The FA women's super league : framing developments in elite women's football

In 2009, the Football Association (FA), the national governing body of football in England, announced its plan to introduce the country's first semi-professional women's elite league. Launched in 2010 as the FA Women's Super League (FA WSL), its introduction provided both an opportunity to research whether this evidenced a change of position for the women's elite game within footballing narratives and also to examine the place of the FA within these. This study adopted a critical sociological feminist approach to deconstruct the assumptions, values and practices that frame the female game and the introduction of the FA WSL, while providing new insights into the role of the sport's governing organisation in defining elite women's football. Through observations at matches and interviews with people working within the women's game, an examination of the development and introduction of the FA WSL was undertaken, with valuable early insights provided into the first three years of the new League. The study identified that the introduction of the FA WSL was impacted upon by the complex, closed and gendered nature of the FA's organisational structure. The new League adhered to traditional societal concepts of hegemonic masculinity, heteronormativity and liberal approaches to gender equality. The study also found that the new elite women's structures required the clubs who gained entry into the FA WSL to adhere to commercialised, spectacularised and commodified values which dominate the men's game and neo liberal societal narratives. The increased inclusion of females into elite football structures did not profoundly disrupt traditional discourses or provide evidence of a fundamental challenge to gender inequality in the game

Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors

<p>The small GTPase Rac1 is implicated in various cellular processes that are essential for normal cell function. Deregulation of Rac1 signaling has also been linked to a number of diseases, including cancer. The diversity of Rac1 functioning in cells is mainly attributed to its ability to bind to a multitude of downstream effectors following activation by Guanine nucleotide Exchange Factors (GEFs). Despite the identification of a large number of Rac1 binding partners, factors influencing downstream specificity are poorly defined, thus hindering the detailed understanding of both Rac1's normal and pathological functions. In a recent study, we demonstrated a role for 2 Rac-specific GEFs, Tiam1 and P-Rex1, in mediating Rac1 anti- versus pro-migratory effects, respectively. Importantly, via conducting a quantitative proteomic screen, we identified distinct changes in the Rac1 interactome following activation by either GEF, indicating that these opposing effects are mediated through GEF modulation of the Rac1 interactome. Here, we present the full list of identified Rac1 interactors together with functional annotation of the differentially regulated Rac1 binding partners. In light of this data, we also provide additional insights into known and novel signaling cascades that might account for the GEF-mediated Rac1-driven cellular effects.</p

Global Dynamics of the <i>Escherichia coli</i> Proteome and Phosphoproteome During Growth in Minimal Medium

Recent phosphoproteomics studies have generated relatively large data sets of bacterial proteins phosphorylated on serine, threonine, and tyrosine, implicating this type of phosphorylation in the regulation of vital processes of a bacterial cell; however, most phosphoproteomics studies in bacteria were so far qualitative. Here we applied stable isotope labeling by amino acids in cell culture (SILAC) to perform a quantitative analysis of proteome and phosphoproteome dynamics of Escherichia coli during five distinct phases of growth in the minimal medium. Combining two triple-SILAC experiments, we detected a total of 2118 proteins and quantified relative dynamics of 1984 proteins in all measured phases of growth, including 570 proteins associated with cell wall and membrane. In the phosphoproteomic experiment, we detected 150 Ser/Thr/Tyr phosphorylation events, of which 108 were localized to a specific amino acid residue and 76 were quantified in all phases of growth. Clustering analysis of SILAC ratios revealed distinct sets of coregulated proteins for each analyzed phase of growth and overrepresentation of membrane proteins in transition between exponential and stationary phases. The proteomics data indicated that proteins related to stress response typically associated with the stationary phase, including RpoS-dependent proteins, had increasing levels already during earlier phases of growth. Application of SILAC enabled us to measure median occupancies of phosphorylation sites, which were generally low (<12%). Interestingly, the phosphoproteome analysis showed a global increase of protein phosphorylation levels in the late stationary phase, pointing to a likely role of this modification in later phases of growth

Global Dynamics of the <i>Escherichia coli</i> Proteome and Phosphoproteome During Growth in Minimal Medium

Recent phosphoproteomics studies have generated relatively large data sets of bacterial proteins phosphorylated on serine, threonine, and tyrosine, implicating this type of phosphorylation in the regulation of vital processes of a bacterial cell; however, most phosphoproteomics studies in bacteria were so far qualitative. Here we applied stable isotope labeling by amino acids in cell culture (SILAC) to perform a quantitative analysis of proteome and phosphoproteome dynamics of Escherichia coli during five distinct phases of growth in the minimal medium. Combining two triple-SILAC experiments, we detected a total of 2118 proteins and quantified relative dynamics of 1984 proteins in all measured phases of growth, including 570 proteins associated with cell wall and membrane. In the phosphoproteomic experiment, we detected 150 Ser/Thr/Tyr phosphorylation events, of which 108 were localized to a specific amino acid residue and 76 were quantified in all phases of growth. Clustering analysis of SILAC ratios revealed distinct sets of coregulated proteins for each analyzed phase of growth and overrepresentation of membrane proteins in transition between exponential and stationary phases. The proteomics data indicated that proteins related to stress response typically associated with the stationary phase, including RpoS-dependent proteins, had increasing levels already during earlier phases of growth. Application of SILAC enabled us to measure median occupancies of phosphorylation sites, which were generally low (<12%). Interestingly, the phosphoproteome analysis showed a global increase of protein phosphorylation levels in the late stationary phase, pointing to a likely role of this modification in later phases of growth

Global Dynamics of the <i>Escherichia coli</i> Proteome and Phosphoproteome During Growth in Minimal Medium

Recent phosphoproteomics studies have generated relatively large data sets of bacterial proteins phosphorylated on serine, threonine, and tyrosine, implicating this type of phosphorylation in the regulation of vital processes of a bacterial cell; however, most phosphoproteomics studies in bacteria were so far qualitative. Here we applied stable isotope labeling by amino acids in cell culture (SILAC) to perform a quantitative analysis of proteome and phosphoproteome dynamics of Escherichia coli during five distinct phases of growth in the minimal medium. Combining two triple-SILAC experiments, we detected a total of 2118 proteins and quantified relative dynamics of 1984 proteins in all measured phases of growth, including 570 proteins associated with cell wall and membrane. In the phosphoproteomic experiment, we detected 150 Ser/Thr/Tyr phosphorylation events, of which 108 were localized to a specific amino acid residue and 76 were quantified in all phases of growth. Clustering analysis of SILAC ratios revealed distinct sets of coregulated proteins for each analyzed phase of growth and overrepresentation of membrane proteins in transition between exponential and stationary phases. The proteomics data indicated that proteins related to stress response typically associated with the stationary phase, including RpoS-dependent proteins, had increasing levels already during earlier phases of growth. Application of SILAC enabled us to measure median occupancies of phosphorylation sites, which were generally low (<12%). Interestingly, the phosphoproteome analysis showed a global increase of protein phosphorylation levels in the late stationary phase, pointing to a likely role of this modification in later phases of growth

Quantitative Phosphoproteomics of Murine <i>Fmr1</i>-KO Cell Lines Provides New Insights into FMRP-Dependent Signal Transduction Mechanisms

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that has a major effect on neuronal protein synthesis. Transcriptional silencing of the <i>FMR1</i> gene leads to loss of FMRP and development of Fragile X syndrome (FXS), the most common known hereditary cause of intellectual impairment and autism. Here we utilize SILAC-based quantitative phosphoproteomics to analyze murine <i>FMR1</i>^– and <i>FMR1</i>⁺ fibroblastic cell lines derived from <i>FMR1</i>-KO embryos to identify proteins and phosphorylation sites dysregulated as a consequence of FMRP loss. We quantify FMRP-related changes in the levels of 5,023 proteins and 6,133 phosphorylation events and map them onto major signal transduction pathways. Our study confirms global downregulation of the MAPK/ERK pathway and decrease in phosphorylation level of ERK1/2 in the absence of FMRP, which is connected to attenuation of long-term potentiation. We detect differential expression of several key proteins from the p53 pathway, pointing to the involvement of p53 signaling in dysregulated cell cycle control in FXS. Finally, we detect differential expression and phosphorylation of proteins involved in pre-mRNA processing and nuclear transport, as well as Wnt and calcium signaling, such as PLC, PKC, NFAT, and cPLA2. We postulate that calcium homeostasis is likely affected in molecular pathogenesis of FXS

Effect of overbalancing phosphorylation activity on the integrity of spore envelopes.

<p>Live-dead staining of spore chains of the eSTPK mutants NLΔPkaI and NLΔ4775–4779 (A) revealed the presence of dead spores (red) or spores without DNA (black). In contrast, spore chains of the parental M145 strain (A) only contained viable spores (green). Expression of a second copy of any eSTPK gene of cluster <i>SCO4775-4779</i> (B-F) caused a similar sporulation defect in <i>S</i>. <i>coelicolor</i> M145, NLΔPkaI, or NLΔ4775–4779. None of the eSTPK genes was able to complement aberrant sporulation of the five-fold mutant NLΔ4775–4779. A, no plasmid integrated; B, :: pSET152-pkaH; C, :: pSET152-SCO4776; D, :: pSET152-pkaD; E, :: pSET152-pkaI; F, :: pSET152-pkaJ. Bar = 5 μm.</p

Effect of Ser/Thr kinases on the viability of spores (A) and the resistance of germinating spores to vancomycin (B).

<p>Spore chains were stained with the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes) and observed by fluorescence microscopy. Percentage of viable (green), dead (red) and spores without DNA (black) is given for each strain. Spores of the different strains were plated onto LB agar and filter discs containing 5 μg vancomycin were applied. Whereas, M145 and the <i>pkaI</i> mutant NLΔPkaI were resistant, NLΔ4775–4779 spores showed vancomycin sensitivity, suggesting an impaired spore wall. Vancomycin sensitivity of M145 or NLΔPkaI was also caused by expressing a second copy of each kinase gene, with the exception of <i>pkaI</i>. Supplementation of the agar plates with 3 mM MgCl_2, known to rescue mutants impaired in cell wall synthesis restored vancomycin resistance to all strains. A, no plasmid integrated; B, :: pSET152-pkaH; C, :: pSET152-SCO4776; D, :: pSET152-pkaD; E, :: pSET152-pkaI; F, :: pSET152-pkaJ.</p

Effect of overbalancing phosphorylation activity on proper sporulation of <i>S</i>. <i>coelicolor</i>.

<p>Phase contrast microscopy of spore chains revealed the presence of aberrant spores (arrows) in eSTPK mutants NLΔPkaI and NLΔ4775–4779 (A). In contrast, spore chains of the parental M145 strain (A) and the complemented mutant NLΔPkaI::pSET-pkaI (E) contain mainly regular ovoid spores. Not only deletion, but also expression of a second copy of any eSTPK gene of cluster <i>SCO4775-4779</i> causes a similar sporulation defect (B-F, white arrows) in <i>S</i>. <i>coelicolor</i> M145, NLΔPkaI, or NLΔ4775–4779. None of the eSTPK genes is able to complement aberrant sporulation of the five-fold mutant NLΔ4775–4779. A, no plasmid integrated; B, :: pSET152-pkaH; C, :: pSET152-SCO4776; D, :: pSET152-pkaD; E, :: pSET152-pkaI; F, :: pSET152-pkaJ. Bar = 5 μm.</p

Phosphorylation of PBP2 by PkaI.

<p><i>S</i>. <i>coelicolor pbp2</i> with a S-tag encoding sequence was co-expressed with <i>pkaI</i> in <i>E</i>. <i>coli</i>. PBP2_S-tag was purified under denaturing conditions by affinity chromatography. PkaI with an N-terminal His-tag was purified by Ni-NTA chromatography under native conditions. Purified His_PkaI and PBP2_S-tag proteins (bold letters and underlining indicate, which protein was purified) were separated on an SDS polyacrylamide gel and stained with Coomassie blue (<b>A</b>). Phosphorylated proteins were identified by ProQ Diamond staining. The white arrow indicates auto-phosphorylated His_PkaI, while the black arrow marks phosphorylated PBP2_S-tag. Immunoblotting with Anti-S-tag antibodies confirmed the identity of PBP2_S-tag. Domain architecture of PBP2 and positions of the most likely phosphosites (<b>B</b>). Predicted Pfam domains (PBP dimerization, dark grey, PBP transpeptidase, light grey), a transmembrane helix (TM) and the positions of phosphorylated S/T residues, identified by LC-MS/MS, are indicated.</p