localization of PKC in IKKε-deficient cells can be due to either defects in PKC maturation or the excess activation of PKC. These two scenarios can be distinguished by examining PKC activity. In the former model, defects in PKC maturation should lead to decreased PKC activity in IKKε-deficient cells, whereas in the latter scenario PKC activity should be increased in IKKε-deficient cells.
We therefore tested the impact of IKKε on PKC activity by expressing a Förster (or fluorescence) resonance energy transfer (FRET) biosensor for PKC (Eevee-PKC) (Komatsu et al., 2011) in S2 cells. Eevee-PKC is an intramolecular FRET biosensor for PKC, and consists of a donor and acceptor fluorescent protein separated by a phosphate-binding domain, a long and flexible linker, and a PKC substrate peptide. Upon phosphorylation of the substrate peptide by PKC, the biosensor changes its conformation, resulting in an increase in FRET/CFP value (Komatsu et al., 2011). The Eevee-PKC FRET biosensor was able to report PKC activity in S2 cells, as its FRET/CFP signal was increased and enriched in the plasma membrane-associated region upon treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (Fig. 7A-D). FRET analysis revealed that expression of IKKε[WT] suppressed PKC activity (Fig. 7E,F,I), whereas expression of dominant-negative IKKε[K41A] activated PKC (Fig. 7G,I). The cytoplasmic FRET/CFP signals could be due to the diffusion of the FRET biosensor upon prolonged expression of IKKε[K41A]. The increase in FRET/CFP signal in IKKε[K41A]-expressing cells was significantly reduced by pretreatment with the PKC inhibitor bisindolylmaleimide GF 109203X (Toullec et al., 1991), which blocks phosphorylation of the FRET biosensor by PKC (Fig. 7H,I). These results demonstrate that PKC is activated in IKKε[K41A]-expressing cells, supporting the excess activation model. Taken together, these results suggest that IKKε inhibits PKC.
IKKε inhibits PKC. (A,B) Eevee-PKC reports PKC activity in Drosophila S2 cells. Eevee-PKC-expressing S2 cells were treated with 1 µM TPA, resulting in an acute increase in FRET signals at the membrane. (C,D) Quantification of FRET signals in TPA-treated cells. FRET signal is increased upon addition of TPA. FRET ratio was normalized against the mean FRET ratio prior to the addition of TPA (−10 to 0 min). *P<0.05 by two-tailed paired t-test. n=4. (E-H) IKKε regulates PKC activity. FRET signal is reduced in IKKε[WT]-expressing cells (F) compared with the control (E), but is increased in IKKε[K41A]-expressing cells (G). Pretreatment with PKC inhibitor (GF 109203X) reduces the FRET signal in IKKε[K41A]-expressing cells (H). (I) Quantification of FRET analysis. n=24 for IKKε[K41A], n=20 for IKKε[K41A]+GF 109203X, n=25 for others. P-values determined by the Wilcoxon rank-sum test. IMD, intensity modulated display. Scale bars: 5 µm.
IKKε prevents excess PKC activation
We have shown that IKKε inhibits PKC so as to protect Fascin from inhibitory phosphorylation, thereby promoting actin bundling during bristle morphogenesis (Fig. 8). PKC is responsible for the inhibition of Fascin-dependent actin bundling in IKKε-deficient bristles, whereas knockdown of PKC in a wild-type background yields no visible phenotype. These results suggest that IKKε prevents excess activation of PKC in wild-type bristles. How PKC is activated in IKKε mutant bristles remains to be characterized, but it should be noted that several signaling molecules that can potentially activate PKC are known to be involved in pupal thorax development (including Fz, Dpp, PVR) (Adler et al., 1994; Martin-Blanco et al., 2000; Ishimaru et al., 2004). IKKε could act as a switch to activate Fascin-dependent actin bundling by turning off PKC signaling during bristle morphogenesis. Given the widespread involvement of PKC in various signaling pathways, it is likely that IKKε-dependent PKC repression plays important roles in other systems. Of note, gene amplification of IKKε has been linked to oncogenesis (Boehm et al., 2007), and PKC functions as a tumor suppressor (Antal et al., 2015). Whether IKKε-dependent suppression of PKC plays any role in oncogenesis remains to be clarified in the future.
IKKε prevents excess activation of PKC. Model for IKKε-dependent regulation of actin bundling. In wild-type bristles, IKKε prevents excess PKC activation, thereby protecting Fascin from inhibitory phosphorylation to promote actin bundling (left). In IKKε mutant bristles, excess activation of PKC occurs, resulting in inhibition of Fascin-dependent actin bundling (right).
PKC membrane translocation and activation is controlled by its interaction with lipids through the C1 and C2 domains (Newton, 2003). Phosphorylation of the C2 domain of the novel PKC in Aplysia can reduce its membrane translocation (Farah et al., 2012). Although the molecular mechanisms underlying IKKε-dependent inhibition of PKC remain unknown, it would be interesting to determine whether IKKε can phosphorylate PKC to regulate its membrane translocation and activation.
Possible roles of Rab35 in bristle morphogenesis
In contrast to previous findings (Zhang et al., 2009), our results suggest that Rab35 does not play a significant role in regulating Fascin localization in bristle morphogenesis. These differences might be due to the different Gal4 drivers used: prospero-Gal4 in the previous study versus scabrous-Gal4 in this study. As prospero-Gal4 drives expression throughout the peripheral nervous system, it is possible that the previously observed bristle morphology defect in Rab35[S22N]-overexpressing flies was due to non-cell-autonomous effects. Nevertheless, overexpression of Rab35[Q67L] can suppress the bristle morphology phenotype of IKKε-deficient bristles, which could be due to the involvement of both IKKε and Rab35 in vesicle trafficking (Kouranti et al., 2006; Sato et al., 2008; Otani et al., 2011). Further studies are required to clarify the role of Rab35 in bristle morphogenesis.
IKKε and actin bundle organization
Our results suggest that IKKε regulates the actin bundling activity of Fascin during bristle morphogenesis. Interestingly, although IKKε is localized at the distal tip of growing bristles (Bitan et al., 2010; Otani et al., 2011, 2015) it can regulate the localization of PKC53E-GFP and Fascin throughout the bristles. As IKKε regulates Fascin via PKC, diffusion of PKC might serve to propagate the inhibitory influence of the tip-localized IKKε signaling on PKC. Our FRAP analysis indicates that Fascin can dynamically exchange within the actin bundles, consistent with the idea that paracrystalline actin bundles are dynamic structures (Hwang et al., 2015). The reversible cross-linking activity of Fascin might allow dynamic regulation of paracrystalline actin bundles during their repair or disassembly (Hwang et al., 2015; Overton, 1967; Tilney et al., 1996).
In addition to its role in regulating actin bundling, IKKε is also required for the cortical localization of actin bundles (Bitan et al., 2010; Otani et al., 2011). Previous studies have demonstrated that Arp2/3 complex-dependent branched actin network structures known as actin 'snarls' play an important role in localizing the actin bundles to the cell cortex (Hopmann et al., 1996; Hopmann and Miller, 2003; Tilney et al., 2003; Frank et al., 2006). Whether IKKε plays any role in regulating the dynamics of actin snarls, and how distinct actin-based structures interact with each other to organize a parallel array of actin bundles, are important issues to investigate in the future.
Drosophila genetics
All Drosophila strains were raised at 25°C on standard corn meal agar food. The following strains were used: y1 w67C21 as a control; IKKε1 (also known as IKKε36), IKKεalice (IKKε mutants recombined with FRT40A were from Kathryn Anderson, Sloan Kettering Institute, New York, USA) (Oshima et al., 2006; Shapiro and Anderson, 2006), y w Ubx-flp; ubi-GFP FRT40A/CyO (Ubx-flp from Jürgen Knoblich, IMB, Vienna, Austria) (Xu and Rubin, 1993; Emery et al., 2005), IKKεRNAi (Oshima et al., 2006), IKKεDN (Oshima et al., 2006), sca-Gal4 (Brand and Perrimon, 1993; de Celis et al., 1999), neuP72-Gal4 (provided by François Schweisguth, Institut Pasteur, Paris, France) (Bellaiche et al., 2001), forked36a (Petersen et al., 1994), singed3 (Paterson and O'Hare, 1991) (forked and singed were from Drosophila Genetic Resource Center), UAS-YFP-Rab35[S22N], UAS-YFP-Rab35[Q67L] (UAS-YFP-Rab35 flies were from Bloomington Stock Center) (Zhang et al., 2007), UAS-Fascin[WT]-GFP, UAS-Fascin[S52A]-GFP, UAS-Fascin[S52E]-GFP (Fascin-GFP flies were from François Payre, Serge Plaza, and Jennifer Zanet, Université de Toulouse, Toulouse, France) (Zanet et al., 2009), UAS-PKC53ERNAi[TRiP.JF02641], UAS-PKCδRNAi[TRiP.JF02991], UAS-PKC98ERNAi[TRiP.JF02470], UAS-aPKCRNAi[TRiP.JF01966] and UAS-PKNRNAi[TRiP.JF02970] (UAS-PKCRNAi flies were from Bloomington Stock Center). UAS-PKC53E-GFP transgenic flies were generated by standard P-element-mediated transgenesis. See Table S1 for the genotypes used in each experiment.
The following antibodies were used: mouse anti-Fascin monoclonal (Developmental Studies Hybridoma Bank, clone sn 7c; 1:5) (Cant et al., 1994); rabbit anti-Forked polyclonal antiserum (from Greg Guild, University of Pennsylvania, Philadelphia, USA; 1:500) (Guild et al., 2003); mouse anti-GFP (Roche, clones 7.1 and 13.1, #11814460001; 1:500); rabbit anti-GFP (MBL, #598; 1:500); anti-GFP HRP-DirecT (MBL, #598-7; 1:20,000); and anti-myc HRP-DirecT (MBL, #M047-7; 1:5000). The following secondary antibodies and detection reagents were used: Alexa 488-conjugated anti-mouse IgG (Molecular Probes, #A-11001; 1:200); Alexa 568-conjugated anti-mouse IgG (Molecular Probes, #A-11031; 1:200); Alexa 488-conjugated anti-rabbit IgG (Molecular Probes, #A-11008; 1:200); Alexa 568-conjugated anti-rabbit IgG (Molecular Probes, #A-11011; 1:200).
Pkc53E cDNA was from the Drosophila Genomics Resource Center (clone GH03188). IKKε[WT] and IKKε[K41A] constructs were characterized previously (Oshima et al., 2006). Fusion constructs were generated by subcloning Pkc53E or IKKε into pUAST-EGFP-N, pUAST-HA-Ctag, pUAST-myc-Ntag or pUAST-IRES-mKO vectors. The PKC FRET biosensor Eevee-PKC (3599NES) expression vector was provided by Kazuhiro Aoki and Michiyuki Matsuda (Kyoto University, Kyoto, Japan) (Komatsu et al., 2011) and was subcloned into the pUbi-attB vector (Kondo and Hayashi, 2013).
Pupae were fixed as described previously (Otani et al., 2011). Blocking was performed in 0.1% BSA, 0.2% Triton X-100 and 0.2% Tween 20 in PBS overnight at 4°C. Anti-Fascin, anti-Forked, mouse anti-GFP, secondary antibodies and Alexa 488/568-conjugated Phalloidin were diluted (as indicated above) in blocking solution, and antibody incubation was performed overnight at 4°C with gentle agitation. Washing with 0.1% Triton X-100 in PBS was performed three times after each antibody incubation step. After the final wash, the thorax specimens were mounted dorsal side up on glass slides in Vectashield mounting medium (Vector Labs) using a cover glass (0.12-0.17 mm thickness, Matsunami) as a spacer.
Cell culture and immunofluorescence
Drosophila S2 cells (provided by Shin-ichi Yanagawa, Kyoto University, Kyoto, Japan) (Schneider, 1972; Yanagawa et al., 1998) were cultured in Schneider's Insect Medium (Gibco) supplemented with 10% FCS and antibiotics at 25°C. Transfection was performed using Effectene (Qiagen) according to the manufacturer's instructions, and cells were harvested 36-48 h after transfection.
For RNAi, dsRNA against lacZ (control) or IKKε (Oshima et al., 2006) was added to the medium to a final concentration of 37 nM, and 36-48 h later the cells were transferred and transfected with PKC53E-GFP as described above. Cells were harvested 36-48 h after plasmid transfection (3-4 days after dsRNA addition).
For immunofluorescence, cells were fixed in 4% PFA in PBS for 20 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 min, and blocked with 5% skimmed milk in TBS (Tris-buffered saline). Rabbit anti-GFP primary antibody and secondary antibodies were diluted (as indicated above) in blocking solution, and antibody incubation was performed for 1 h at room temperature. After each antibody incubation, the coverslips were washed three times with 0.1% Triton X-100 in PBS. The cells were mounted in Vectashield mounting medium.
Confocal microscopy was performed using an FV1000 laser scanning confocal microscope mounted on a BX61 microscope using a UPlanSApo 60×/NA 1.35 objective or UPLSAPO 40×2/NA 0.95 objective (all Olympus) and argon (488 nm) and helium-neon (543 nm) lasers. Macrochaetes were examined for all of the samples, and image acquisition was performed using Fluoview software (Olympus). Fascin-GFP still images were obtained from living samples without fixation, and were captured using an FV1000 laser scanning confocal microscope mounted on an IX81 inverted microscope with a PlanApo N 60×/NA 1.42 objective (all Olympus) and an argon (488 nm) laser, and image acquisition was performed using Fluoview software. A Gaussian blur filter was applied to the acquired stacked images. PKC53E-GFP images from macrochaetes and Fascin/F-actin images in PKCRNAi bristles were obtained using a TCS-SPE laser scanning confocal microscope mounted on a DMI4000B inverted microscope with an HCX PL APO 63×/NA 1.40 objective (all Leica) and diode laser lines (488/532 nm), and image acquisition was performed using LAS AF software (Leica). Image processing (z-stacking, Gaussian blur filter application, brightness and contrast adjustments and generation of plot profiles) were performed using ImageJ (NIH) or Photoshop (Adobe) without any nonlinear adjustments. z-stacks of Fascin immunostaining were generated by maximum intensity projections of cortical sections. PKC53E-GFP plasma membrane localization was quantified by measuring the ratio between plasma membrane intensity (obtained by line scan) and total intensity of individual cells using ImageJ. Cells that express PKC53E-GFP at relatively low levels were analyzed to avoid artifacts caused by overexpression. Statistical significance was evaluated by t-test (two-tailed) using Excel (Microsoft).
For FRAP analysis, pupal cases were removed with fine forceps and the pupae were placed dorsal side down in glass-bottom dishes (Iwaki). Two pieces of 3MM filter paper were placed lateral to the pupae, and water was added to cover the pupae to avoid drying. Images of pupae were captured using the FV1000 mounted on an IX81 with the PlanApo N 60×/NA 1.42 objective and argon (488 nm) laser. GFP fluorescence was captured at 3 s intervals with z-sectioning. Photobleaching was performed using a diode (405 nm) laser. Fluorescence intensity of the selected region of interest (ROI) was measured using ImageJ and normalized by setting the pre-bleach level to 100% and the post-bleach level (0 s after photobleaching) to 0%. Statistical significance was evaluated by t-test (two-tailed) using Excel.
For FRET analysis, S2 cells were cotransfected with pUbi-Eevee-PKC, actin5Ce-Gal4 driver, and pUAST-IRES-mKO or pUAST-IKKε[WT]-IRES-mKO or pUAST-IKKε[K41A]-IRES-mKO, and plated on glass-bottom dishes. mKO-expressing cells were imaged using the FV1000 mounted on an IX81 with the PlanApo N 60×/NA1.42 objective. FRET probe was excited by a diode laser (440 nm), and the ratio between the CFP and FRET signals was calculated using ImageJ. The intensity modulated display (IMD) ratio images were generated using an in-house-written ImageJ plugin (developed by Housei Wada). For TPA treatment, TPA (Sigma) was added to the culture medium at 1 µM final concentration and immediately imaged. FRET ratio was normalized against the mean FRET ratio prior to the addition of TPA (−10 to 0 min), and statistical significance was evaluated by paired t-test (two-tailed) using Excel. For GF 109203X hydrochloride (Sigma) treatment, cells were pretreated with 30 nM or 300 nM GF 109203X prior to transfection. The GF 109203X concentration did not affect the results, and data for 30 nM treatment are presented. Multiple comparisons of FRET ratio were performed by Kruskal–Wallis test, and P-values for pairwise comparisons were calculated using the Wilcoxon rank-sum test using R statistical software.
Scanning electron microscopy (SEM)
Adult flies were anesthetized with CO2 and their legs and wings were removed using fine forceps. The dissected flies were mounted dorsal side up, sputter-coated with platinum (JFC-1600, JEOL) or osmium (Neoc-STB, Meiwafosis), and viewed by SEM (JSM-5600-LV, JEOL) under low vacuum (30 Pa) using an acceleration voltage of 10 kV.
Pupal cases were removed with fine forceps and the pupae dissected in fixative (2.0% glutaraldehyde in 0.05 M phosphate buffer, pH 6.8; as described by Tilney and Tilney, 1994). The dorsal thorax explant was then transferred to fresh fixative. Pupae were fixed at room temperature for 2-4 h and then transferred to 4°C. After three 10 min washes with 0.05 M phosphate buffer (pH 6.8) on ice, pupae were postfixed with 1% OsO4 in 0.05 M phosphate buffer (pH 6.8) for 45 min on ice. The fixed pupae were then washed three times for 10 min in water and stained en bloc with 0.5% aqueous uranyl acetate overnight on ice. After washing for 5 min with water, pupae were dehydrated in a graded acetone series (25, 50, 70, 80, 90, 95 and 99.5%) for 15 min at each concentration and transferred to 100% acetone for two 20 min incubations. Polybed 812 (Polysciences) or Quetol 651 (NEM) was used as the resin. When Polybed 812 was used, pupae were incubated in propylene oxide for 20 min twice, infiltrated with a 1:1 mixture of propylene oxide:Polybed 812, and finally placed into 100% Polybed 812. The resin was polymerized for more than 48 h at 60°C. For Quetol 651, pupae were incubated in a 1:1 mixture of n-butyl glycidyl ether (QY1):acetone for 20 min, incubated in 100% QY1 for 20 min, infiltrated with a 1:1 mixture of QY1:Quetol 651 for 2 h, and finally placed into 100% Quetol 651. The resin was polymerized for more than 48 h at 60°C. Semi-thin (0.4-0.7 µm) sections were cut from the anterior side of the pupae and examined using Toluidine Blue staining to confirm orientation. Based on the appearance of the dorsal thoracic microchaetes, the orientation of the pupae was carefully readjusted to obtain a perfect transverse section of the bristle. After confirming orientation, ultrathin sections (∼60 nm) were cut and mounted on 50-mesh Formvar-coated copper grids. The sections were stained with 4% aqueous uranyl acetate in the dark for 10 min, stained with Reynold's lead citrate for 3 min, and then coated with a thin layer of carbon. Observations were performed using a JEM-1010 transmission electron microscope (JEOL) at 100 kV accelerating voltage, and images were captured by a Gatan Bioscan 792 digital camera.
Quantification of hexagonal packing of actin filaments
To quantify the hexagonal packing of actin filaments, a Gaussian blur filter was applied to the original TEM images to reduce noise, and binary images of the centroids of the actin filaments were generated with the Find Maxima command of ImageJ. The image was subsequently subtracted with a binary image generated from the Gaussian-filtered image by thresholding to reduce erroneous detection of actin filament centroids. Radial distribution functions were measured by calculating the density of actin filaments centroids within a distance of r and r+dr away from a given centroid. Calculation was performed for r=0.6-59.4 nm with dr=1.2 nm and Δr=0.4 nm. The centroids used for the measurement were manually selected so that the actin filament centroids at the perimeter of the bundles were not included within the 60 nm diameter. Data from all measurable centroids were averaged to obtain the final radial distribution. Voronoi tessellation was performed using the Voronoi commands of ImageJ. The shapes of the polygons were analyzed using in-house-written programs (available upon request). Statistical analyses by two-tailed t-test were performed using Excel.
Quantification of the bristle phenotype
Bristle morphology was examined using a Stemi 2000 dissection microscope (Zeiss) and the phenotype of the scutellar bristles was scored. The number and morphology of the scutellar bristles of each animal were recorded, and bristles with a branched, hooked, bifurcated or bulged morphology were classified as 'abnormal'. The mean percentage of normal bristles was calculated, and two-tailed t-test performed using Excel. All data are presented as mean±s.d.
Transfected S2 cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM DTT). The lysates were incubated for 30 min at 4°C, then cleared by centrifugation at 20,000 g for 10 min at 4°C. 20 µl anti-GFP beads (MBL) were added to the supernatant and the samples incubated with rotation for 2 h at 4°C. The beads were rapidly washed three times with lysis buffer, and the complexes were eluted by boiling in 2×Laemmli sample buffer supplemented with 10% β-mercaptoethanol. SDS-PAGE was performed by standard methods using 5-20% SuperSep Ace polyacrylamide gels (Wako). Western blotting was performed as described previously (Otani et al., 2011). Anti-GFP HRP-DirecT and anti-myc HRP-DirecT were diluted (as indicated above) in Can Get Signal (Toyobo).
We thank Hisao Honda for suggestions on the radial distribution function analysis; Kenzi Oshima and Michiko Takeda for providing reagents; Housei Wada for developing an ImageJ plugin; Yasushi Izumi for comments on the manuscript; Yasushi Izumi and Mikio Furuse for their generous support of the revision experiments; Greg Guild, François Payre, Serge Plaza, Jennifer Zanet, Kathryn Anderson, François Schweisguth, Jürgen Knoblich, Kazuhiro Aoki and Michiyuki Matsuda for kindly sharing antibodies, fly stocks and DNA clones; and the Bloomington Stock Center, Drosophila Genetic Resource Center, Drosophila Genomics Resource Center, and Developmental Studies Hybridoma Bank for providing fly stocks, cDNA clones and antibodies.
The authors declare no competing or financial interests.
T.O. and S.H. conceived the study. T.O. obtained the majority of experimental data with the assistance of A.K. Y.O. performed the FRET analysis. K.M. and S.Y. performed the TEM analysis. T.M. performed the quantitative TEM image analysis. T.O. and S.H. wrote the paper and all authors read and commented on the manuscript.
This work was supported by MEXT/Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Young Scientists (B) [grant number 24770197 to T.O.] and Grant-in-Aid for Scientific Research on Innovative Areas [grant number 22111007 to S.H.]. Deposited in PMC for immediate release.
Supplementary information available online at http://dev.biologists.org/lookup/doi/10.1242/dev.138495.supplemental
Received April 8, 2016.
© 2016. Published by The Company of Biologists Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
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(1998). Changes in the F-actin cytoskeleton during neurosensory bristle development in Drosophila: the role of singed and forked proteins. Cell Motil. Cytoskelet. 40, 119-132. doi:10.1002/(SICI)1097-0169(1998)40:2<119::AID-CM2%3.0.CO;2-A
Xu, T. and
Rubin, G. M.
(1993). Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117, 1223-1237.
Matsumura, F. and
Yamashiro, S.
(1996). Phosphorylation of human fascin inhibits its actin binding and bundling activities. J. Biol. Chem. 271, 12632-12638. doi:10.1074/jbc.271.21.12632
Yanagawa, S.-i.,
Lee, J.-S. and
Ishimoto, A.
(1998). Identification and characterization of a novel line of Drosophila Schneider S2 cells that respond to wingless signaling. J. Biol. Chem. 273, 32353-32359. doi:10.1074/jbc.273.48.32353
Zanet, J.,
Stramer, B.,
Millard, T.,
Martin, P.,
Payre, F. and
Plaza, S.
(2009). Fascin is required for blood cell migration during Drosophila embryogenesis. Development 136, 2557-2565. doi:10.1242/dev.036517
Zhang, J.,
Schulze, K. L.,
Hiesinger, P. R.,
Suyama, K.,
Wang, S.,
Fish, M.,
Acar, M.,
Hoskins, R. A.,
Bellen, H. J. and
Scott, M. P.
(2007). Thirty-one flavors of Drosophila rab proteins. Genetics 176, 1307-1322. doi:10.1534/genetics.106.066761
Fonovic, M.,
Bogyo, M. and
(2009). Rab35 controls actin bundling by recruiting fascin as an effector protein. Science 325, 1250-1254. doi:10.1126/science.1174921
Actin
Fascin
IKKε
Bristle morphogenesis
You are going to email the following IKKε inhibits PKC to promote Fascin-dependent actin bundling
The RNA helicase DDX3 induces neural crest by promoting AKT activity
Loss of ESRP1 blocks mouse oocyte development and leads to female infertility
C. elegans establishes germline versus soma by balancing inherited histone methylation
Show more RESEARCH ARTICLE

redpajama_set_name: RedPajamaCommonCrawl


text:
Coaches Hot Seat Quotes of the Day – Tuesday, December 6, 2022 – Bud Wilkinson
"Football in its purest form remains a physical fight. As in any fight, if you don't want to fight, it's impossible to win."
"Morale and attitude are the fundamental ingredients to success."
"If a team is to reach its potential, each player must willingly subordinate his own personal goals to the good of the team."
"We compete, not so much against an opponent, but against ourselves. The real test is this: Did I make my best effort on every play?"
"I feel more strongly about this than anything else in coaching: Anybody who lacks discipline, who doesn't want to be part of the team, who doesn't want to meet the requirements – has to go. It's that simple."
"The man who tried his best and failed is superior to the man who never tried."
"Losing is easy. It's not enjoyable, but it's easy."
"If you are going to be a champion, you must be willing to pay a greater price."
Wikipedia: Bud Wilkinson
Bud Wilkinson, Coaches Hot Seat, Coaches Hot Seat Rankings


This information is intended to be helpful to the voters of Windsor Lake. For official information, consult the Elections NL website here https://www.elections.gov.nl.ca/elections/ or call Elections NL at 729-7987 or 1-877-729-7987.
How do I find out if I'm in Windsor Lake District?
You can find out if you're a resident of the district by calling Elections NL at 729-7987 or 1-877-729-7987 or by clicking here.
You can also call us at 552-8120.
How do I find out if I'm registered to vote?
Call Elections NL at 729-7987 or 1-877-729-7987.
You DON'T need a Voter Information Card from Elections NL.
Polls are open 8 am to 8 pm.
All employees are entitled to 4 consecutive hours to vote on Election Day.
When voting in the Advance Polls or on Election Day, you must bring identification / documents with your name and address.


text:
AMA Blog
5 Lessons from 'Writing Characters That Inspire'
by Shanee Edwards on June 25, 2021
Do you know how to write a character that inspires?
Character development is one of the toughest tasks a screenwriter is faced with when sitting down to write. However, ScreenCraft recently held a virtual panel that featured some of TV and film's biggest heavy hitters and they shared some expert-level advice on how to create dimensional, intriguing, and inspiring characters.
The panel included TV writer/producer Javier Grillo-Marxuach (Lost, The Dark Crystal, Cowboy Bebop), screenwriter Peter Chiarelli (Crazy Rich Asians, Now You See Me 2, The Proposal), and screenwriters/producers Rick Jaffa and Amanda Silver (Rise of the Planet of the Apes, Jurassic World, Mulan, Avatar 2 and 3).
Hosted by ScreenCraft's own Emily Dell (How I Met Your Murderer), this lively discussion touched an everything, including conflict, character, and going the extra mile as a screenwriter. Here are five highlights from the event.
Secrets to Creating Memorable Characters
"Irreconcilable Contradiction"
But Grillo-Marxuach went a little bit deeper. His advice is to create an "irreconcilable contradiction" inside the heart of the character. Coming from the TV world, Grillo-Marxuach used Don Draper from TV's Mad Men as an example.
"Don Draper is a character trying to live up to an ideal that doesn't exist, but he's a very different person than that ideal and his soul is always trying to sabotage him… Ultimately, he doesn't resolve it, he stays the same guy."
Sometimes, that "irreconcilable contradiction" in a character can play out as the spine, or theme for the entire show, movie, or even film franchise.
Don Draper in 'Mad Men'
Smart Person + Impossible Challenge
Why do we remember some characters for years while others are forgettable? There's no perfect equation to creating memorable characters, but putting some serious thought into their wants, flaws, and backstory can get you closer.
Chiarelli summed up creating memorable characters like this: Have a smart person face what seems like an impossible challenge.
"Watching them get through it and solve it at the same time – and then, the old character arc thing, they learn a little something about themselves. It's as simple as that."
Case Study: Caesar from Planet of the Apes
Amanda Silver said this was certainly the case for Planet of the Apes. She cited the central contradiction within the protagonist, Caesar.
"It's kind of an umbrella for the whole Planet of the Apes franchise, this Pinocchio thing where you've got this little boy ape who wants to be a real boy. That's what endeared us to Caesar when he was first born. But of course, he's got to grow over time to embrace his real tribe from there, but you can see it in subsequent sequels – this idea of this central conflict within him. He was raised by humans, he has this thing for humans yet he's an ape. He's the leader of the apes. There's so much great fuel in that."
Caesar's "irreconcilable contradiction" is that he wants to be something other than who he is. This is a problem many of us struggle with in real life thereby making this ape surprisingly relatable.
A good writer would give Caesar the arc of finally accepting his true self. But Silver and Rick Jaffa are great writers and went a step further.
Jaffa said the pitch for Planet of the Apes was simple: "From Pinocchio to Moses." Yes, Moses from the bible. Over the course of the franchise, Caesar would go from "wanting to be a real boy" like Pinocchio to accepting his true self to becoming the leader of the apes and avenging his kind. This is some epic storytelling that all started with a simple "irreconcilable contradiction" inside one character.
Caesar in 'Dawn of the Planet of the Apes'
Finding a Character's 'True North'
As you write a character and create their "irreconcilable contradiction," it's easy to put it into action – at first. As you get into the weeds of act II (in a film script) there's plot, other characters, and set pieces (particularly in a comedy or action film), all of which can distract from the protagonist's true objective and flaw.
But having written the screenplay for Crazy Rich Asians, which has numerous characters and locations, Chiarelli summarized exactly what the story is about.
"The governing principle of the whole thing is, can Rachel win over Eleanor (the mom) just a little bit? That's the through-line that everything else orbits around. All the flash and whatever, it's all going toward that problem. Ultimately the person who changes in the movie is Eleanor, not Rachel as much. That was my true north. You need that true north character line, or you get lost."
Chiarelli said that his guide for choosing which characters from the book (Crazy Rich Asians by Kevin Kwan) to include in the movie, was Rachel's "true north" principal. If a character could help Rachel win over Eleanor in a significant way, they made the cut. If they didn't – no matter how likable or funny they were – they didn't make it into the screenplay.
Once you discover your protagonist's "true north," the writers suggested writing it down and keeping it near your computer to use as a compass for the times you get off course.
More info coming soon | Remind Me
Crash-Test Your Characters
We've all heard the phrase, "Torture your protagonist," but many beginning writers struggle to do this consistently. Grillo-Marxuach said to give yourself this directive:
"You must first put your characters in the one, singular situation that most challenges their true self."
He went on to say he once had a playwriting teacher who summed up writing conflict this way:
"A dramatist is like an insane train conductor who's trying to get all the trains to collide with each other."
This makes sense considering every train crash-tests your protagonist, pushes them to dig deep, and makes for great drama. Start small and build up to an epic crash in your screenplay. By the end, if your protagonist survives, you will know exactly who they are.
Jack from 'Lost'
It's Good to Get Personal with a Character
Should characters be purely fictional, or should the writer bring their own self to the character? Silver said that she and Jaffa bring their own wants, needs, and issues.
"For us, it's always personal. That's the shocking thing I discovered when I started writing. The audience might not recognize it is personal or think that Rick and I would have anything in common with Caesar, but we kind of gave him our heart and soul."
She said that your characters don't need to be autobiographical, but a writer should look for ways to put parts of themself into a character.
"Be vulnerable, even when you're trying to be funny. For me, the barometer is when we hand a script in, I can't bear that someone's going to read it. That's how personal it is. I want to hide under my bed all weekend."
Antagonists Need Love Too
Writers often get so preoccupied with their hero that they often neglect their antagonist or villain. This could be in part because writers don't like to think they have much in common with someone who's evil, or at the very least, bad. Chiarelli had an excellent suggestion for overcoming this obstacle.
"I want to believe my villain thinks they are right the whole time. I've never written a sociopath, but you write versions of it, the idea that they have a firm footing, and they are absolutely dedicated to what they are doing makes complete sense. That's good writing versus bad writing."
Jaffa agreed, adding:
"[The villain] is a direct reflection of the protagonist – the journey, the need."
One way to think about your antagonist is that they are the shadow version of your protagonist. They are two similar souls, but one character goes to the light, while the other goes to the darkness. Remember that the function of your antagonist is to force your protagonist to change. It's always a painful process that's never pretty, but it is necessary for your story.
Eleanor, Nick, and Rachel in 'Crazy Rich Asians'
So, as you begin to work on your characters, keep these great tips in mind. Make them memorable, give them an irreconcilable contradiction, put your heart and soul into them, and then put them through the gauntlet.
And don't forget about your villains!
Shanee Edwards is a screenwriter, journalist and author. After receiving her MFA in Screenwriting from UCLA, she was hired to adapt various stories for the screen including Apes or Angels, the true story of naturalist Charles Darwin, and Three Wishes, based on the New York Times best selfing novel by Kristen Ashley. You can listen to her interview Oscar-winning screenwriters on The Script Lab Podcast, or read her book Ada Lovelace: the Countess who Dreamed in Numbers. Follow her on Twitter: @ShaneeEdwards
101 Terrifying Horror Story Prompts
99 Archetypes and Stock Characters Screenwriters Can Mold
101 Great Plot Twist Ideas to Elevate Your Script


Chicago Audubon Declaration of Support for the Paris Climate Accord; article about the dangers of fishing line for birds; winners of the Second Annual Chicago Audubon photo contest.
Birdathon! 2017, May 13 and 14 -- Join a team and have some fun counting birds, raise money for Chicago Audubon programs and goals! Read about Chicago Audubon's 2nd Annual Spring Photo Contest, and enjoy a book review about a wonderful book, Flora of the Chicago Region.
Details of the 2017 Environmental Awards Banquet, Membership Meeting and evening's program; upcoming Chicago Audubon Program on Jackson Park; bird monitor training.
Read about the 117th Annual Christmas Bird Count, Why Native Plants are Better for Birds and People, and upcoming events.


text:
Philip Facchina
Phil is a Principal of SurgCenter and serves as Chief Strategy Officer. Phil has spent the last two decades in the Public and Private Capital Markets, where he directly managed public and private capital transactions of equity and debt, lead M&A and special advisory processes including take-privates and played a role in activist investor resolutions. Most recently Phil was Partner, Co-Portfolio Manager and the Chief Operating Officer of Ramsey Asset Management, an investment management firm. Prior to that he served as Senior Managing Director and Group Head of Technology, Media and Telecom and Healthcare Investment Banking within Friedman, Billings, Ramsey and FBR Capital Markets, an investment banking firm, where he also served on the Commitment Committee and as Head of Financial Sponsors, interfacing with a myriad of private equity and venture investors. Phil has served in various C-level roles of both public and private companies, holds an MBA from the University of Pennsylvania's Wharton School of Business and a B.S. degree in Accounting from the University of Maryland.


If you're looking to add more potential mishaps to your imaginary list of things that could go wrong in a presentation, check out this list of real-life presentation disasters.
Some pretty hilarious stuff. It makes you realize that no disaster is so bad you can't recover from it!
This from ScottGu's blog, where he also shares a story that's pretty funny.
Anyone have a good story to share that you can laugh about now?


Have a wonderful Beaity and Health Wednesday. I will be having it on the flight to Buenos Aires. I'm so excited to go to Argentina!
Rosemariy Hydrosol and Cleansing Powder will help me stay refreshed & relaxed in transit :).


text:
Q: (Congruence) Why does the std:: before the first thread make it print second? #include <iostream>
#include <thread>

void foo(){
    std::cout << "Thread 1 \n";
}

void bar(){
    std::cout<< "Thread 2 \n";
}
using namespace std;
int main()
{
    std::thread t1(foo);
    thread t2(bar);

    t1.join();
    t2.join();

    return 0;
}

The above code gives me an output of Thread 1 \n Thread 2. However, when I change std::thread t1(foo); to thread t1(foo);, my output is Thread 2 \n Thread 1. Why is this?
The compiler I'm using for this is: https://www.onlinegdb.com/online_c++_compiler

A: Using std:: has absolutely no impact here. You simply do not have any control over in which order the threads will be run so you can get any order.

redpajama_set_name: RedPajamaStackExchange


text:
AM Music Entertainment
Hire the hottest talent for your next event or party
Book This Artist
Flo Rida is an American rapper, singer and composer from Carol City, Florida. His 2008 breakout single "Low" was number one for 10 weeks in the United States and broke the record for digital download sales at the time of its release.
Flo Rida's debut studio album, 2008's Mail on Sunday, reached number four in the US. The album was succeeded by R.O.O.T.S., the next year. His subsequent albums, 2010's Only One Flo (Part 1) and 2012's Wild Ones, also charted on the US Billboard 200 chart. Flo Rida has sold over 80 million records worldwide, making him one of the best-selling music artists. His catalog includes the international hit singles "Right Round", "Club Can't Handle Me", "Good Feeling", "Wild Ones", "Whistle", "I Cry", "G.D.F.R.", and "My House".
His brother-in-law was a hype man for local rap group 2 Live Crew, and while in ninth grade, Flo Rida joined an amateur rap group called the GroundHoggz. The GroundHoggz had been a three-man group, with members who lived in the same apartment complex as Flo Rida. They started recording at underground studios in Carver Ranches. The GroundHoggz, in this four-man incarnation, remained together for eight years.
Flo Rida's work with 2 Live Crew member Fresh Kid Ice, attracted the attention of DeVante Swing, a member of the band Jodeci. However, he was rejected by several of the major labels, so he sought many other jobs outside of music. After graduating from high school in 1998, he studied international business management at the University of Nevada, Las Vegas for two months and attended Barry University for two months. He returned to Florida to continue pursuing his music career after receiving a phone call from a representative of the independent label Poe Boy Entertainment.
Flo Rida signed with Hot Boyz, in 2000 and with Poe Boy, in 2004 and began affiliating himself for Cash Money with other Southern hip hop artists, such as Rick Ross, Trina, T-Pain, and Trick Daddy. A promotional single entitled "Birthday", featuring Rick Ross, was his first significant release. He made his debut guest appearance with the song "Bitch I'm From Dade County" on DJ Khaled's album We the Best (2007), which also features Trick Daddy, Trina, Rick Ross, Brisco, C-Ride, and Dre.
Old School Rap / Hip-Hop
Copyright © 2021 · AM Entertainment LLC · Built by Hampson


Request a price Home Visit Store Customer Reinforce EOTech Holographic Sight Mossy Oak 512.MO SCOPELIST SKU – 512.MOMPN – 512.A65MO Magnification Range:1xPower Supply:AA BatteryDimensions:5.4″x2″x2.25″ (137×50.8×57 mm)Weight:10.9 oz (309 grams)Battery Life:1,000 continuous hours (lithium) at nominal setting 12; 600 continuous with AA alkaline Why SCOPELIST Scopelist strives to offer best professional optics and accessories along side odd customer service. We stock up on premium optics brands like Nightforce, Vortex, Trijicon, Schmidt Bender, Swarovski, Leica, Zeiss, Leupold, Meopta and more. We're constantly expanding our selection of premium European optics and accessories based on customer feedback and requirements. But even so, purchasing from Scopelist has a host of perks accompanying it. Not only do you get total shopping satisfaction but also buyer's assurance and many exclusive offers. We welcome you with:FREE and FAST Shipping : Shop for $500 or above and get free shipping to addresses in the United States. Place your order before 4:00PM Monday through Friday EST, and we can ship the same day. Any order processed after that time will be shipped the following business day. Make an Offer on Many Brands : Our customers can make an offer and get the lowest prices on many brands. We give the best deals based on our inventory, margin and pricing standards as set by our suppliers. Lowest Price Guarantee : We just remember to get the Lowest Price on the market when you buy from us. Shop at Scopelist and stay assured you've got the best deal. 15 DAYS Hassle-FREE Return Policy : We offer 15 DAYS Hassle-FREE Returns and Money Back Guarantee. Return the item to us in the same condition as it was when received and we can issue you a full refund within 15 Days from the date of receiving your parcel. Paypal Accepted : You enjoy Paypal Buyer Protection which in other words means 100% protection for unauthorized payments sent from your account. It is extremely safe15 Day Money back Guarantee : We give a 15 Day money back guarantee if you want to return a product. This translates into a full credit refund for items returned in new condition.Inventory Guarantee : If you have placed an order and completed its transaction, and it has gone out of stock after that, we give you $25 Scopelist Gift Card. Please note that we may have to cancel this order as backorder is not conceivable on ebay.Price Error Guarantee : If as a result of some unavoidable reason there is an error or change in the pricing without notice, we give you $25 Scopelist Gift Card. Refer a Friend and Save BIG Offer : Refer a friend or group of friends to Scopelist and instantly get $25 referral reward when your referrals purchase items worth of $500 and above. Just make sure whoever you are referring to Scopelist has to be the first time buyer at Scopelist Marketplace. Write about us and earn $25 instantly : Write about your shopping experience with Scopelist on eBay, or write honest product reviews on items purchased from us, mentioning Scopelist in a positive light. Publish your work in forums, your own blog, social media, youtube or in any other relevant places. After a short verification of authenticity we can give you $25 Scopelist Gift Card. Please note that the content should be substantial to receive this gift cart.No Tax : No Tax for 49 states except there is 6% tax if the item is shipped to PA. For Clothing and Boots, there is not any PA tax! Payment Information Payment is due immediately. We reserve the right to open an unpaid item dispute at our convenience. PayPal is our preferred method of payment since it provides security for both the buyer and the seller. We also accept major credit cards as well as e-checks.Shipping InformationAll orders that are processed before 4:00PM EST will be shipped the same day. Any order processed after that time will be shipped the following business day. Please allow 1 to 7 business days for items shipped to addresses within the United States. Please contact us for quicker delivery rates.Return InformationMost of our items offer a 14 day return policy. We expect the returned item to be in the same condition as it was when received. If the item purchased is a scope please do not mount it. If the returned scope is found to have been mounted a minimum fee of 20% will be assessed. Your satisfaction is important to us! If the item does not satisfy your needs in any way please contact us.Please NoteWe reserve the right to change shipping carriers. In the event of the buyer upgrading shipping the cost will be the responsibility of the buyer. All shipping times are ESTIMATED. When the product leaves our warehouse it becomes the carriers responsibility to get the product to you.We encourage you to contact us if you have any concerns regarding our products or return policy, find shipping discrepancies, need assistance to connect with the manufacturer or have other queries.*International CustomersPlease contact us before placing your order.


At Royal Roofing, we are committed to providing you with quality and affordable roofing for your home. For low-cost, great value domestic roofing in Eastry, we are one of the reputable providers. In Eastry, our team of experienced roofers are always developing innovative techniques so that we can provide better roofing options for our customers. For more than a decade, we have provided excellent roofing services for our Eastry customers, earning us credibility and helping u to learn everything we know about roofing. By creating innovative ways to carry out domestic roofing services, we strive to take a pioneering role in the roofing industry; always looking to improve and use best practice equipment and methods. Because of our rapid response policy, Royal Roofing stands out among other service providers; offering emergency services at affordable prices.
Customer satisfaction is a key part of our service policy.Whenever you need us, there is an available team of expert roofers in Eastry ready to provide solutions for your roofing needs.
With over a decade of experience in domestic roofing, Royal Roofing is a leading provider of quality roofing services in Eastry.
Our customers are assured of complete satisfaction in our service because we pride ourselves in delivering quality service. With our team of specialist roofers, Eastry get the best domestic roofing experience possible.
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Royal Roofing is rated high as a domestic roofing contractor on the TrustaTrader website. We offer our customers excellent service and professionalism, which is why they choose us for domestic roofing services. As shown on TrustaTraders, Royal Roofing has established a status in Eastry and its neighbouring towns as a reliable company.
The customer is at the centre of our service here in Royal RoofingOur customer-centred approach means your request are treated with utmost urgency.Our client interests come first because we consider them a key part of our business.We have a full insurance policy to cover our clients in Eastry from incurring any liability on the job.
Quality is our watchword at Royal Roofing. Our customers in Eastry can have peace of mind that they will receive good quality domestic roofing service. We aim to provide satisfactory services for our customers and the best standards of domestic roofing they need. We want our customers to feel safe in their homes, so our technicians at Royal Roofing make our roofs rigid and secure. Our team of roofers are not only skilled but well-trained to excel at their tasks.
Royal Roofing has been around for over 10 years delivering domestic roofing of the highest quality. We are highly qualified to provide useful tips on domestic roofing services. Within and around Eastry, we are experts are providing domestic roofing services.
We expect our clients to feel secure in their residence. This is why we have set our rates at prices our customers can afford. Because of our transparent pricing methods, customers trust our quotation; we have no hidden charges. Our policy at Royal Roofing ensures we have an open pricing system that puts the customers first before other issues.


text:
"use strict";
function __export(m) {
    for (var p in m) if (!exports.hasOwnProperty(p)) exports[p] = m[p];
}
__export(require("./blockScopeAwareRuleWalker"));
__export(require("./programAwareRuleWalker"));
__export(require("./ruleWalker"));
__export(require("./scopeAwareRuleWalker"));
__export(require("./skippableTokenAwareRuleWalker"));
__export(require("./syntaxWalker"));

redpajama_set_name: RedPajamaGithub


text:
https://www.sfgate.com/sports/article/3-Bay-Area-runners-win-class-titles-3300933.php
3 Bay Area runners win class titles
CROSS COUNTRY STATE MEET
By Mitch Stephens
Published 4:00 am PST, Sunday, November 25, 2007
A two-year Bay Area drought ended with three rather remarkable CIF State Cross Country individual titles Saturday at Woodward Park.
St. Ignatius junior Katy Daly (Division III), Healdsburg senior Sarah Sumpter (Div. IV) and Dublin senior James Attarian (Div. III), each winning titles they never could have envisioned, gave the Bay Area three titlists for the first time since 2001.
On an unseasonably warm day that featured nearly 2,000 runners, senior German Fernandez of Riverbank (Stanislaus County) finished in 14 minutes, 24 seconds to shatter the course mark by 14 seconds and the state meet record by 21 seconds.
But Attarian had the meet's second most stunning performance when he passed defending champion Mohamed Abdalla in the final 100 meters to win in 15:17. Abdalla (15:20) was second followed by, among others, Acalanes-Lafayette senior Nate Beach (fourth, 15:36) and Jefferson-Daly City senior Gambileg Bor (sixth, 15:43).
Abdalla, a Willow Glen-San Jose senior, led from the 800-meter mark over the rolling 3.1-mile course but couldn't hold off the third-year runner, who last year finished 24th in 16:16. Dublin coach Ken Grace, who coached at City College of San Francisco from 1980 to 1995, said Attarian trailed Abdalla by 14 seconds at the halfway point.
"I just kept trying to cut into the lead," Attarian said. "When I finally saw him in the final straightaway, I knew I had a shot. When I was on his heels, I pretty much knew I had it. I figured I'd come this far so I went for it."
Like Attarian, Sumpter made huge strides in the offseason after placing 11th last season in 18:49. On Saturday, she was calm and in control throughout, blistering the field in the day's fourth best female time of 17:26, well ahead of runner-up Jennifer Bergman (18:10) of Valley Christian-San Jose.
"If you would have told me a couple of years ago I would someday win the state title in that time, I would have just laughed," she said. "I couldn't have imagined this."
An injury on the soccer field three years ago led Daly to running and she hit the pinnacle with her first state title after placing third and ninth, respectively, the last two years. Daly, whose 18:13 beat her previous best by 10 seconds, said, "I was just way more relaxed today. I never thought about winning, but just running."
The day's other local highlights included a second-place finish (Div. II) by Casa Grande-Petaluma sophomore Jacque Taylor (Div. II), fourth-place finishes by Carondelet sophomore Nicole Hood (Div. II), St. Mary's-Berkeley senior Matt Duffy (Div. IV) and Chinese Christian-San Leandro senior Victor Ho (Div. V), and a fifth-place finish from Mountain View sophomore Garrett Rowe.
There were no local team titles - top ranked (Div. II) Carondelet-Concord placed fifth - but the boys and girls teams at St. Mary's, University boys (Div. V) and Petaluma boys (Div. II) all placed second.


Brooklyn Fare is currently hiring part-time and full-time staff at all of our three locations. Culinary and grocery experience is preferred, but we will train the right individual as necessary.
Active openings are listed below, please click through for more information.
For Chef's Table at Brooklyn Fare employment opportunities, please send an email with a cover letter, 3 references, and CV in .pdf or .doc form to: careers@brooklynfare.com.
Please note that our Chef's Table team is highly competitive and, thereby, we cannot guarantee immediate response or availability.


text:
Use these free tools and learn from the gurus to build an inclusive community
Categories: Inclusion
Tags: diversity & inclusion, equity, global, identity, inspiration, intercultural, value of difference
For a whole world of reasons, organizations and people are focused on building an inclusive community in the workplace. Are we late to the game? You bet. Is it a long haul? You bet. Self-education and sustainable change require consistency. However, it begins with a single committed step forward. To learn, embrace, and include others who are different across a variety of spectrums. This includes disability, race, gender, age, sexual preference, and other equity-related identity areas.
In October, I launched a three-part series showcasing the best of the best across my blog themes. I'm including my top tools, resources, communities, and leaders within each theme Last month it was branding; this month it's inclusion.
Tools to help you build an inclusive community
Photo by "My Life Through A Lens" on Unsplash
Creating accessible content for the disabled. If you produce work communications, are you mindful of all audiences? For example, do you think about enabling sound for the sight-impaired? Likewise, do you provide descriptions for the hearing impaired? Both Microsoft and Google provide built-in accessibility tools. Whether you're creating a document, email, spreadsheet, or presentation, accessibility is a click away. Learn more about Microsoft's Accessibility Checker and Google's Accessibility Products.
Guiding the career path for marginalized job seekers. Jennifer Tardy is pioneering new trails across a variety of digital platforms to support job seekers. I subscribe to the But What About Me podcast, which highlights the challenges marginalized groups often face. For instance, groups like veterans, transgender people, previously incarcerated persons, and adults aged 50 and over, among many others. Across all groups, Tardy shares how to elevate your strengths and expertise.
Acquiring skills to be an inclusive community builder. Earlier this year, I completed the USF certificate program, DEI in the Workplace. Beyond educational, the course is layered with honest dialogue and practical application. Not only will you learn about developing business practices, but you'll also learn how to increase company revenue and improve performance.
Guiding respectful communication across our borders. Do you interact with international colleagues? If so, It's important to understand cultural norms. For example, which cultures rely on group discussion for decision-making? Which opt for individual thinking? Which cultures don't mind you standing close and which prefer distance? Geert Hofstede developed the cultural compass to navigate these cultural preferences.
Diversifying voices in the advertising world. When it comes to the agency world, we lack diversity. I know; I've worked in the space for 20 years. Equally, we've lacked diversity in advertising as well. That said, I do believe the ad space is changing and evolving. Kudos to membership groups like AIR: Allies in Recruiting for bringing formidable voices to the table to drive change. For "giving grace to all who are doing the work to actively evolve."
Make it known and…
Photo by Mitchell Griest on Unsplash
In addition to these tools, there are amazing, intuitive leaders carrying others as they climb. Standing in the gap and having the conversations. Providing education and advocating relentlessly. Below is a shortlist of my heroes and gurus.
Amplifying voice for the disabled. Jenny Lay-Flurrie is clear in her mission: "act with a bolder ambition to open doors and empower disabled talent." As the Chief Accessibility Officer for Microsoft and a member of the disabled community, she's boots on the ground authentic.
Understanding what unconscious bias means. Along with Flurrie, Valerie Alexander was a favorite instructor of mine in the USF DEI program. Want to understand what unconscious bias means? Alexander's TED Talk will explain it in relatable terms. She demonstrates how lived experiences affect how we see ourselves in the world – and with others.
The art of storytelling. I discovered the brilliance of René Carayol as a guest on the Inclusion Works podcast. He's a king when it comes to storytelling. Rather than showcasing program success, Carayol says share lived experiences. Most mportantly, he's an advocate for truth-telling. Real and raw.
The world isn't flat. With today's global economy, many of us work with colleagues beyond our borders. The goal of SIETAR USA is to facilitate respectful communication with people from other cultures. Equally, the organization provides knowledge and expertise through conferences, webinars, and online content.
Going there, being bold. Often, we avoid dialogue with people different from us because we don't want to say the wrong thing. Likewise, we feel everything we say is the wrong thing. This is where the learning gets traction! Emmanuel Accho, a former NFL linebacker goes right to the heart of race matters in his YouTube series.
Meet my brothers
My Sudanese brothers from left to right: Santino, Malou, and Yai. Not pictured, Mabior.
Besides the tools and gurus mentioned above, I'm also sharing my favorite blog post. It's short and sweet – unlike my journey with my Sudanese brothers. It's been long, bittersweet, and heart-wrenching at times. Consequently, I've learned a lifetime across the years since we first met on 9/11. As a result, I'm an enlightened sister and a better world citizen for knowing them. I'm educated in new ways on struggle, perseverance, humility, and love.
I hope you find these tools and experts insightful and helpful. Please explore! I invite you to share your favorite inclusion resources and leaders in the comments section.
Debbie Schallock is a content and brand strategist, DEI ally, communication thought leader, and content marketing partner to agencies. She's the founder of Seachange Branding, arming startups and SMBs with the right tools to clarify business positioning and carve a competitive advantage. She blogs on the topics of branding and inclusion. Her research has been published in the International Journal of Diversity in Organisations, Communities & Nations – A Space for Gray: The Value of Difference.


Do you know someone who deserves a Smile Ticket Treat?
Local community groups and disadvantaged fans can apply for Posh Smile Tickets so they can enjoy watching a game of football free of charge, which perhaps they would otherwise be unable to enjoy.
Fans can also nominate individuals or groups for a special Posh Smile Ticket Treat as a means of acknowledging hard work, selfless acts and a commitment to their local community – this could be anything from cleaning up litter to helping an elderly person, or indeed a treat for someone who is seriously ill and their Carer.
The number of tickets available for matches governed by the amount of money in the Posh Smile Tickets fund.
Fansbet are bookmakers with a difference as it gives back 50% of its net profit to supporters groups, such as the PST.
The PST advocates responsible gambling, so we don't wish to encourage people to start betting if they don't already do so, but if you do already enjoy a flutter, then why not use Fansbet instead of your usual online betting service.
What is a Posh Smile Ticket?
It is a free ticket to attend a Peterborough United home match.
Who is entitled to apply for a Posh Smile Ticket?
Local community groups and disadvantaged fans can apply. You can also nominate individuals or groups for a Posh Smile Ticket Treat as a means of acknowledging hard work, selfless acts and devotion in the local community etc. – this could be anything from cleaning up litter to helping an elderly person, a treat for someone who is seriously ill or indeed their hard working Carer.
How many Posh Smile Tickets are available?
The number of tickets available for each match is limited only by the amount of money in the Smile Ticket Fund. The Posh Supporters Trust launched the scheme with a £500 donation and Fansbet sponsor the scheme so we receive regular income from them to keep the scheme going, however your donations / fund raising can help to make the Posh Smile Tickets scheme work even better!
Can you offer some Sponsorship to our scheme – and receive some great publicity!
Participating Businesses will receive acknowledgement on our Website and in our Newsletters.


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Obviously one of the biggest reasons anyone choices any field of work is the potential salary, of course there's other reasons that may come into play such as a desire for that line of work, but salary is very important. It's nothing to feel bad about, people get jobs to make money in order to provide for themselves and/or their families, so worrying about salary is very normal.
Here you'll learn what the very lowest rates to be expected as a Pharmacy Technician are, as well as more of the average middle of the road rates, and then what the most successful Pharmacy Technicians are able to make. It all comes down to education, experience, and usually more importantly – finding the right job that will pay you what you're worth.
Unfortunately, there's no solid answer to how much a Pharmacy Technician will make, since this varies greatly from state to state as well as from job to job. The very minimum a PT can expect to make is somewhere around $9/hr working inside of a Kmart, but this job is usually no experience or education required, and on par with pretty much any other low-end job. The upside to working as a Pharmacy Technician at Kmart as opposed to a retail associate, is that is going to look a lot better on a resume.
Don't be scared off by the low pay-rate of a Pharmacy Tech working at Kmart, because there's a lot of entry-level Pharmacy Tech positions that pay upwards of $12/hr, which is pretty good considering there may be no training or education required.
If the above pay-rates are somewhat of a step down from what you're making now, or something you just can't afford to get into, there's still plenty of hope for you becoming a Pharmacy Technician. With some moderate training, you can really increase the amount you can earn with this job position. The most you can expect to make with this job is $50,000 a year and beyond, but generally that only will occur with a 4 year degree and years of experience.
For those who are wanting a quick jump into a career earning decent money, it is very possible as a Pharmacy Technician. This varies from state to state, but a safe guess is with one or two certifications passed (this may require taking a class, but could also be done with self-research) you should easily make between $27,000 – $33,000 a year.
Besides education, the best way to ensure the best pay rate as a Pharmacy Tech is to try landing a job in a Hospital Pharmacy. Hospitals will pay much higher and offer better benefits for their PTs as opposed to Pharmacies located inside of Grocery Stores or Convenience Stores, not to mention the room for advancement is much greater.
What are the job duties of a Pharmacy Technician?


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