Journal: STAR Protocols

Article Title: Immunofluorescence staining of phosphoinositides in primary mouse hippocampal neurons in dissociated culture

doi: 10.1016/j.xpro.2022.101549

Figure Lengend Snippet: Immunofluorescence staining of intracellular PIPs and Rab8/TGN46 in mouse hippocampal neurons Mouse hippocampal neurons were fixed on DIV12 and co-immnunostained for intracellular PI3P, PI4P or PI(4,5)P 2 and Rab8 or TGN46. DAPI-stained cell nuclei are shown in blue. (A–F) Shown are representative confocal microscopy images of intracellular PI3P and Rab8 (A), intracellular PI4P and Rab8 (B), intracellular PI(4,5)P 2 and Rab8 (C), intracellular PI3P and TGN46 (D), intracellular PI4P and TGN46 (E), intracellular PI(4,5)P 2 and TGN46 (F). Rab8: protein marker for secretory endosomes, TGN46: protein marker for the trans -Golgi network. Lower panels are magnifications of outlined regions in the top panels. Scale bars: 5 μm.

Article Snippet: Mouse anti-PI(4,5)P 2 , IgM (1:50 - 1:150) , Echelon Biosciences , Cat# Z-A045, RRID: AB_427211.

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Marker

Journal: STAR Protocols

Article Title: Immunofluorescence staining of phosphoinositides in primary mouse hippocampal neurons in dissociated culture

doi: 10.1016/j.xpro.2022.101549

Figure Lengend Snippet: Immunofluorescence staining of PM PIPs and EEN1 in mouse hippocampal neurons Mouse hippocampal neurons were transfected with pAOV-CaMKIIα-mCherry-2A-3Flag on DIV5 to express mCherry as volume marker ( Note : the CaMKIIα promoter drives gene expression specifically in excitatory neurons but not inhibitory neurons) ( Kohara et al., 2020 ), fixed on DIV8 and immnunostained for PM PI3P, PI4P or PI(4,5)P 2 . DAPI-stained cell nuclei are shown in blue. (A–D) Shown are representative confocal microscopy images of PM PI3P (A), PM PI4P (B), PM PI(4,5)P 2 (C), and costaining of PM PI(4,5)P 2 and PM EEN1 (D). Lower panels are magnifications of boxed regions in the top panels. Scale bars: 5 μm.

Article Snippet: Mouse anti-PI(4,5)P 2 , IgM (1:50 - 1:150) , Echelon Biosciences , Cat# Z-A045, RRID: AB_427211.

Techniques: Immunofluorescence, Staining, Transfection, Marker, Expressing, Confocal Microscopy

Journal: STAR Protocols

Article Title: Immunofluorescence staining of phosphoinositides in primary mouse hippocampal neurons in dissociated culture

doi: 10.1016/j.xpro.2022.101549

Figure Lengend Snippet:

Article Snippet: Mouse anti-PI(4,5)P 2 , IgM (1:50 - 1:150) , Echelon Biosciences , Cat# Z-A045, RRID: AB_427211.

Techniques: Recombinant, Electron Microscopy, Software, Microscopy, Cell Culture, In Vitro, Cell Counting

Journal: JCI Insight

Article Title: Mutations in OSBPL2 cause hearing loss associated with primary cilia defects via sonic hedgehog signaling

doi: 10.1172/jci.insight.149626

Figure Lengend Snippet: ( A ) Immunofluorescence staining of cilia in Osbpl2 –/– HEI-OC1 cells transiently expressing Flag-ΔFFAT, Flag-ΔORD, and Flag-OSBPL2. Cells were stained with anti-acetylated tubulin (red), anti–gamma-tubulin (green), and DAPI (blue). Scale bar: 5 μm. ( B ) The ciliary length of Osbpl2 –/– HEI-OC1 cells transiently expressing Flag-ΔFFAT, Flag-ΔORD, and Flag-OSBPL2 (50 cells per genotype, each dot represents a cell in a microscope field. * P < 0.05; ** P < 0.01; ns: not significant; tested by 1-way ANOVA). ( C ) The proportion of ciliated cells in Osbpl2 –/– HEI-OC1 cells transiently expressing Flag-ΔFFAT, Flag-ΔORD, and Flag-OSBPL2 (each dot represents the proportion of ciliated cells in a microscope field; ns: not significant; tested by 1-way ANOVA). ( D ) Immunofluorescence staining of HEI-OC1 cells with anti-PI(4,5)P 2 (green), anti-acetylated tubulin (red), and DAPI (blue). Dashed frames denote the locally zoomed regions in solid frames (bottom right). Scale bar: 5 μm. ( E ) Quantification of ciliary PI(4,5)P 2 intensity in HEI-OC1 cells (50 cells per genotype, each dot represents a cell. *** P < 0.05 by 2-tailed Student’s t test). ( F ) The proportion of PI(4,5)P 2 positive cilia in Osbpl2 –/– HEI-OC1 cells (each dot represents the proportion of PI(4,5)P 2 positive cilia in a microscope field. ns: not significant by 2-tailed Student’s t test). ( G ) Immunofluorescence staining of HEI-OC1 cells with anti-PI4P (green), anti-acetylated tubulin (red), and DAPI (blue). PI4P showed no significant difference in Osbpl2 –/– and WT HEI-OC1 cells. Dashed frames denote the locally zoomed regions in solid frames (bottom right). Scale bar: 5 μm. ( H ) Quantification of ciliary PIP4 intensity in HEI-OC1 cells (50 cells per genotype, each dot represents a cell. ns: not significant by 2-tailed Student’s t test). ( I ) The proportion of PI4P positive cilia in Osbpl2 –/– HEI-OC1 cells (each dot represents the proportion of PI4P positive cilia in a microscope field. ns: not significant by 2-tailed Student’s t test).

Article Snippet: The antibodies used in this study included anti-acetylated tubulin (Sigma-Aldrich, T7451 and Cell Signaling Technology, 5335); anti-ARL13B (Proteintech, 17711-1-AP); anti-gamma tubulin (Abcam, ab179503); anti-INPP5E (Proteintech, 17797-1-AP); anti-OSBPL2 (Proteintech, 14751-1-AP and Abclonal, A14199); anti-FLAG (Sigma-Aldrich, F1804); anti-HA (Cell Signaling Technology, 3724); anti-GAPDH (Cell Signaling Technology, 5174); anti–PI(4,5)P 2 (Echelon, Z-P045); anti–PI4P (Echelon, Z-P004); anti-SMO (Santa Cruz, sc-166685); anti-Gli3 (Abcam, ab6050 and Proteintech, 19949-1-AP); anti-Gli1 (Proteintech, 66905-1-Ig); Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (Invitrogen, A31570); Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (Invitrogen, A10040); Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A21202); Donkey F(ab′)2 Anti-Rabbit IgG H&L, Alexa Fluor 647 (Abcam, ab181347); IRDye 800CW Secondary Antibody (LI-COR, 925-32211); and IRDye 680LT Secondary Antibody (LI-COR, 925-68020).

Techniques: Immunofluorescence, Staining, Expressing, Microscopy

Journal: JCI Insight

Article Title: Mutations in OSBPL2 cause hearing loss associated with primary cilia defects via sonic hedgehog signaling

doi: 10.1172/jci.insight.149626

Figure Lengend Snippet: ( A ) Immunofluorescence staining of Osbpl2 –/– HEI-OC1 cells with anti-SMO (green), anti-acetylated tubulin (red), and DAPI (blue). In Osbpl2 –/– HEI-OC1 cells expressing 5HT 6 -HA-INPP5E, the localization of ciliary SMO was partially rescued. Dashed frames denote the locally zoomed regions in solid frames (bottom right). Scale bar: 5 μm. ( B ) Quantification of ciliary SMO intensity in Osbpl2 –/– HEI-OC1 cells with or without INPP5E expression (at least 30 cells from a microscope field, each dot represents a cell. ** P < 0.01 by 2-tailed Student’s t test). ( C ) Immunofluorescence staining of Osbpl2 –/– HEI-OC1 cells with anti-GLI3 (purple), anti-acetylated tubulin (green), and anti–gamma-tubulin (red). In Osbpl2 –/– HEI-OC1 cells expressing 5HT 6 -HA-INPP5E, the localization of ciliary GLI3 was partially rescued. Dashed frames denote the locally zoomed regions in solid frames (bottom right). Scale bar: 5 μm. ( D ) Quantification of ciliary GLI3 intensity in Osbpl2 –/– HEI-OC1 cells with or without INPP5E expression (at least 30 cells from a microscope field, each dot represents a cell; *** P < 0.001 by 2-tailed Student’s t test). ( E ) Schematic diagram of OSBPL2 regulating ciliogenesis and Shh signaling transduction in auditory cells. OSBPL2 was localized at the base of the cilia and regulated the homeostasis of ciliary PI(4,5)P 2 , which affected the ciliogenesis and thereby influenced transduction of the Shh signaling pathway. OSBPL2 deficiency led to dyshomeostasis of ciliary PI(4,5)P 2 , which was responsible for ciliary defects and inhibited Shh signal transduction.

Article Snippet: The antibodies used in this study included anti-acetylated tubulin (Sigma-Aldrich, T7451 and Cell Signaling Technology, 5335); anti-ARL13B (Proteintech, 17711-1-AP); anti-gamma tubulin (Abcam, ab179503); anti-INPP5E (Proteintech, 17797-1-AP); anti-OSBPL2 (Proteintech, 14751-1-AP and Abclonal, A14199); anti-FLAG (Sigma-Aldrich, F1804); anti-HA (Cell Signaling Technology, 3724); anti-GAPDH (Cell Signaling Technology, 5174); anti–PI(4,5)P 2 (Echelon, Z-P045); anti–PI4P (Echelon, Z-P004); anti-SMO (Santa Cruz, sc-166685); anti-Gli3 (Abcam, ab6050 and Proteintech, 19949-1-AP); anti-Gli1 (Proteintech, 66905-1-Ig); Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (Invitrogen, A31570); Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (Invitrogen, A10040); Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A21202); Donkey F(ab′)2 Anti-Rabbit IgG H&L, Alexa Fluor 647 (Abcam, ab181347); IRDye 800CW Secondary Antibody (LI-COR, 925-32211); and IRDye 680LT Secondary Antibody (LI-COR, 925-68020).

Techniques: Immunofluorescence, Staining, Expressing, Microscopy, Transduction

Journal: Life

Article Title: Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P 2 and PI4P in Human Platelets

doi: 10.3390/life11121331

Figure Lengend Snippet: Intracellular and PM localization of PI(4,5)P 2 and PI4P in HEK293T and BALB3T3 cell lines. HEK293T cells were fixed 24 h after seeding and were stained for ( A ) the intracellular pool or ( B ) the PM pool of PI(4,5)P 2 and PI4P. The cells were co-stained for actin and the nucleus. BALB3T3 cells were fixed 24h after seeding and were stained for ( C ) the intracellular pool or ( D ) the PM pool of PI(4,5)P 2 and PI4P. The cells were co-stained for actin and the nucleus. Representative images display a single confocal optical section. The scale bar of the images is 50 μm, while the scale bar of the inserts is 5 μm.

Article Snippet: Antibodies were obtained from the following resources: monoclonal mouse anti-PI(4,5)P 2 (Z-P045) and anti-PI4P (Z-P004), both IgM, were from Echelon Biosciences, polyclonal rabbit CD42b/anti-GPIbα was from Novus Biological (NBP2-89128), phalloidin (A12379) conjugated with Alexa Fluor (AF)-488 was from Invitrogen, monoclonal mouse IgM (MAB1326, R&D Systems,, Abigdon, UK) was a kind gift from Dr. Jelena Ban, (Laboratory of Molecular Neurobiology, Department of Biotechnology, University of Rijeka), polyclonal rabbit α-tubulin (SAB4500087, Sigma Aldrich, Taufkirchen, Germany) was a kind gift from Dr. Iva Tolić (Laboratory of Cell Biophysics, Ruđer Bošković Institute), and DAPI (D9542) was from Sigma Aldrich.

Techniques: Staining

Journal: Life

Article Title: Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P 2 and PI4P in Human Platelets

doi: 10.3390/life11121331

Figure Lengend Snippet: The intracellular localization of PI(4,5)P 2 and PI4P in resting and activated PLTs. PLTs were isolated from human peripheral blood, ( A ) fixed immediately or ( B ) spread on glass for 45 min stained for the intracellular pools of PI(4,5)P 2 and PI4P, co-stained for actin, and imaged with a confocal microscope. Representative images display a single confocal optical section. The scale bar of the images is 20 μm, while the scale bar of the inserts is 2 μm for resting, and 5 μm for activated PLTs.

Article Snippet: Antibodies were obtained from the following resources: monoclonal mouse anti-PI(4,5)P 2 (Z-P045) and anti-PI4P (Z-P004), both IgM, were from Echelon Biosciences, polyclonal rabbit CD42b/anti-GPIbα was from Novus Biological (NBP2-89128), phalloidin (A12379) conjugated with Alexa Fluor (AF)-488 was from Invitrogen, monoclonal mouse IgM (MAB1326, R&D Systems,, Abigdon, UK) was a kind gift from Dr. Jelena Ban, (Laboratory of Molecular Neurobiology, Department of Biotechnology, University of Rijeka), polyclonal rabbit α-tubulin (SAB4500087, Sigma Aldrich, Taufkirchen, Germany) was a kind gift from Dr. Iva Tolić (Laboratory of Cell Biophysics, Ruđer Bošković Institute), and DAPI (D9542) was from Sigma Aldrich.

Techniques: Isolation, Staining, Microscopy

Journal: Life

Article Title: Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P 2 and PI4P in Human Platelets

doi: 10.3390/life11121331

Figure Lengend Snippet: The plasma membrane localization of PI(4,5)P 2 and PI4P in resting and activated PLTs. PLTs were isolated from human peripheral blood, ( A ) fixed immediately or ( B ) spread on glass for 45 min, fixed and stained for the plasma membrane pools of PI(4,5)P 2 and PI4P, co-stained for actin, and imaged with a confocal microscope. Representative images display a single confocal optical section. The scale bar of the images is 20 μm, while the scale bar of the inserts is 2 μm for resting and 5 μm for activated PLTs.

Article Snippet: Antibodies were obtained from the following resources: monoclonal mouse anti-PI(4,5)P 2 (Z-P045) and anti-PI4P (Z-P004), both IgM, were from Echelon Biosciences, polyclonal rabbit CD42b/anti-GPIbα was from Novus Biological (NBP2-89128), phalloidin (A12379) conjugated with Alexa Fluor (AF)-488 was from Invitrogen, monoclonal mouse IgM (MAB1326, R&D Systems,, Abigdon, UK) was a kind gift from Dr. Jelena Ban, (Laboratory of Molecular Neurobiology, Department of Biotechnology, University of Rijeka), polyclonal rabbit α-tubulin (SAB4500087, Sigma Aldrich, Taufkirchen, Germany) was a kind gift from Dr. Iva Tolić (Laboratory of Cell Biophysics, Ruđer Bošković Institute), and DAPI (D9542) was from Sigma Aldrich.

Techniques: Isolation, Staining, Microscopy

Journal: Life

Article Title: Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P 2 and PI4P in Human Platelets

doi: 10.3390/life11121331

Figure Lengend Snippet: Modulation of the PM staining of PI(4,5)P 2 and PI4P with 1 h and 30 min of permeabilization and different saponin concentrations. HEK293T cells were fixed 24 h after seeding and were stained for the PM pool of PI(4,5)P 2 and PI4P, and co-stained for actin. PLTs were isolated from human peripheral blood, spread on glass for 45 min, fixed, stained for the PM pools of PI(4,5)P 2 and PI4P, co-stained for actin, and imaged with a confocal microscope. ( A – C ) HEK293T cells and human PLTs were permeabilized for 1 h with ( A , G ) 0.5% saponin, ( B , H ) 0.8% saponin, and ( C , I ) 1% saponin and stained for ( A – C ) PI(4,5)P 2 or ( G – I ) PI4P. ( A – C ) HEK293T cells and human PLTs were permeabilized for 30 min with ( D , J ) 0.5% saponin, ( E , K ) 0.8% saponin, and ( F , L ) 1% saponin and stained for ( D – F ) PI(4,5)P 2 or ( J – L ) PI4P. Representative images display a single confocal optical section. The scale bar of the images is 20 μm, while the scale bar of the inserts is 5 μm.

Article Snippet: Antibodies were obtained from the following resources: monoclonal mouse anti-PI(4,5)P 2 (Z-P045) and anti-PI4P (Z-P004), both IgM, were from Echelon Biosciences, polyclonal rabbit CD42b/anti-GPIbα was from Novus Biological (NBP2-89128), phalloidin (A12379) conjugated with Alexa Fluor (AF)-488 was from Invitrogen, monoclonal mouse IgM (MAB1326, R&D Systems,, Abigdon, UK) was a kind gift from Dr. Jelena Ban, (Laboratory of Molecular Neurobiology, Department of Biotechnology, University of Rijeka), polyclonal rabbit α-tubulin (SAB4500087, Sigma Aldrich, Taufkirchen, Germany) was a kind gift from Dr. Iva Tolić (Laboratory of Cell Biophysics, Ruđer Bošković Institute), and DAPI (D9542) was from Sigma Aldrich.

Techniques: Staining, Isolation, Microscopy

Journal: Life

Article Title: Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P 2 and PI4P in Human Platelets

doi: 10.3390/life11121331

Figure Lengend Snippet: Modulation of the PM staining of PI(4,5)P 2 and PI4P with 5 min of permeabilization and different saponin concentrations. HEK293T cells were fixed 24 h after seeding and were stained for the PM pool of PI(4,5)P 2 and PI4P, and co-stained for actin. PLTs were isolated from human peripheral blood, spread on glass for 45 min, fixed, stained for the PM pools of PI(4,5)P 2 and PI4P, co-stained for actin, and imaged with a confocal microscope. ( A – F ) HEK293T cells and human PLTs were permeabilized for 5 min with ( A , D ) 0.5% saponin, ( B , E ) 0.8% saponin, and ( C , F ) 1% saponin and stained for ( A – C ) PI(4,5)P 2 or ( D – F ) PI4P. Representative images display a single confocal optical section. The scale bar of the images is 20 μm, while the scale bar of the inserts is 5 μm.

Article Snippet: Antibodies were obtained from the following resources: monoclonal mouse anti-PI(4,5)P 2 (Z-P045) and anti-PI4P (Z-P004), both IgM, were from Echelon Biosciences, polyclonal rabbit CD42b/anti-GPIbα was from Novus Biological (NBP2-89128), phalloidin (A12379) conjugated with Alexa Fluor (AF)-488 was from Invitrogen, monoclonal mouse IgM (MAB1326, R&D Systems,, Abigdon, UK) was a kind gift from Dr. Jelena Ban, (Laboratory of Molecular Neurobiology, Department of Biotechnology, University of Rijeka), polyclonal rabbit α-tubulin (SAB4500087, Sigma Aldrich, Taufkirchen, Germany) was a kind gift from Dr. Iva Tolić (Laboratory of Cell Biophysics, Ruđer Bošković Institute), and DAPI (D9542) was from Sigma Aldrich.

Techniques: Staining, Isolation, Microscopy

Journal: Life

Article Title: Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P 2 and PI4P in Human Platelets

doi: 10.3390/life11121331

Figure Lengend Snippet: PM localization of PI(4,5)P 2 and PI4P with the modified staining protocol. PLTs were isolated from human peripheral blood, ( A ) fixed or ( B ) spread on glass for 45 min, stained for the PM pools of PI(4,5)P 2 and PI4P, co-stained for GPIbα, and imaged with a confocal microscope. Representative images display a single confocal optical section. The scale bar of the images is 20 μm, while the scale bar of the inserts is 2 μm for resting and 5 μm for activated PLTs.

Article Snippet: Antibodies were obtained from the following resources: monoclonal mouse anti-PI(4,5)P 2 (Z-P045) and anti-PI4P (Z-P004), both IgM, were from Echelon Biosciences, polyclonal rabbit CD42b/anti-GPIbα was from Novus Biological (NBP2-89128), phalloidin (A12379) conjugated with Alexa Fluor (AF)-488 was from Invitrogen, monoclonal mouse IgM (MAB1326, R&D Systems,, Abigdon, UK) was a kind gift from Dr. Jelena Ban, (Laboratory of Molecular Neurobiology, Department of Biotechnology, University of Rijeka), polyclonal rabbit α-tubulin (SAB4500087, Sigma Aldrich, Taufkirchen, Germany) was a kind gift from Dr. Iva Tolić (Laboratory of Cell Biophysics, Ruđer Bošković Institute), and DAPI (D9542) was from Sigma Aldrich.

Techniques: Modification, Staining, Isolation, Microscopy

Journal: Life

Article Title: Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P 2 and PI4P in Human Platelets

doi: 10.3390/life11121331

Figure Lengend Snippet: The intracellular and PM localization of PI(4,5)P 2 and PI4P, visualized with the optimized protocol, in resting PLTs and their modulation by OCRL and PI4KIIIα inhibitors. PLTs were isolated from human peripheral blood, fixed, stained for the ( A , C ) intracellular or ( E , G ) PM pools of PI(4,5)P 2 and PI4P, co-stained for actin, and imaged with a confocal microscope. The dephosphorylation of PI(4,5)P 2 was inhibited by 10 µM OCRL inhibitor YU142670 and the production of PI4P was inhibited by 100 nM of PI4KIIIα inhibitor GSK-A1. Representative images display a single confocal optical section. The scale bar of the images is 20 μm, while the scale bar of the inserts is 2 μm. ( B , D , F , H ) Images were analyzed with Fiji ImageJ software to measure the mean fluorescence intensity of PI(4,5)P 2 and PI4P. The graphs show the mean fluorescence intensity of PI(4,5)P 2 and PI4P. Results in the graphs are presented as means, error bars denote ± SEM from 3 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant.

Article Snippet: Antibodies were obtained from the following resources: monoclonal mouse anti-PI(4,5)P 2 (Z-P045) and anti-PI4P (Z-P004), both IgM, were from Echelon Biosciences, polyclonal rabbit CD42b/anti-GPIbα was from Novus Biological (NBP2-89128), phalloidin (A12379) conjugated with Alexa Fluor (AF)-488 was from Invitrogen, monoclonal mouse IgM (MAB1326, R&D Systems,, Abigdon, UK) was a kind gift from Dr. Jelena Ban, (Laboratory of Molecular Neurobiology, Department of Biotechnology, University of Rijeka), polyclonal rabbit α-tubulin (SAB4500087, Sigma Aldrich, Taufkirchen, Germany) was a kind gift from Dr. Iva Tolić (Laboratory of Cell Biophysics, Ruđer Bošković Institute), and DAPI (D9542) was from Sigma Aldrich.

Techniques: Isolation, Staining, Microscopy, De-Phosphorylation Assay, Software, Fluorescence

Journal: Life

Article Title: Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P 2 and PI4P in Human Platelets

doi: 10.3390/life11121331

Figure Lengend Snippet: The intracellular and PM localization of PI(4,5)P 2 and PI4P, visualized with the optimized protocol, in activated PLTs and their modulation by OCRL and PI4KIIIα inhibitors. PLTs were isolated from human peripheral blood, fixed, stained for the ( A – D ) intracellular or (E-H) PM pools of PI(4,5)P 2 and PI4P, co-stained for actin, and imaged with a confocal microscope. The dephosphorylation of PI(4,5)P 2 was inhibited by 10 µM of OCRL inhibitor YU142670, and the production of PI4P was inhibited by 100 nM of PI4KIIIα inhibitor GSK-A1. Representative images display a single confocal optical section. The scale bar of the images is 20 μm, while the scale bar of the inserts is 5 μm. ( B , D , F , H ) Images were analyzed with Fiji ImageJ software to measure the mean fluorescence intensity of PI(4,5)P 2 and PI4P. The graphs show the mean fluorescence intensity of PI4P and PI(4,5)P 2 . Results in the graphs are presented as means, and error bars denote ± SEM from 3 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant.

Article Snippet: Antibodies were obtained from the following resources: monoclonal mouse anti-PI(4,5)P 2 (Z-P045) and anti-PI4P (Z-P004), both IgM, were from Echelon Biosciences, polyclonal rabbit CD42b/anti-GPIbα was from Novus Biological (NBP2-89128), phalloidin (A12379) conjugated with Alexa Fluor (AF)-488 was from Invitrogen, monoclonal mouse IgM (MAB1326, R&D Systems,, Abigdon, UK) was a kind gift from Dr. Jelena Ban, (Laboratory of Molecular Neurobiology, Department of Biotechnology, University of Rijeka), polyclonal rabbit α-tubulin (SAB4500087, Sigma Aldrich, Taufkirchen, Germany) was a kind gift from Dr. Iva Tolić (Laboratory of Cell Biophysics, Ruđer Bošković Institute), and DAPI (D9542) was from Sigma Aldrich.

Techniques: Isolation, Staining, Microscopy, De-Phosphorylation Assay, Software, Fluorescence

Journal: eLife

Article Title: The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

doi: 10.7554/eLife.68215

Figure Lengend Snippet: ( A ) Domain structures of the constructs used in this study. The structure of the soluble C2AB domains was rendered using PyMol, from PDB: 5kj7 . The orientations of the C2A and C2B domains relative to each other are not known in the presence of SNAREs and membranes. Conserved aspartate residues coordinating calcium ions are depicted in orange. Calcium ions are shown as orange spheres. A poly-lysine motif on the side of C2B (K324,K325,K326,K327 in the rat sequence) that preferentially interacts with PI(4,5)P 2 is highlighted in cyan. ( B ) Incorporation of exogenous PI(4,5)P 2 into the outer leaflet of flipped t-SNARE cells. Top: cells were incubated with diC8-PI(4,5)P 2 for 20 min, rinsed, and immunolabeled for PI(4,5)P 2 at the indicated time points. Only control cells that were permeabilized with saponin showed immunostaining, confirming absence of PI(4,5)P 2 in the outer leaflet, and providing a reference value for inner-leaflet PI(4,5)P 2 levels ( a and b ). Cells incubated with diC8-PI(4,5)P 2 showed immunofluorescence in the absence of permeabilization, indicating successful incorporation of PI(4,5)P 2 into the outer leaflet of the cell membrane ( c–e ). The signal was comparable to endogenous inner-leaflet PI(4,5)P 2 levels, and persisted at least for 80 min (lower panel). Cells processed similarly, but not treated with saponin or diC8-PI(4,5)P 2 served as negative controls ( a ). One-way analysis of variance (ANOVA) followed by multiple comparison test was used to compare the signals from the endogenous PI(4,5)P 2 sample ( b ) with all others. *, **, *** indicate p<0.05, 0.01, and 0.001, respectively. ( C ) Schematic of the single-pore nanodisc-cell fusion assay. A glass micropipette forms a tight seal on a patch of the plasma membrane of a cell expressing ‘flipped’ t-SNARE proteins on its surface. NLPs co-reconstituted with Syt1 and VAMP2 are included in the pipette solution (left). NLP-cell fusion results in a fusion pore connecting the cytosol to the cell’s exterior (right). Under voltage clamp, direct-currents passing through the pore report pore dynamics. With ~25 nm NLPs, the scaffolding ring does not hinder pore expansion up to at least 10 nm diameter. Exogenous PI(4,5)P 2 can be added to the cell’s outer leaflet as in B, and calcium in the pipette is controlled using calcium buffers. ( D ) Representative currents that were recorded during vsNLP-tCell fusion, for the indicated conditions. PI(4,5)P 2 indicates cells were pre-treated with diC8-PI(4,5)P 2 . Tetanus neurotoxin (TeNT) light chain cleaves VAMP2 and blocks exocytosis. Currents were larger when all components were present (SNAREs, Syt1, exogenous PI(4,5)P 2 and calcium). ( E ) Similar to D, but instead of full-length Syt1, 10 μM soluble Syt1 C2AB domains were used together with NLPs carrying ~4 copies of VAMP2 per face.

Article Snippet: Mouse monoclonal anti-PI(4,5)P 2 primary antibodies (Echelon Biosciences Inc, Utah) were added to the cells at time points of 0, 40, and 80 min and incubated 1 hr at 37°C.

Techniques: Construct, Sequencing, Incubation, Immunolabeling, Immunostaining, Immunofluorescence, Cell Fusion Assay, Expressing, Transferring, Scaffolding

Journal: eLife

Article Title: The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

doi: 10.7554/eLife.68215

Figure Lengend Snippet: ( A ) The rate at which current bursts appeared (pore nucleation rate) for the conditions indicated (error bars represent ± S.E.M.). SNARE-induced pores appeared more frequently in the presence of Syt1 or C2AB, when both calcium and PI(4,5)P 2 were also present. Student's t-test (one-tailed) was used to assess significant differences between the 'no Syt1' group and the rest. *, **, *** indicate p<0.05, 0.01, and 0.001, respectively. There is no difference between the Syt1 and C2AB groups in the presence of calcium and exogenous PI(4,5)P 2 (Student’s t-test: p = 0.18 ). ( B ) Mean single fusion pore conductance, ⟨ G p o ⟩ , for different conditions as indicated (± S.E.M.). ⟨ G p o ⟩ was three-fold larger in the presence of Syt1 or C2AB, when both calcium and PI(4,5)P 2 were also present. Two-sample Kolmogorov-Smirnov test was used to assess significant differences between the 'no Syt1' group and the rest. The same asterisk notation as in A was used. There is no difference between the Syt1 and C2AB groups in the presence of calcium and exogenous PI(4,5)P 2 (two-sample Kolmogorov-Smirnov test: p = 0.29 ). ( C ) Probability density functions (PDFs) for point-by-point open-pore conductances (see Materials and methods) for pores induced in the presence of Syt1, PI(4,5)P 2 and with 0 or 100 μM calcium. Notice the higher density at larger conductance values in the presence of 100 μM calcium. ( D ) Probability density functions for pore radii, calculated from the conductance PDFs in C, assuming a 15-nm long cylindrical pore . ( E ) Apparent free energy profiles for Syt1 and soluble Syt1 C2AB domains in the absence or presence of calcium. These profiles were calculated from the pore radii PDFs as in D (see text and Materials and methods) . The profiles were shifted vertically for clarity. ( F ) Cumulative density functions (CDFs) for mean single-pore conductances for the conditions indicated. Soluble C2AB recapitulated effects of full-length Syt1 co-reconstituted into NLPs.

Article Snippet: Mouse monoclonal anti-PI(4,5)P 2 primary antibodies (Echelon Biosciences Inc, Utah) were added to the cells at time points of 0, 40, and 80 min and incubated 1 hr at 37°C.

Techniques: One-tailed Test

Journal: eLife

Article Title: The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

doi: 10.7554/eLife.68215

Figure Lengend Snippet: Open-pore conductance fluctuations relative to mean ( A ), average flicker rate during a burst ( B ), average open-pore probability, P o , during a current burst (fraction of time pore is in the open state during a burst) ( C ), and average burst lifetime, T o , ( D ) for the indicated conditions. ( E ) Distributions of the number of flickers per burst, N f l i c k e r s , for the indicated conditions. Fits to geometric distributions are shown in red, y = p 1 - p n - 1 , n = 1 , 2 , 3 , … . Best fit parameters (with ± 95% confidence intervals) are p = 0.072 ( 0.053 , 0.092 ) (no Syt1, 100 μM Ca 2+ , averaged over 49 individual fusion pores from 10 cells, mean N f l i c k e r s = 12.8 ), 0.083(0.051,0115) (Syt1, 0 μM Ca 2+ ; averaged over 24 individual fusion pores from 11 cells, mean N f l i c k e r s = 11.0 ), 0.053(0.044,0.063) (Syt1, 100 μM Ca 2+ ; averaged over 123 individual fusion pores from 20 cells, mean N f l i c k e r s = 17.7 ). ( F ) Distribution of burst lifetimes, T o for the indicated conditions. Best fits to single exponentials are shown as red curves, with means (and 95% confidence intervals) as follows. No Syt1, 100 μM Ca 2+ : 6.1 s (4.7 to 8.3 s, 49 fusion pores from 10 cells), Syt1, 0 μM Ca 2+ : 6.5 s (4.5 to 10.1 s, 24 fusion pores from 11 cells), Syt1, 100 μM Ca 2+ : 16 s (13.5 to 19.3 s, 123 fusion pores from 20 cells). In A-D, the two-sample Kolmogorov-Smirnov test was used to assess significant differences between the "no C2AB" group and the rest. *, **, *** indicate p<0.05, 0.01, and 0.001, respectively. Comparison between Syt1 and C2AB in the presence of Ca 2+ and PI(4,5)P 2 are also indicated (using the two-sample Kolmogorov-Smirnov test).

Article Snippet: Mouse monoclonal anti-PI(4,5)P 2 primary antibodies (Echelon Biosciences Inc, Utah) were added to the cells at time points of 0, 40, and 80 min and incubated 1 hr at 37°C.

Techniques:

Journal: eLife

Article Title: The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

doi: 10.7554/eLife.68215

Figure Lengend Snippet: ( A–D ) Probability density function (PDF) for point-by-point open-pore conductance values for the indicated conditions. Substantial density is present for G p o ≳ 500 pS only when C2AB, calcium, and PI(4,5)P 2 were all present. ( F–I ) PDFs for open-pore radii corresponding to the conductance distributions in A-D, assuming pores are 15 nm long cylinders. Data were from 49 fusion pores/10 cells (SNARE only), 44 fusion pores/12 cells (0 µM Ca 2+ ), 84 fusion pores/19 cells (no PI(4,5)P 2 ) and 98 fusion pores/17 cells (100 µM Ca 2+ plus PI(4,5)P 2 ).

Article Snippet: Mouse monoclonal anti-PI(4,5)P 2 primary antibodies (Echelon Biosciences Inc, Utah) were added to the cells at time points of 0, 40, and 80 min and incubated 1 hr at 37°C.

Techniques:

Journal: eLife

Article Title: The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

doi: 10.7554/eLife.68215

Figure Lengend Snippet: ( A ) Overview of the Syt1-SNARE complex . The electrostatic potential of PDB 5kj7 was rendered using Pymol. The sites mutated in this work are marked by boxes labeled 1–3 on the left and shown in the panels to the right. D309 is a key calcium-binding residue (1), K326, K327 interact with acidic lipids (2), and R398,R399 (3) interact with the t-SNAREs SNAP 25 (E51, E52, and E55) and syntaxin 1A (D231, E234, and E238). VAMP2 is shown in blue, SNAP25 in yellow, and syntaxin 1A in red. ( B ) Pore nucleation rates (+/- SEM) for the indicated conditions. All conditions included 100 μM free calcium and pre-incubation of tCells with exogenous PI(4,5)P 2 . Pores appeared two to three times less frequently with the mutated proteins compared to wild-type Syt1 C2AB. Student's t-test was used to assess significant differences between the ‘no C2AB’ group and the rest. ( C ) Mean single open-pore conductance values (± SEM) for the same conditions as in B. Disrupting binding to calcium (D309N), acidic lipids (K326A, K327A), or the SNARE complex (R398, R399) resulted in ~3-fold smaller mean conductance compared to wild-type C2AB, abrogating the effects of Syt1 C2AB. Two-sample Kolmogorov-Smirnov test was used to assess significant differences between the ‘no C2AB’ group and the rest. *, **, *** indicate p<0.05, 0.01, and 0.001, respectively.

Article Snippet: Mouse monoclonal anti-PI(4,5)P 2 primary antibodies (Echelon Biosciences Inc, Utah) were added to the cells at time points of 0, 40, and 80 min and incubated 1 hr at 37°C.

Techniques: Labeling, Binding Assay, Incubation

Journal: eLife

Article Title: The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

doi: 10.7554/eLife.68215

Figure Lengend Snippet: ( A ) Schematic depiction of Syt1 C2B domain’s calcium-dependent interactions with membranes. Calcium-free C2B interacts with acidic lipids through its poly-lysine motif (highlighted in cyan as in ). Upon binding to calcium, hydrophobic residues (V304 and I367 on C2B) insert into the membrane, causing C2B to reorient and inducing membrane curvature ( ; ). In the presence of PI(4,5)P 2 , the calcium-bound C2B assumes a tilted conformation with respect to the membrane . M173 and F234 on C2A top loops similarly insert into membranes in a calcium-dependent manner, with similar effect on orientation and curvature generation (not shown). A mutant with the membrane-inserting residues replaced with tryptophans (M173W, F234W, V304W, and I367W, ‘4W’) binds membranes more avidly, resulting in more membrane tubulation activity, whereas alanine substitution of the same residues (‘4A’) abolishes membrane penetration and curvature induction . ( B ) Pore nucleation rate (mean ± S.E.M) in the presence of wildtype, 4W and 4A mutants. Student's t-test was used to assess significant differences between the ‘no C2AB’ group and the rest. ( C ) Mean open-pore conductance (± S.E.M) for the conditions indicated. Two-sample Kolmogorov-Smirnov test was used to assess significant differences between the ‘no C2AB’ group and the rest. ( D ) Cumulative density functions for mean open-pore conductances for wild-type Syt1 C2AB, 4W and 4A mutants. In A, calcium-free C2B was rendered from PDB 5w5d and calcium-bound C2B was rendered from 5kj7 . *, **, *** indicate p<0.05, 0.01, and 0.001, respectively.

Article Snippet: Mouse monoclonal anti-PI(4,5)P 2 primary antibodies (Echelon Biosciences Inc, Utah) were added to the cells at time points of 0, 40, and 80 min and incubated 1 hr at 37°C.

Techniques: Binding Assay, Mutagenesis, Activity Assay

Journal: eLife

Article Title: The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

doi: 10.7554/eLife.68215

Figure Lengend Snippet: ( A ) Schematic of model. The membrane free energy has contributions from membrane tension and bending energy. SNARE complexes may be unzippered and free to roam laterally, or zippered and confined to the pore waist. Crowding among zippered SNARE complexes generates entropic forces that tend to enlarge the pore (top view, shown lower right). The Syt1 C2B domain (green ellipsoid) has a SNARE-binding region, a polybasic patch and Ca 2+ -binding loops. ( B ) Free energy-minimizing fusion pore shapes determined by solving the membrane shape equation in the presence and absence of constraints applied by the SNARE-C2B complex (see Appendix 1). The C2B calcium-binding loops may either be unburied (top panel) or buried (lower panel) in the membrane. In the buried state the SNARE complex tilts upwards, expanding the fusion pore. The membrane shape constraint is evaluated using the SNARE-C2B complex crystal structure in a space filling representation. Both upper and lower panels depict situations in the presence of Ca 2+ . The model predicts the tilted configuration is strongly favored at high [ C a 2 + ] following equilibration, while the untilted configuration is relevant to the kinetics that establish this equilibrium, and to experiments using low [ C a 2 + ] . VAMP2, syntaxin, SNAP25 and the C2B domain are shown blue, red, yellow, and green, respectively. The C2B hydrophobic membrane-inserting residues (V304, I367), polybasic patch (K326, K327) and SNARE-binding region (R398, R399) are shown orange, cyan, and purple, respectively. The protein structure was generated with PyMOL using the SNARE-C2B crystal structure (PDB ID 5ccg) . The TMD of the SNARE complex (PDB ID 3hd7) was incorporated using UCSF chimera software . ( C ) Model-predicted free energy and experimental apparent free energy versus pore radius without calcium and in the presence of excess calcium. ( D ) Model-predicted normalized conductances shown with experimentally measured values for comparison. Experimental data taken from experiments including Ca 2+ and PI(4,5)P 2 . ( E ) Pore dilation mechanism emerging from the model. Under conditions of low calcium concentration, the C2B domain is unburied, the SNARE complex lies parallel to the membrane and the membrane separation is set by the maximum thickness of the SNARE-C2B complex. At high calcium concentrations, the calcium binding loops penetrate the plasma membrane, rotating the C2B domain and the entire SNARE-C2B complex which exerts force (red arrows) on the upper and lower membranes of the fusion pore in a lever-like action. These forces increase the fusion pore height, which is coupled by membrane energetics to fusion pore dilation.

Article Snippet: Mouse monoclonal anti-PI(4,5)P 2 primary antibodies (Echelon Biosciences Inc, Utah) were added to the cells at time points of 0, 40, and 80 min and incubated 1 hr at 37°C.

Techniques: Binding Assay, Generated, Software, Concentration Assay