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5 results for “Phosphatidylinositol 4,5-bisphosphate”
Deconvolved STED nanoscopy images of the nuclear phosphatidylinositol 4,5-bisphosphate and nuclear speckle marker SON together with deconvolved confocal images of DAPI stained nuclei in human formalin-fixed paraffin-embedded skin warts sections
<p>The collection and analysis of formalin-fixed paraffin-embedded (FFPE) human skin sections was approved by the local ethics-committee at the Department of Pathology, University of Cologne, Germany. Written informed consentwas obtained from all patients in accordance with the Declaration of Helsinki. For biopsy materials from archival paraffin blocks of human skin, an informed consent was obtained from all the subjects and ethical approval obtained from the Ethics Committee at the University of Cologne. Surgically removed human FFPE skin biopsies were sectioned into 4 µm sections. Sections were dewaxed, and indirectly immunofluorescently labeled against nuclear phosphatidylinositol 4,5-bisphosphate (nPI(4,5)P2) using 5 µg/mL rabbit primary polyclonal antibody (Echelon Biosciences Inc. Z-A045, clone 2C11). The primary antibody against nPI(4,5)P2 was recognized by the goat secondary antibody conjugated with Abberrior Star 635P (Abberior 2-0002-007-5). Sections were indirectly immunofluorescently labeled against nuclear speckle marker SON using 1 µg/mL rabbit primary polyclonal antibody (Abcam ab121759). The primary antibody against SON was recognized by the goat secondary antibody conjugated with Abberrior Star 580 (Abberrior ST580-1002). Sections were co-stained by DAPI 1:1000 in PBS for 5 min.</p> <p>Imaging of nPI(4,5)P2-635P channel was performed on Leica TCS SP8 STED 3x inverted DMi8 microscope with pulsed white light laser 470-640 nm 1.5 mW and 775 nm pulse STED laser >1.5 W controlled by Leica Application Suite X software and equipped with HC PL APO CS2 100x/1.40 OIL objective used with Leica Type F immersion oil n=1.518. Unidirectional xyz scanning speed was 400 Hz, line accumulation 8. Pixel size was 20 nm in X and Y. Channel settings: 7% 633 nm laser; 775 Notch filter; 50% 775 nm STED laser; 30% 3D STED; HyD 639-698 nm, photon-counting mode, gain 100, gating 0.3-10 ns. Imaging of SON-580 channel was performed on Leica TCS SP8 STED 3x inverted DMi8 microscope with pulsed white light laser 470-640 nm 1.5 mW and 775 nm pulse STED laser >1.5 W controlled by Leica Application Suite X software and equipped with HC PL APO CS2 100x/1.40 OIL objective used with Leica Type F immersion oil n=1.518. Unidirectional xyz scanning speed was 400 Hz, line accumulation 8. Pixel size was 20 nm in X and Y. Channel settings: 10% 585 nm laser; 775 Notch filter; 80% 775 nm STED laser, 30% 3D STED; Hybrid detector (HyD) 589-616 nm, photon-counting mode, gain 100, gating 0.4-10 ns.</p> <p>Z-stacks of STED images were deconvolved using Huygens Professional 22.10 software (Scientific Imaging B.V.). Data sets were processed using Workflow Processor. The workflow consisted of selecting images, setting up the microscopy and deconvolution parameters and saving deconvolved images as 8-bit TIFF single files for individual channels (which were later used for the quantitative analyses; see below). Microscopy parameters were optimized and set as follows. Sampling intervals were ≤20 nm in X and Y and ≤20 nm in Z. Numerical aperture was 1.4; refractive indexes of the lens immersion oil was 1.518 and of the embedding media 1.458; objective quality was good, coverslip position was 0 µm and imaging direction was downward. For nPI(4,5)P2-635P STED channel the backprojected pinhole was 216 nm; excitation (ex.) and emission (em.) wavelengths (λ) were 633 and 651 nm, resp., ex. fill factor 2. STED depletion mode was pulsed, saturation factor 25, STED λ = 775, STED immunity factor 10 and STED 3X was 30%. Classic MLE algorithm with stabilization of Z-slices was used and signal-to-noise ratio was 5.1. For SON-580 STED channel the backprojected pinhole was 195 nm; excitation (ex.) and emission (em.) wavelengths (λ) were 585 and 602 nm, resp., ex. fill factor 2. STED depletion mode was pulsed, saturation factor 20, STED λ = 775, STED immunity factor 10 and STED 3X was 30%. Classic MLE algorithm with stabilization of Z-slices was used and signal-to-noise ratio was 4.</p>
NMR data in 'Ca2+-dependent release of Synaptotagmin-1 from the SNARE complex on phosphatidylinositol 4,5-bisphosphate-containing membranes' by Voleti, Jaczynska and Rizo, eLife 2020
<p>The Ca<sup>2+</sup> sensor synaptotagmin-1 and the SNARE complex cooperate to trigger neurotransmitter release. Structural studies elucidated three distinct synaptotagmin-1-SNARE complex binding modes involving 'polybasic', 'primary' and 'tripartite' interfaces of synaptotagmin-1. We investigated these interactions using NMR and fluorescence spectroscopy. Synaptotagmin-1 binds to the SNARE complex through the polybasic and primary interfaces in solution. Ca<sup>2+</sup>-free synaptotagmin-1 binds to SNARE complexes anchored on PIP<sub>2</sub>-containing nanodiscs. R398Q/R399Q and E295A/Y338W mutations at the primary interface, which strongly impair neurotransmitter release, disrupt and enhance synaptotagmin-1-SNARE complex binding, respectively. Ca<sup>2+</sup> induces tight binding of synaptotagmin-1 to PIP<sub>2</sub>-containing nanodiscs, releasing synaptotagmin-1-SNARE interactions. Specific effects of mutations in the polybasic region on Ca<sup>2+</sup>-dependent synaptotagmin-1-PIP<sub>2</sub>-membrane interactions correlate with their effects on release. Our data suggest that synaptotagmin-1 binds to the SNARE complex through the primary interface and that Ca<sup>2+</sup> releases this interaction, inducing PIP<sub>2</sub>/membrane binding and allowing cooperation between synaptotagmin-1 and the SNAREs in membrane fusion to trigger release.</p>
Deconvolved STED nanoscopy images of the nuclear phosphatidylinositol 4,5-bisphosphate and nuclear speckle marker SON together with deconvolved confocal images of DAPI stained nuclei in human formalin-fixed paraffin-embedded skin sections
<p>The collection and analysis of formalin-fixed paraffin-embedded (FFPE) human skin sections was approved by the local ethics-committee at the Department of Pathology, University of Cologne, Germany. Written informed consentwas obtained from all patients in accordance with the Declaration of Helsinki. For biopsy materials from archival paraffin blocks of human skin, an informed consent was obtained from all the subjects and ethical approval obtained from the Ethics Committee at the University of Cologne. Surgically removed human FFPE skin biopsies were sectioned into 4 µm sections. Sections were dewaxed, and indirectly immunofluorescently labeled against nuclear phosphatidylinositol 4,5-bisphosphate (nPI(4,5)P2) using 5 µg/mL rabbit primary polyclonal antibody (Echelon Biosciences Inc. Z-A045, clone 2C11). The primary antibody against nPI(4,5)P2 was recognized by the goat secondary antibody conjugated with Abberrior Star 635P (Abberior 2-0002-007-5). Sections were indirectly immunofluorescently labeled against nuclear speckle marker SON using 1 µg/mL rabbit primary polyclonal antibody (Abcam ab121759). The primary antibody against SON was recognized by the goat secondary antibody conjugated with Abberrior Star 580 (Abberrior ST580-1002). Sections were co-stained by DAPI 1:1000 in PBS for 5 min.</p> <p>Imaging of nPI(4,5)P2-635P channel was performed on Leica TCS SP8 STED 3x inverted DMi8 microscope with pulsed white light laser 470-640 nm 1.5 mW and 775 nm pulse STED laser >1.5 W controlled by Leica Application Suite X software and equipped with HC PL APO CS2 100x/1.40 OIL objective used with Leica Type F immersion oil n=1.518. Unidirectional xyz scanning speed was 400 Hz, line accumulation 8. Pixel size was 20 nm in X and Y. Channel settings: 7% 633 nm laser; 775 Notch filter; 50% 775 nm STED laser; 30% 3D STED; HyD 639-698 nm, photon-counting mode, gain 100, gating 0.3-10 ns. Imaging of SON-580 channel was performed on Leica TCS SP8 STED 3x inverted DMi8 microscope with pulsed white light laser 470-640 nm 1.5 mW and 775 nm pulse STED laser >1.5 W controlled by Leica Application Suite X software and equipped with HC PL APO CS2 100x/1.40 OIL objective used with Leica Type F immersion oil n=1.518. Unidirectional xyz scanning speed was 400 Hz, line accumulation 8. Pixel size was 20 nm in X and Y. Channel settings: 10% 585 nm laser; 775 Notch filter; 80% 775 nm STED laser, 30% 3D STED; Hybrid detector (HyD) 589-616 nm, photon-counting mode, gain 100, gating 0.4-10 ns.</p> <p>Z-stacks of STED images were deconvolved using Huygens Professional 22.10 software (Scientific Imaging B.V.). Data sets were processed using Workflow Processor. The workflow consisted of selecting images, setting up the microscopy and deconvolution parameters and saving deconvolved images as 8-bit TIFF single files for individual channels (which were later used for the quantitative analyses; see below). Microscopy parameters were optimized and set as follows. Sampling intervals were ≤20 nm in X and Y and ≤20 nm in Z. Numerical aperture was 1.4; refractive indexes of the lens immersion oil was 1.518 and of the embedding media 1.458; objective quality was good, coverslip position was 0 µm and imaging direction was downward. For nPI(4,5)P2-635P STED channel the backprojected pinhole was 216 nm; excitation (ex.) and emission (em.) wavelengths (λ) were 633 and 651 nm, resp., ex. fill factor 2. STED depletion mode was pulsed, saturation factor 25, STED λ = 775, STED immunity factor 10 and STED 3X was 30%. Classic MLE algorithm with stabilization of Z-slices was used and signal-to-noise ratio was 5.1. For SON-580 STED channel the backprojected pinhole was 195 nm; excitation (ex.) and emission (em.) wavelengths (λ) were 585 and 602 nm, resp., ex. fill factor 2. STED depletion mode was pulsed, saturation factor 20, STED λ = 775, STED immunity factor 10 and STED 3X was 30%. Classic MLE algorithm with stabilization of Z-slices was used and signal-to-noise ratio was 4.</p>
NMR data in 'Ca2+-dependent release of Synaptotagmin-1 from the SNARE complex on phosphatidylinositol 4,5-bisphosphate-containing membranes' by Voleti, Jaczynska and Rizo, eLife 2020
Open the record for dataset details and reuse information.
Phosphatidylinositol 4,5-bisphosphate (PIP2) facilitates norepinephrine transporter dimerization and modulates substrate efflux
<p>Source data supporting the findings of the study.</p>
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