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

Journal: Molecular Biology of the Cell

Article Title: PLCζ causes Ca 2+ oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P 2

doi: 10.1091/mbc.E11-08-0687

Figure Lengend Snippet: Plasma membrane PI(4,5)P 2 changes monitored with GFP fused to the PLCδ1 PH domain (GFP-PH). Calcium and plasma membrane PI(4,5)P 2 changes (ratio of pmGFP/cytoGFP) in eggs caused by PLCζ or PLCδ1 were recorded (A–G) starting ∼20 min after injection of RNA. Injection of PLCζ cRNA (0.002 μg/μl) caused Ca 2+ oscillations and plasma membrane PI(4,5)P 2 increases following each Ca 2+ spike (A). A slight decrease (4.8 ± 2.6%) in resting level of PI(4,5)P 2 was detected (A, H). Keeping eggs in medium containing 10 μg/ml cytochalasin B had little effect on changes in plasma membrane PI(4,5)P 2 in PLCζ-injected eggs (B, H). PLCδ1 (cRNA, >10 μg/μl) triggered Ca 2+ oscillations and plasma membrane PI(4,5)P 2 increases (D). During 2 h of imaging the resting level of PI(4,5)P 2 showed little change in eggs with Ca 2+ oscillations caused by PLCδ1 (C, H). In contrast, when eggs were treated with cytochalasin B, a dramatic consumption of plasma membrane PI(4,5)P 2 was detected (22.0 ± 6.9%, E and H) as judged by the percentage change in signal over the 2 h from the start to end of the recording. A clear consumption of plasma membrane PI(4,5)P 2 was also detected (12.7 ± 3.0%) in eggs injected with reduced amounts of PLCδ1(5 μg/μl), which was insufficient to cause Ca 2+ oscillations. A summary of the data is shown in H, with egg numbers indicated for each condition above the traces in A–G. Images of GFP-PH distribution in eggs injected with PLCζ or PLCδ1 and kept in cytochalasin B for 4 h are shown in C and F. Bar, 10 μm.

Article Snippet: Mouse monoclonal anti-PI(4,5)P 2 antibody (clone 2C11; immunoglobulin M [IgM]) was purchased from Echelon Bioscience (Salt Lake City, UT) and used at 2.5 μg/μl.

Techniques: Injection, Imaging

Journal: Molecular Biology of the Cell

Article Title: PLCζ causes Ca 2+ oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P 2

doi: 10.1091/mbc.E11-08-0687

Figure Lengend Snippet: Inhibition of Ca 2+ oscillations following plasma membrane PI(4,5)P 2 depletion using Lyn-GFP-Inp54p (LynPs). The majority of LynPs accumulated at plasma membrane (A). Expression of LynPs blocked Ca 2+ oscillations caused by PLCδ1 (B, 100%). However, LynPs did not affect Ca 2+ oscillations in PLCζ-injected eggs (C) or in IVF eggs (D). Bar, 10 μm.

Article Snippet: Mouse monoclonal anti-PI(4,5)P 2 antibody (clone 2C11; immunoglobulin M [IgM]) was purchased from Echelon Bioscience (Salt Lake City, UT) and used at 2.5 μg/μl.

Techniques: Inhibition, Expressing, Injection

Journal: Molecular Biology of the Cell

Article Title: PLCζ causes Ca 2+ oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P 2

doi: 10.1091/mbc.E11-08-0687

Figure Lengend Snippet: Block of calcium oscillation by PI(4,5)P 2 depletion with overdose of LynPs.

Article Snippet: Mouse monoclonal anti-PI(4,5)P 2 antibody (clone 2C11; immunoglobulin M [IgM]) was purchased from Echelon Bioscience (Salt Lake City, UT) and used at 2.5 μg/μl.

Techniques: Blocking Assay

Journal: Molecular Biology of the Cell

Article Title: PLCζ causes Ca 2+ oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P 2

doi: 10.1091/mbc.E11-08-0687

Figure Lengend Snippet: The distribution of PI(4,5)P 2 in MII eggs. (A) Both ring-like plasma membrane PI(4,5)P 2 and intracellular PI(4,5)P 2 were detected in MII eggs. The cortical section and magnification illustrate the presence of intracellular PI(4,5)P 2 in vesicular structures, which aggregate into patch-like areas (ii and iii). (B) A negative control for PI(4,5)P 2 staining in which PI(4,5)P 2 antibody was incubated with PI(4,5)P 2 for 20 min at room temperature; the only weak staining seen is in the zona pellucida. Bar, 1 μm in magnification views; 10 μm in all other images. (C) Distinctive nuclear PI(4,5)P 2 was detected in CHO cells, but very few intracellular vesicles were seen to contain PI(4,5)P 2 . The only vesicles seen are pointed to by the white arrow and one can been seen more clearly in the inset. Bar, 10 μm.

Article Snippet: Mouse monoclonal anti-PI(4,5)P 2 antibody (clone 2C11; immunoglobulin M [IgM]) was purchased from Echelon Bioscience (Salt Lake City, UT) and used at 2.5 μg/μl.

Techniques: Negative Control, Staining, Incubation

Journal: Molecular Biology of the Cell

Article Title: PLCζ causes Ca 2+ oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P 2

doi: 10.1091/mbc.E11-08-0687

Figure Lengend Snippet: Differential changes in intracellular PI(4,5)P 2 and plasma membrane PI(4,5)P 2 upon PLCζ and PLCδ1 expression. Each egg is shown in low resolution with a higher-resolution insert. Plasma membrane PI(4,5)P 2 (A) or intracellular PI(4,5)P 2 (C) was specifically preserved by different fixation protocols as described in Materials and Methods . In the presence of cytochalasin B, PLCδ1 caused ∼30% reduction of plasma membrane PI(4,5)P 2 (fluorescence intensities measured in the whole of each egg cortex; A, B), in contrast to the 10% plasma membrane PI(4,5)P 2 reduction caused by PLCζ (A, B; p < 0.01, measured in the whole egg). However, PLCζ caused higher reduction of intracellular PI(4,5)P 2 than PLCδ1 regardless of cytochalasin B treatment (C, D). Bar, 10 μm for main image; 1 μm for insets. The numbers of eggs analyzed for each treatment are indicated above the bars in B and D.

Article Snippet: Mouse monoclonal anti-PI(4,5)P 2 antibody (clone 2C11; immunoglobulin M [IgM]) was purchased from Echelon Bioscience (Salt Lake City, UT) and used at 2.5 μg/μl.

Techniques: Expressing, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: PLCζ causes Ca 2+ oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P 2

doi: 10.1091/mbc.E11-08-0687

Figure Lengend Snippet: Block of calcium oscillations following PI(4,5)P 2 depletion with ciPLCζ-GFP-Inp54p, which distributed throughout the cytoplasm without any accumulation in the plasma membrane (A). Expression of this construct did not affect Ca 2+ oscillations caused by PLCδ1 (B). However, Ca 2+ oscillations were completely blocked in 35% of PLCζ-expressing eggs (C, i) and 24% of fertilized eggs (D, i). In eggs in which Ca 2+ oscillations were observed using PLCζ (C) or sperm (D), they were greatly attenuated. Bar, 10 μm.

Article Snippet: Mouse monoclonal anti-PI(4,5)P 2 antibody (clone 2C11; immunoglobulin M [IgM]) was purchased from Echelon Bioscience (Salt Lake City, UT) and used at 2.5 μg/μl.

Techniques: Blocking Assay, Expressing, Construct

Journal: The Journal of General Physiology

Article Title: Voltage- and temperature-dependent activation of TRPV3 channels is potentiated by receptor-mediated PI(4,5)P 2 hydrolysis

doi: 10.1085/jgp.200910388

Figure Lengend Snippet: PI(4,5)P 2 depletion potentiates 2-APB–activated TRPV3 currents. HM1 cells expressing WT TRPV3 were treated for 2 min (CCh) or 5 min (PAO and WM) with the indicated drug, and currents were elicited in the presence of 10 µM 2-APB. (A) Representative currents resulting from voltage steps to +100 and −100 mV after treatment with the indicated drugs. (B) Representative I TAIL –V relations from the same cells as current traces shown in A. (C) Average fold change in step current at +80 mV (open bars) and −80 mV (gray bars) elicited by treatment with the indicated drugs or vehicle control. (D and E) Average changes in V 0.5 (D) and VIF (E) under the indicated conditions. In D and E, bar shading represents CCh alone (open), PAO (diagonally hatched), CCh plus PAO (gray), WM (crosshatched), or vehicle control (black). Final drug concentrations in A–E: CCh, 100 µM; PAO, 30 µM; WM, 10 nM. For C–E, data represent means ± SEM from n = 5–8 cells. See for a summary of the data.

Article Snippet: ATP was from Roche, purified bacterial phosphatidylinositol-specific PLC (PI-PLC) was obtained from Invitrogen, and diC8-PIPs and anti-PI(4,5)P 2 mouse monoclonal antiserum (mAb) were from Echelon Biosciences.

Techniques: Expressing

Journal: The Journal of General Physiology

Article Title: Voltage- and temperature-dependent activation of TRPV3 channels is potentiated by receptor-mediated PI(4,5)P 2 hydrolysis

doi: 10.1085/jgp.200910388

Figure Lengend Snippet: TRPV3 channel activity in excised patches is stimulated by PI(4,5)P 2 depletion. Inside-out patches pulled from HM1 cells expressing WT TRPV3 were exposed to either anti-PI(4,5)P 2 mAb or PI-PLC for 5 min, or OAG for 2 min (V m = +80 mV). (A) A representative current record (left) showing the potentiating effect of mAb (1:200 dilution) on TRPV3 channel activity. Open boxes before ( a ) and after ( b ) reagent addition indicate time segments used for determination of NP OPEN . Expanded current traces (right) during the time period shown in box a or b . The level of current associated with the indicated number of open TRPV3 channels is shown on the left. (B) Selected all-points histograms showing TRPV3 channel activity in patches before and after treatment with vehicle control, mAb (1:200 dilution; data from A), 100 µM OAG, or 0.5 U/ml PI-PLC. (C) Mean calculated NP OPEN values before and after reagent addition in patches treated with vehicle control ( n = 7), mAb ( n = 7), OAG ( n = 11), or PI-PLC ( n = 29). Error bars represent SEM; *, P < 0.05; **, P < 0.001 versus control after vehicle addition by Student’s nonpaired t test.

Article Snippet: ATP was from Roche, purified bacterial phosphatidylinositol-specific PLC (PI-PLC) was obtained from Invitrogen, and diC8-PIPs and anti-PI(4,5)P 2 mouse monoclonal antiserum (mAb) were from Echelon Biosciences.

Techniques: Activity Assay, Expressing

Journal: The Journal of General Physiology

Article Title: Voltage- and temperature-dependent activation of TRPV3 channels is potentiated by receptor-mediated PI(4,5)P 2 hydrolysis

doi: 10.1085/jgp.200910388

Figure Lengend Snippet: PI(4,5)P 2 decreases TRPV3 single-channel open probability. Inside-out patches excised from HM1 cells expressing WT TRPV3 (V m = +80 mV) were treated with 0.5 U/ml PI-PLC (open bar), followed by water-soluble diC8-phosphatidylinositol phosphate analogues. (A and B) Representative current records showing the [diC8-PI(4,5)P 2 ]–dependent decrease in TRPV3 channel activity (A) and lack of similar effect in vehicle-treated patches (B). Filled bars in B represent serial dilutions of vehicle that mimic those used to achieve the indicated [diC8-PI(4,5)P 2 ] shown in A. (C) A representative patch recording in which TRPV3 activity was stimulated by mAb and reversibly inhibited by 30 µM diC8-PI(4,5)P 2 . (D) The average diC8-PI(4,5)P 2 concentration–response relation (data represent means ± SEM; n = 5 patches). Solid line represents a Hill fit to the data for 1–60 µM diC8-PI(4,5)P 2 (IC 50 = 10.0 ± 4.5 µM). (E) Average change in NP OPEN induced by the addition of the indicated diC8-phosphatidylinositol phosphates. Data represent means ± SEM from n = 3–6 patches.

Article Snippet: ATP was from Roche, purified bacterial phosphatidylinositol-specific PLC (PI-PLC) was obtained from Invitrogen, and diC8-PIPs and anti-PI(4,5)P 2 mouse monoclonal antiserum (mAb) were from Echelon Biosciences.

Techniques: Expressing, Activity Assay, Concentration Assay

Journal: The Journal of General Physiology

Article Title: Voltage- and temperature-dependent activation of TRPV3 channels is potentiated by receptor-mediated PI(4,5)P 2 hydrolysis

doi: 10.1085/jgp.200910388

Figure Lengend Snippet: Effects of G q/11 -coupled receptor activation are attenuated in TRP domain mutants. (A) Sequence alignment of TRP domains from TRPV1, TRPV3, and TRPV5. Numbers indicate starting position in the amino acid sequence of human TRPV1 (NCBI Protein database accession no. NM_080705 ), human TRPV3 ( AF514998.1 ), and human TRPV5 ( NM_019841 ). Residues that are identical to TRPV3 are shown in gray, and basic residues previously identified as being important for PI(4,5)P 2 -dependent modulation of TRPV1 and TRPV5 that are conserved in TRPV3 are shown within open boxes. Asterisks denote positions of TRPV3 mutations reported here. Whole cell currents were recorded from HM1 cells transfected with TRPV3 R696A (B–F) or K705A (G–K). M 1 was activated by bath application of 100 µM CCh in the presence (CCh + Ca 2+ ) or absence (CCh − Ca 2+ ) of buffered free [Ca 2+ ] i , as described in . Cells were incubated with 30 µM PAO for 5 min between the first and second 2-APB applications to inhibit PI 4 kinase activity. The 2-APB concentrations used for R696A and K705A were 3 and 6 µM, respectively. (B and G) Representative current records (bottom) elicited by steps to the indicated voltages (top) recorded under the following conditions: CCh + Ca 2+ (black circles), CCh − Ca 2+ (gray squares), and PAO (gray triangles). The zero current level is indicated by a solid line. (C and H) Representative I TAIL –V relations from the cells shown in B and G under the following conditions: CCh + Ca 2+ (circles), CCh − Ca 2+ (squares), and PAO (triangles). Lines represent fits to a Boltzmann function. (D and I) Average fold change in current amplitude at +80 mV (open bars) and −80 mV (black bars) elicited by treatment under the indicated conditions. (E and J) Average change in V 0.5 (ΔV 0.5 = ΔV 0.5 POST − ΔV 0.5 PRE ) measured in cells treated under the indicated conditions. (F and K) Average change in VIF (VIF POST − VIF PRE ) before and after treatment under the following conditions: CCh + Ca 2+ (open bars), CCh − Ca 2+ (diagonally hatched bars), and PAO (crosshatched bars). For D–F and I–K, data represent means ± SEM from n = 5–7 cells.

Article Snippet: ATP was from Roche, purified bacterial phosphatidylinositol-specific PLC (PI-PLC) was obtained from Invitrogen, and diC8-PIPs and anti-PI(4,5)P 2 mouse monoclonal antiserum (mAb) were from Echelon Biosciences.

Techniques: Activation Assay, Sequencing, Transfection, Incubation, Activity Assay

Journal: The Journal of General Physiology

Article Title: Voltage- and temperature-dependent activation of TRPV3 channels is potentiated by receptor-mediated PI(4,5)P 2 hydrolysis

doi: 10.1085/jgp.200910388

Figure Lengend Snippet: The TRP domain mutants R696A and K705A are insensitive to changes in PI(4,5)P 2 . Inside-out patches were excised from HM1 cells expressing TRPV3 R696A or K705A, and voltage was clamped at +80 mV. (A and D) Representative current records from inside-out patches exposed to mAb (1:200 dilution; black bars, left). Open boxes indicate the time segments used for analysis of single-channel properties ( a , before exposure to mAb; b , after exposure to mAb). Expanded current traces (right) during the time period shown in box a or b . (B and E) All-points histograms of raw data shown in A and D and similar recordings (see Fig. S3) in nontreated controls or patches treated with 0.5 U/ml PI-PLC and 30 µM diC8-PI(4,5)P 2 . (C and F) Average NP OPEN in patches expressing R696A or K705A either before (open bars) or after (black bars) the addition of mAb or PI-PLC, or in nontreated control recordings. Data represent means ± SEM from n = 3–5 patches.

Article Snippet: ATP was from Roche, purified bacterial phosphatidylinositol-specific PLC (PI-PLC) was obtained from Invitrogen, and diC8-PIPs and anti-PI(4,5)P 2 mouse monoclonal antiserum (mAb) were from Echelon Biosciences.

Techniques: Expressing