Journal: The Journal of Neuroscience

Article Title: Titration of Syntaxin1 in Mammalian Synapses Reveals Multiple Roles in Vesicle Docking, Priming, and Release Probability

doi: 10.1523/JNEUROSCI.0187-13.2013

Figure Lengend Snippet: Hypomorphic expression of a Stx1Byfp fusion knock-in allele. A, Cloning strategy for the Stx1Byfp fusion allele. Double asterisks denote a modified exon 7 described in Gerber et. al. (2008). B, Diagrams depicting the orientations of plasma membrane-anchored wild-type (top) and EYFP-fused Stx1B (bottom). C, RT-PCR amplification of a full ∼1.6 kb transcript encoded by the Stx1Byfp allele from Stx1B+/yfp mRNA. D, Immunoblot showing severely reduced levels of Stx1B–YFP in Stx1Byfp/yfp;Stx1A−/− embryonic brain lysates compared with lysates from control littermates. β-tubulin was used as a loading control. E, Immunofluorescent staining of hippocampal mass cultures for extracellularly localized YFP. Stx1Byfp/yfp, but not wild-type controls transduced with intracellular fluorescent protein, is positive for surface expression of YFP. Scale bar, 10 μm. F, Peroxidase immunostaining of Stx1B–YFP fusion protein in sagittal sections of P14 hippocampus and cerebellum. Antibodies to synaptotagmin 1 (Syn1) and Rab3A were used as positive controls, while PBS corresponds to negative control. DG, Dentate gyrus; MF, hippocampal mossy fiber synapse. G, Immunoblot showing expression levels of SNARE proteins, Munc18-1, and other synaptic proteins in wild-type, Stx1Byfp/yfp, and Stx1A−/− P14 mice. Valosin-containing protein (VCP) was used as a loading control. Asterisks mark proteins with significant changes in expression levels.

Article Snippet: For RNAi knock-down experiments, a Stx1B -specific short hairpin RNA (shRNA) target sequence (5′-GAT CCC AGG CAC AAT GAG ATC ATC AAA-3′) was obtained using Genscript siRNA Target Finder ( http://www.genscript.com ).

Techniques: Expressing, Knock-In, Cloning, Modification, Clinical Proteomics, Membrane, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Control, Staining, Transduction, Immunostaining, Negative Control

Journal: The Journal of Neuroscience

Article Title: Titration of Syntaxin1 in Mammalian Synapses Reveals Multiple Roles in Vesicle Docking, Priming, and Release Probability

doi: 10.1523/JNEUROSCI.0187-13.2013

Figure Lengend Snippet: Stx1Byfp/yfp;Stx1A−/− double mutants show premature lethality, gross morphological defects, and diminished Ca2+-evoked and spontaneous release. A, Weight curves for the Stx1Byfp mouse line. Stx1A+/+;Stx1Byfp/yfp and StxA+/−; Stx1Byfp/yfp show impaired growth and development before death at 3 weeks. B, Survival curves for the Stx1Byfp mouse line. Stx1A+/+;Stx1Byfp/yfp and Stx1A+/−;Stx1Byfp/yfp die postnatally after 3 weeks. Stx1A−/−;Stx1Byfp/yfp are not shown because they die at birth. C, Gross morphological abnormalities in Stx1Byfp/yfp;Stx1A−/− embryos compared with Stx1B+/yfp;Stx1A−/− and Stx1B+/+;Stx1A−/− littermates at E18.5. D, Sample traces of EPSCs from Stx1B+/+;Stx1A−/− (black), Stx1B+/yfp;Stx1A−/− (blue), and Stx1Byfp/yfp;Stx1A−/− (red) autaptic neurons. E, Normalized summary plot of EPSC peak amplitudes (***p < 0.0001). F, Normalized summary plot of EPSC rise times (***p < 0.0001). G, Sample traces of mEPSCs recorded at −70 mV from Stx1B+/+;Stx1A−/− (black) and Stx1Byfp/yfp;Stx1A−/− (red) neurons. H, Normalized summary plot of mEPSC frequency (***p < 0.0001).

Article Snippet: For RNAi knock-down experiments, a Stx1B -specific short hairpin RNA (shRNA) target sequence (5′-GAT CCC AGG CAC AAT GAG ATC ATC AAA-3′) was obtained using Genscript siRNA Target Finder ( http://www.genscript.com ).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Titration of Syntaxin1 in Mammalian Synapses Reveals Multiple Roles in Vesicle Docking, Priming, and Release Probability

doi: 10.1523/JNEUROSCI.0187-13.2013

Figure Lengend Snippet: Stx1Byfp/yfp;Stx1A−/− double mutants have a smaller RRP of vesicles and slower RRP refilling rate. A, Sample traces of release induced by hyperosmotic stimulation from Stx1B+/+;Stx1A−/− (black), Stx1B+/yfp;Stx1A−/− (blue), and Stx1Byfp/yfp;Stx1A−/− (red) autaptic neurons. B, Normalized summary plot of RRP charge (***p < 0.0001). C, Refilling of the RRP as measured by paired applications of 0.5 m sucrose in Stx1B+/+;Stx1A−/− (black) and Stx1Byfp/yfp;Stx1A−/− (red). D, Fraction of the RRP refilled 2, 5, and 10 s after the first hyperosmotic stimulus (**p < 0.001).

Article Snippet: For RNAi knock-down experiments, a Stx1B -specific short hairpin RNA (shRNA) target sequence (5′-GAT CCC AGG CAC AAT GAG ATC ATC AAA-3′) was obtained using Genscript siRNA Target Finder ( http://www.genscript.com ).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Titration of Syntaxin1 in Mammalian Synapses Reveals Multiple Roles in Vesicle Docking, Priming, and Release Probability

doi: 10.1523/JNEUROSCI.0187-13.2013

Figure Lengend Snippet: Vesicle fusogenicity is impaired in Stx1Byfp/yfp;Stx1A−/− neurons. A, Sample traces of evoked responses from Stx1B+/+;Stx1A−/− (black) and Stx1Byfp/yfp;Stx1A−/− (red) neurons at different external Ca2+ concentrations. B, Ca2+ dose–response curves for Stx1B+/+;Stx1A−/− (black) and Stx1Byfp/yfp;Stx1A−/− (red) neurons. C, Normalized summary plot of Pvr, measured as the ratio of the EPSC charge and RRP charge, in Stx1B+/+;Stx1A−/− (black), Stx1B+/yfp;Stx1A−/− (blue), and Stx1Byfp/yfp;Stx1A−/− (red) neurons (***p < 0.0001). D, Short-term plasticity behavior during 10 Hz stimulation. E, Sample traces of release induced by different (hyperosmotic) sucrose concentrations. F, Dose–response curves from hypertonic sucrose stimulation. G, Spontaneous release rate, calculated as the fraction of the RRP released spontaneously per second (*p < 0.05).

Article Snippet: For RNAi knock-down experiments, a Stx1B -specific short hairpin RNA (shRNA) target sequence (5′-GAT CCC AGG CAC AAT GAG ATC ATC AAA-3′) was obtained using Genscript siRNA Target Finder ( http://www.genscript.com ).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Titration of Syntaxin1 in Mammalian Synapses Reveals Multiple Roles in Vesicle Docking, Priming, and Release Probability

doi: 10.1523/JNEUROSCI.0187-13.2013

Figure Lengend Snippet: Presynaptic Ca2+ influx is not altered in Stx1Byfp/yfp;Stx1A−/− neurons. A, Example baseline fluorescence (left; average of three images; F0) and peak 5 AP fluorescence (right; average two images; ΔF) for an Stx1Byfp/yfp;Stx1A−/− autaptic neuron. B, Average ΔF/F0 for ROIs selected from neuron shown in A (n = 41) in response to 2, 3, and 5 AP at 20 Hz. Arrow indicates initiation of stimulation. C, Average ΔF/F0 responses per cell for Stx1Byfp/yfp;Stx1A−/− (red; n = 35 cells) and control (black; Stx1B+/+;Stx1A−/− and Stx1B+/yfp;Stx1A−/− pooled; n = 33) for 2, 3, 5, and 10 AP at 20 Hz. D, E, Data were fitted with a linear regression analysis (red and black lines, respectively). Average paired-pulse ratio (D; 50 ms interstimulus interval; ***p < 0.0001) and EPSC amplitude (E; ***p < 0.0001) for Stx1Byfp/yfp;Stx1A−/− and control cells from C. All error bars represent SEM.

Article Snippet: For RNAi knock-down experiments, a Stx1B -specific short hairpin RNA (shRNA) target sequence (5′-GAT CCC AGG CAC AAT GAG ATC ATC AAA-3′) was obtained using Genscript siRNA Target Finder ( http://www.genscript.com ).

Techniques: Fluorescence, Control

Journal: The Journal of Neuroscience

Article Title: Titration of Syntaxin1 in Mammalian Synapses Reveals Multiple Roles in Vesicle Docking, Priming, and Release Probability

doi: 10.1523/JNEUROSCI.0187-13.2013

Figure Lengend Snippet: Knockdown of Stx1B–YFP further impaired release efficiency. A, Immunoblot showing knockdown of both Stx1B (in wild-type background), and Stx1B–YFP fusion protein (in Stx1Byfp/yfp;Stx1A−/− background), but not Stx1A, using a Stx1B sequence-specific shRNA. B, Sample traces of EPSCs from Stx1B+/+;Stx1A−/− (black), Stx1Byfp/yfp;Stx1A−/− untransduced (red), transduced with lentiviral vector only (Lois et al., 2002), and transduced with knock-down construct (purple). C, Sample traces of RRP released by hyperosmotic stimulation. D, Normalized summary plot of EPSC amplitudes (***p < 0.0001). E, Normalized summary plot of RRP charge (*p < 0.05, ***p < 0.001). F, Normalized summary plot of vesicular release probability. G, Short-term plasticity behavior during 10 Hz stimulation.

Article Snippet: For RNAi knock-down experiments, a Stx1B -specific short hairpin RNA (shRNA) target sequence (5′-GAT CCC AGG CAC AAT GAG ATC ATC AAA-3′) was obtained using Genscript siRNA Target Finder ( http://www.genscript.com ).

Techniques: Knockdown, Western Blot, Sequencing, shRNA, Transduction, Plasmid Preparation, Construct

Journal: The Journal of Neuroscience

Article Title: Titration of Syntaxin1 in Mammalian Synapses Reveals Multiple Roles in Vesicle Docking, Priming, and Release Probability

doi: 10.1523/JNEUROSCI.0187-13.2013

Figure Lengend Snippet: Knockdown of wild-type Stx1B phenocopied Stx1Byfp/yfp;Stx1A−/− hypomorphs. A, Sample traces of EPSCs from Stx1B+/+;Stx1A−/− neurons untransduced (black), transduced with lentiviral vector only (blue), or transduced with a Stx1B sequence-specific shRNA (green). B, Normalized summary plot of EPSC amplitudes (***p < 0.0001). C, Sample traces of RRP released by hyperosmotic stimulation. D, Normalized summary plot of RRP charge (***p < 0.0001). E, Normalized summary plot of vesicular release probability (**p < 0.001). F, Short-term plasticity behavior during 10 Hz stimulation. G, Sample images of autaptic neurons double labeled with the synaptic marker VGluT1 and the dendritic marker MAP2. Scale bar, 10 μm. H, Summary plot of synaptic vesicle density measured as VGluT1 puncta per 100 μm of dendrite length.

Article Snippet: For RNAi knock-down experiments, a Stx1B -specific short hairpin RNA (shRNA) target sequence (5′-GAT CCC AGG CAC AAT GAG ATC ATC AAA-3′) was obtained using Genscript siRNA Target Finder ( http://www.genscript.com ).

Techniques: Knockdown, Transduction, Plasmid Preparation, Sequencing, shRNA, Labeling, Marker

Journal: The Journal of Neuroscience

Article Title: Titration of Syntaxin1 in Mammalian Synapses Reveals Multiple Roles in Vesicle Docking, Priming, and Release Probability

doi: 10.1523/JNEUROSCI.0187-13.2013

Figure Lengend Snippet: Lentiviral expression of either Stx1A or Stx1B, but not Munc18-1, rescues synaptic release in neurons with severe reduction in Stx1 levels. A–C, Normalized summary plots of EPSC peak amplitudes (A), RRP charge (B), and vesicular release probability (C) in control Stx1Byfp/yfp;Stx1A−/− neurons (black), and Stx1Byfp/yfp;Stx1A−/− neurons transduced without (red) or with Stx1A, Stx1B, or Munc18-1 rescue constructs (red outlines; ANOVA with Tukey's test, ***p < 0.001; n.s., not significant). D–F, Normalized summary plots of EPSC peak amplitudes (D), RRP charge (E), and vesicular release probability (F) in Stx1A−/− neurons without (black) or with shRNA-mediated knockdown of Stx1B (teal), and knockdown neurons transduced with Stx1A, Stx1B, or Munc18-1 rescue constructs (teal outlines; ANOVA with Tukey's test, *p < 0.05, ***p < 0.001; n.s., not significant).

Article Snippet: For RNAi knock-down experiments, a Stx1B -specific short hairpin RNA (shRNA) target sequence (5′-GAT CCC AGG CAC AAT GAG ATC ATC AAA-3′) was obtained using Genscript siRNA Target Finder ( http://www.genscript.com ).

Techniques: Expressing, Control, Construct, shRNA, Knockdown, Transduction

Journal: The Journal of Neuroscience

Article Title: Titration of Syntaxin1 in Mammalian Synapses Reveals Multiple Roles in Vesicle Docking, Priming, and Release Probability

doi: 10.1523/JNEUROSCI.0187-13.2013

Figure Lengend Snippet: Vesicle distance from the active zone upon reduction of Stx1 expression. A, Sample tomographic images of presynaptic nerve terminals from Stx1B+/+;Stx1A−/− neurons and Stx1Byfp/yfp;Stx1A−/− neurons that were untransduced and transduced with a Stx1B sequence-specific shRNA. Scale bar, 100 nm. B, Cumulative distribution of vesicle distance from the plasma membrane. C, Summary plot of vesicle distance within 40 nm of the plasma membrane. *p < 0.05. D, Summary plot of presynaptic area. E, Summary plot of total number of synaptic vesicles per synapse. **p < 0.001. F, Summary plot of synaptic vesicle density (ANOVA with Tukey's test, **p < 0.001). G, Summary plot of active zone length.

Article Snippet: For RNAi knock-down experiments, a Stx1B -specific short hairpin RNA (shRNA) target sequence (5′-GAT CCC AGG CAC AAT GAG ATC ATC AAA-3′) was obtained using Genscript siRNA Target Finder ( http://www.genscript.com ).

Techniques: Expressing, Transduction, Sequencing, shRNA, Clinical Proteomics, Membrane

Journal: The Journal of Neuroscience

Article Title: Titration of Syntaxin1 in Mammalian Synapses Reveals Multiple Roles in Vesicle Docking, Priming, and Release Probability

doi: 10.1523/JNEUROSCI.0187-13.2013

Figure Lengend Snippet: Priming and release probability as a function of Stx1 expression levels. A, Normalized summary plot of Stx1B fluorescence intensities. ***p < 0.0001. B, Normalized summary plot of Bassoon fluorescence intensities. C, Sample images from double labeling of autaptic neurons with anti-Stx1B and anti-Bassoon antibodies. Scale bar, 5 μm. D, Summary plot of Stx1B/Bassoon fluorescence intensity ratios measured within regions marked by Bassoon. E, F, Readily releasable pool size (E) and vesicular release probability (F), derived from previously shown data, were plotted as a function of normalized Stx1B expression levels (Stx1B+/+;Stx1A−/−, black; Stx1B+/yfp;Stx1A−/−, blue; Stx1B+/+;Stx1A−/− + k.d., teal; Stx1Byfp/yfp;Stx1A−/−, red; Stx1Byfp/yfp;Stx1A−/− + k.d., purple).

Article Snippet: For RNAi knock-down experiments, a Stx1B -specific short hairpin RNA (shRNA) target sequence (5′-GAT CCC AGG CAC AAT GAG ATC ATC AAA-3′) was obtained using Genscript siRNA Target Finder ( http://www.genscript.com ).

Techniques: Expressing, Fluorescence, Labeling, Derivative Assay

Journal: European Journal of Endocrinology

Article Title: Reassessing the role of the p.(Arg304Gln) missense AIP variant in pituitary tumorigenesis

doi: 10.1093/ejendo/lvaf044

Figure Lengend Snippet: AIP R304Q protein structure and functional assays. A) Three dimensional and B) linear structure of AIP protein with close-up of location of R304Q (insert). The AIP co-chaperone protein (330 amino acids) has a peptidyl-prolyl ci-trans isomerase (PPIase)-like domain at the N-terminus, 3 tetratricopeptide domains arranged as antiparallel alpha helices, and a final α−7 helix at the C-terminus. The helical structures enable AIP to interact with numerous partners, including HSP90, AHR, and phosphodiesterases. C) Position 304 is conserved and occupied by the positively charged residues arginine (R) or lysine (K) in the multiple sequence alignment. D) Model of interaction of AIP variant R304Q with Tomm20 protein. AIP is presented in grey, and part of the Tomm20 AQSLAEDDVE-peptide in yellow. Residue Arg304 is presented in green. E) R304Q maintains its interaction with HSPA8. HEK293 cells were co-transfected with pcDNA3.0-Myc-AIP_ R304Q and pSF-CMV-NH2-HA-EKT-NcoI-HSPA8, and immunoprecipitation was performed using anti-Myc or anti-HA mouse antibodies, or mouse IgG. Eluates were resolved by denaturing polyacrylamide gel electrophoresis, followed by Western blot, using anti-Myc and anti-HA antibodies. Top arrow: HA-HSPA8, bottom arrow: Myc-AIP. Arrowhead: heavy chain of mouse immunoglobulins. F) Fluorescence double immunostaining of GH and PDE4A8 in normal pituitary, a tumour without AIP variant (no AIP Var), a tumour with R304* variant and a tumour with R304Q variant; scale bar 25 µm. **** P < .0001 Kruskal–Wallis followed by Dunn's test. G) Fluorescence double immunostaining of GH and PDE4A4 in normal pituitary, a tumour without AIP variant (No AIP Var), a tumour with R304* variant and a tumour with R304Q variant; scale bar 25 µm. **** P < .0001 Kruskal–Wallis followed by Dunn's test. H) Half-life of wild-type AIP and of the AIP variant proteins R304Q and R304*, overexpressed in HEK293 cells. The degradation speed of the R304Q variant ( K = .0118) was not significantly different to that of the wild-type protein ( K = 0.0145, P = .5644) while the variant R304* showed rapid degradation compared with the wild-type protein ( K = 0.1183, P < .0001). The representative WB images show bands for Myc-AIP wild-type and R304Q (39 kDa), Myc-AIP R304* (35.8 kDa) and ACTB loading control (41.7 kDa); data extracted from. I) Cyp1a1 relative mRNA levels in Aip shRNA-KD GH3 cells treated with 10 nm FICZ for 5 hours (EV: empty vector, n = 3; WT-AIP: wild-type AIP, n = 6; R304*, n = 9; R304Q, n = 6). Error bars indicate SEM. ANOVA followed by Tukey–Kramer honest significant difference post-hoc test (** P < .01). MWM, molecular weight marker; IP, immunoprecipitation.

Article Snippet: We depleted Aip from rat somatotrophinoma GH3 cells (ECACC, Porton Down, UK, 87012603) using lentiviral small hairpin RNA (shRNA) with a non-targeting shRNA as control (SirionBiotech, Germany), as previously described.

Techniques: Functional Assay, Sequencing, Variant Assay, Residue, Transfection, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot, Fluorescence, Double Immunostaining, Control, shRNA, Plasmid Preparation, Molecular Weight, Marker