Journal: Nature Communications

Article Title: In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters

doi: 10.1038/ncomms13660

Figure Lengend Snippet: TeTx/LC was transiently expressed in Sx1A-mEos2-expressing larvae. Single-molecule tracking was then carried out on the motor nerve terminals. ( a ) Representative low-resolution image, sptPALM average intensity, diffusion coefficient and trajectory map of Sx1A-mEos2 in larvae with motor neuron expression of TeTx/LC (1,836 trajectories analysed). Scale bar, 3 μm. ( b ) Low-resolution image, sptPALM average intensity, diffusion coefficient and trajectory map of Sx1A-mEos2 in PC12 cells. ( c – e ) Blocking SNARE complex formation via TeTx/LC expression increased the MSD, the diffusion coefficient distribution and the ratio of the mobile to immobile fractions of Sx1A-mEos2 in live motor nerve terminals (Sx1A-mEos2 at the NMJ, 3.5±0.1; Sx1A-mEos2 with TeTx/LC expression at the NMJ, 8.6±0.8; and Sx1A-mEos2 in PC12 cells, 6.5±0.4). This increase in Sx1A-mEos2 mobility is comparable to that observed in PC12 cells ( n =19 NMJ chains for Sx1A-mEos2-expressing larvae, n =14 NMJ chains for larvae also expressing TeTx/LC and n =23 cells for PC12; average of ∼3,000 trajectories in NMJ chains and ∼11,500 trajectories in PC12 cells). Comparisons were performed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. * P <0.05, *** P <0.001. ( f , g ) TeTx/LC expression significantly decreased the Sx1A-mEos2 cluster size from 83.5±4.2 to 58.4±4.0 nm. The average Sx1A-mEos2 density also decreased significantly from 57.2±7.9 to 21.2±2.9 μm −2 . Statistical tests were performed using the Mann–Whitney U -test *** P <0.001. ( h ) Western blot showed that VAMP2 was cleaved by TeTx/LC expression at 32 °C; at 25 °C, there was no TeTx/LC expression and VAMP2 remained intact. ( i – k ) Excitatory junction potentials in wild-type Drosophila larvae, Sx1A-mEos2 transgenic larvae with tubulin Gal80 suppression of TeTx/LC (25 °C) and Sx1A-mEos2 larvae with TeTx/LC expression (32 °C); the amplitude of evoked release decreased significantly on TeTx/LC expression (wild type, 35.3±0.2 mV; TeTx/LC suppression at 25 °C, 35.2±0.1 mV; and TeTx/LC expression at 32 °C, 2.6±0.2 mV). The quantal content decreased significantly with the expression of TeTx/LC (wild type, 58.1±1.0; TeTx/LC suppression at 25 °C, 46.8±3.2; and TeTx/LC expression at 32 °C, 3.1±0.3). Comparisons were performed using one-way ANOVA with Tukey’s multiple comparison test * P <0.05, *** P <0.001. Mean±s.e.m. are plotted. WT, wild type.

Article Snippet: To quantify VAMP2 cleavage by TeTx/LC, third instar larvae were used for protein extraction and detected with VAMP2 antibody (rabbit anti-VAMP2, #104 202 1:1000 Synaptic Systems).

Techniques: Expressing, Diffusion-based Assay, Blocking Assay, Comparison, MANN-WHITNEY, Western Blot, Transgenic Assay

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: In vivo potentiation of human oestrogen receptor alpha by Cdk7-mediated phosphorylation.

doi: 10.1111/j.1365-2443.2004.00777.x

Figure Lengend Snippet: Figure 1 Ligand dependent transactivation of hERα in Drosophila. (A) Schematic representation of hERα constructs. The DNA binding domain (DBD) is located in the C domain (grey box). The transactivation function-1 (AF-1) region is located in the N-terminal A/B domain (blue box), while the transactivation function-2 (AF-2) region is located in the C-terminal E/F domain (white box) that also contains the ligand binding domain (LBD). (B) Ligand-dependent transactivation of hERα mutants in eye imaginal discs. Expression of hERα mutants in third instar larva eye discs driven by GMR-GAL4 was detected with ERα antibodies (B10 or HC-20) (red). Transactivation of hERα mutants was estimated by GFP expression (green). The anterior is to the right. Bottom panels: hERα and GFP expression in four pairs of adult heads as detected by Western blotting. Fold-activation was calculated using hERα expression levels as normalizing factor. Ligands, 10−3 m 17β-oestradiol (E2), 10−2 m tamoxifen (TAM), and 10−2 m ICI 182.780 (ICI), were added in 100 µL of vehicle on top of 10 mL of the medium before hatching. Flies were kept at 25 °C. (C) Measurement of hERα mutants transactivation in Schneider cells. Schneider cells were transfected with hERα mutant expression plasmids, Actin-GAL4 plasmid, ERE-tk-luc reporter plasmid and pRL-CMV internal control plasmid in the presence or absence of 10−8 m E2, 10−8 m TAM or 10−8 m ICI. Firefly luciferase activity (ERE-tk-luc) was measured and normalized against Renilla activity (pRL-CMV-luc) as an internal control. Data are shown as the average and standard deviation of three independent experiments.

Article Snippet: To confirm hERα and GFP expression in Drosophila, cell lysates from the heads of adult flies of third instar larvae were separated by 15% SDS–PAGE and detected with anti-ERα antibodies (HC20 or B10) and anti-GFP antibody (Santa Cruz Biotechnology), and expression levels measured using Adobe Photoshop software facility.

Techniques: Construct, Binding Assay, Ligand Binding Assay, Expressing, Western Blot, Activation Assay, Transfection, Mutagenesis, Plasmid Preparation, Control, Luciferase, Activity Assay, Standard Deviation

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: In vivo potentiation of human oestrogen receptor alpha by Cdk7-mediated phosphorylation.

doi: 10.1111/j.1365-2443.2004.00777.x

Figure Lengend Snippet: Figure 2 hERα transactivation regulated by Drosophila transcriptional co-activators. hERα expression (red) and transactivation (green) were visualized by immunostaining with ERα antibodies (B10 and HC-20) and GFP expression, respectively, in eye imaginal discs. Fly lines contained single copies of GMR-GAL4, UAS-hERα (HEG0 or HE15) and ERE-GFP with or without heterozygous tai k05809, UAS-tai or nej3.

Article Snippet: To confirm hERα and GFP expression in Drosophila, cell lysates from the heads of adult flies of third instar larvae were separated by 15% SDS–PAGE and detected with anti-ERα antibodies (HC20 or B10) and anti-GFP antibody (Santa Cruz Biotechnology), and expression levels measured using Adobe Photoshop software facility.

Techniques: Expressing, Immunostaining

Journal: PLoS Genetics

Article Title: A homozygous KAT2B variant modulates the clinical phenotype of ADD3 deficiency in humans and flies

doi: 10.1371/journal.pgen.1007386

Figure Lengend Snippet: (A) Pedigree and segregation status of mutations found in ADD3 and KAT2B . Discovery of ADD3 mutations in family B and C was facilitated by GeneMatcher . Half red coloured circles or squares denote patients with neurological defects and half blue coloured symbols denote patients with SRNS and cardiomyopathy. + symbols indicate non-mutated alleles. Mutations and segregation were confirmed by Sanger sequencing. (B) Exon structure of human ADD3 cDNA (long isoform NP_058432) and domains of adducin-γ protein. The relative position of ADD3 mutations to protein domains and exons are indicated (arrows). All mutations also affect the short isoform of ADD3 (NP_001112). Below each mutation, the phylogenetic conservation of the altered amino acid residues is shown. (C) Exon structure of human KAT2B cDNA and domains of KAT2B protein. PCAF-HD, p300/CBP-associated factor homology domain; AT, acetyl transferase domain; B, Bromo domain. The relative position of KAT2B variation to protein domains and exons is indicated (arrow). The phylogenetic conservation of the altered amino acid residue is shown. (D, E) Adducin-γ (D) and KAT2B (E) protein levels in control and patient fibroblasts. Lysates of patient II-3 and II-6 (family A) fibroblasts and age-matched control fibroblasts (Ctrl 1 and 2) were analyzed by western blotting. Results were normalized to the loading control α-tubulin. Each quantification is shown in the lower panel (n = 3 independent experiments, student’s t-test).

Article Snippet: Total cell or third instar larvae total protein extractions were performed and the resolved proteins were probed using the primary antibodies: anti-PCAF rabbit monoclonal (3378, Cell Signaling,1:1000), anti-adducin-γ mouse monoclonal (sc-74474, Santa Cruz, 1:1000) and anti-αtubulin mouse monoclonal (T5168, Sigma Aldrich, 1:5000).

Techniques: Sequencing, Mutagenesis, Western Blot

Journal: PLoS Genetics

Article Title: A homozygous KAT2B variant modulates the clinical phenotype of ADD3 deficiency in humans and flies

doi: 10.1371/journal.pgen.1007386

Figure Lengend Snippet: (A) Viability for hts null hemizygous flies and respective rescues with adducin (Add) construct(s) using tubulin- GAL4 ( tub> ). After 48h of egg laying on standard cornmeal/yeast food, viability was calculated as the percentage of hatching adults of the indicated genotype and normalized to the control. The control corresponds to the viable F1 trans-heterozygous flies obtained from the cross between Df(2R)BSC26 (harbouring the hts gene) and a non-overlapping deficiency on the same chromosome ( Df(2R)247 ). Quantification is for >100 F1 eclosing flies/genotype/experiment in 5 independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni post-test. (B) Negative geotaxis assay for one-day-old adult flies. Flies were transferred to a graduated tube, and after tapping, the length climbed in 8 sec was recorded . Quantification was performed on 6 independent experiments with >38 flies/genotype using one-way ANOVA with Kruskal-Wallis post-test. ns, non significant (see for details on transgenic flies). For all panels: ns, non significant, *p<0.05 **p<0.01, ***p<0.001 (see for details on transgenic flies). (C) Viability for Gcn5 null hemizygous flies and respective rescues with Gcn5 and KAT2A/B construct(s) using daughterless- GAL4 ( da> ). Viability was assessed as described in (A). Human KAT2B F307S and S502F mutations correspond to Gcn5 F304S and S478F mutations, respectively. Gcn5 S502F variant predicted to be deleterious (PolyPhen-2 score of 0.98) was found on a healthy individual at the homozygous state in our in-house exome database. The control corresponds to the viable F1 trans-heterozygous flies obtained from the cross between Df(3L)sex204 (harbouring the Gcn5 gene) and a non-overlapping lethal mutant on the same chromosome ( CG3103 0MI0010 ). Quantification is for >100 F1 eclosing flies/genotype/experiment in 5 independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni post-test. (D) Phenotype of Gcn5 WT, Gcn5 F304S and Gcn5 S478F rescue animals. Pictures correspond to adult flies one day post-eclosion and are representative of the defects found in wings (separated wing blades), legs (femur kinking, arrow) and eye (small and mild rough eye). Scale bars: wings: 500μm, legs: 500 μm, eye: 200μm. (E) Quantification of the defects in wings, legs and eyes found in Gcn5 F304S rescue flies is for >100 F1 eclosing flies/genotype. (F) H3K9 acetylation levels of Gcn5 null and Gcn5 WT and F304S rescue animals. Extracted nuclear proteins from 3 rd instar (= late) larvae were analysed by western blotting normalized to non-acetylated Histone 3 (H3). Quantification is shown in the lower panel (n = 3 independent experiments; one-way ANOVA with Bonferroni post-test). For all panels: ns, non significant, *p<0.05 **p<0.01, ***p<0.001 (see for details on transgenic flies).

Article Snippet: Total cell or third instar larvae total protein extractions were performed and the resolved proteins were probed using the primary antibodies: anti-PCAF rabbit monoclonal (3378, Cell Signaling,1:1000), anti-adducin-γ mouse monoclonal (sc-74474, Santa Cruz, 1:1000) and anti-αtubulin mouse monoclonal (T5168, Sigma Aldrich, 1:5000).

Techniques: Construct, Transgenic Assay, Variant Assay, Mutagenesis, Western Blot

Journal: Journal of cell science

Article Title: Functional analysis of parvin and different modes of IPP-complex assembly at integrin sites during Drosophila development.

doi: 10.1242/jcs.102384

Figure Lengend Snippet: Fig. 4. ILK levels control parvin stability and recruitment at MASs. (A–D) Dorsal oblique muscles of late stage living embryos expressing parvin–GFP. (E) Western blot showing parvin (left panel) or both parvin and tubulin (right panel). Asterisks (*), indicate non-specific bands that appeared in a genotype- dependent manner (compare FMZ–GFP with +/TM3- Ser{act:GFP}). The closed arrow indicates the endogenous parvin and the open arrows the tubulin doublet band. (F) MASs at dorsal oblique muscles from a late embryo expressing parvin–GFP (green, F9) and actin5C–mRFP (red, F99). (G–J) Somatic muscles of late embryos expressing GFP–CHShot (green, G) or high levels of parvin–GFP (green, H) and F-actin visualized with Rhodamine-labeled phalloidin (red) in wild type (G,H), ilk mutants (I) or pinch mutants (J). (K–P) Third instar larvae muscles expressing parvin– GFP with mef2Gal4 (K) together with UAS::ILK (L), UAS::Paxillin (M), UAS::PINCH (N), UAS::ILKE356K

Article Snippet: Imaginal wing discs were dissected from third-instar larvae and fixed in phosphate buffered saline containing 4% formaldehyde for 30 min. Primary antibodies were against: parvin (rabbit polyclonal; 1:500), PINCH (rabbit polyclonal; 1:500) (Clark et al., 2003), paxillin (rabbit polyclonal; 1:500) (Chen et al., 2005), pY397-FAK (rabbit polyclonal; 1:500, Cell Signaling), MHC (myosin heavy chain) (mouse monoclonal; 1:60) (Kiehart et al., 1990), and bPS (mouse monoclonal CF.6G11; 1:10) (Brower et al., 1984).

Techniques: Control, Muscles, Expressing, Western Blot, Labeling