Structured Review

AVESTIN 100 nm pore diameter
, with the following parameters: Δφ m = 14.2 ± 1.3 mV, K 1 = (2.6 ± 0.4) × 10 7 M −1 , K 2 = (3.7 ± 0.3) × 10 6 M −1 , and R 2 = 0.998. The inset shows the kinetics of the PD change from <t>100</t> nM protein added in one (red) or two (blue) steps. The arrow indicates the moment of the second addition of protein.
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Images

1) Product Images from "pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion"

Article Title: pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion

Journal: Journal of Virology

doi: 10.1128/JVI.01539-15

, with the following parameters: Δφ m = 14.2 ± 1.3 mV, K 1 = (2.6 ± 0.4) × 10 7 M −1 , K 2 = (3.7 ± 0.3) × 10 6 M −1 , and R 2 = 0.998. The inset shows the kinetics of the PD change from 100 nM protein added in one (red) or two (blue) steps. The arrow indicates the moment of the second addition of protein.
Figure Legend Snippet: , with the following parameters: Δφ m = 14.2 ± 1.3 mV, K 1 = (2.6 ± 0.4) × 10 7 M −1 , K 2 = (3.7 ± 0.3) × 10 6 M −1 , and R 2 = 0.998. The inset shows the kinetics of the PD change from 100 nM protein added in one (red) or two (blue) steps. The arrow indicates the moment of the second addition of protein.

Techniques Used:

AFM topography image of the protein layer adsorbed at an M1 bulk concentration of 100 nM with a removed square patch of the protein layer with dimensions of 0.7 by 0.7 μm 2 . The full z -axis scale is 10 nm.
Figure Legend Snippet: AFM topography image of the protein layer adsorbed at an M1 bulk concentration of 100 nM with a removed square patch of the protein layer with dimensions of 0.7 by 0.7 μm 2 . The full z -axis scale is 10 nm.

Techniques Used: Concentration Assay

AFM topography images of M1 protein layer structures on supported BLMs (A to C) and mica (D to F). (A, D) Changes in M1 layer structure upon a pH drop from 7.1 to 5.0 or pH transitions from 7.1 to 6.0 to 5.0. The bright spots are protein aggregates. (B, E) M1 initially adsorbed at pH 6.0. (C, F) M1 initially adsorbed at pH 5.0. The protein bulk concentration was 70 nM. The working buffer was 100 mM KCl–50 mM MES. The full z -axis scale is 10 nm in panels A to D and 5 nm in panels E and F.
Figure Legend Snippet: AFM topography images of M1 protein layer structures on supported BLMs (A to C) and mica (D to F). (A, D) Changes in M1 layer structure upon a pH drop from 7.1 to 5.0 or pH transitions from 7.1 to 6.0 to 5.0. The bright spots are protein aggregates. (B, E) M1 initially adsorbed at pH 6.0. (C, F) M1 initially adsorbed at pH 5.0. The protein bulk concentration was 70 nM. The working buffer was 100 mM KCl–50 mM MES. The full z -axis scale is 10 nm in panels A to D and 5 nm in panels E and F.

Techniques Used: Concentration Assay

AFM topography images of M1 adsorbed at pH 7.1 to supported BLMs (A to C) and mica (D to F) at the following bulk concentrations: A and D, 10 nM; B and E, 50 nM; C and F, 70 nM. The full z -axis scale is 5 nm for panels A and D to F and 10 nm for panels B and C. The bright spots in panels B and C are protein aggregates. The working buffer was 100 mM KCl–50 mM MES. The inset in panel C is a 300-by-300-nm 2 enlargement of the image. The full z -axis scale of the inset is 5 nm.
Figure Legend Snippet: AFM topography images of M1 adsorbed at pH 7.1 to supported BLMs (A to C) and mica (D to F) at the following bulk concentrations: A and D, 10 nM; B and E, 50 nM; C and F, 70 nM. The full z -axis scale is 5 nm for panels A and D to F and 10 nm for panels B and C. The bright spots in panels B and C are protein aggregates. The working buffer was 100 mM KCl–50 mM MES. The inset in panel C is a 300-by-300-nm 2 enlargement of the image. The full z -axis scale of the inset is 5 nm.

Techniques Used:

2) Product Images from "Analysis of Molecular Concentration and Brightness from Fluorescence Fluctuation Data with an Electron Multiplied CCD Camera"

Article Title: Analysis of Molecular Concentration and Brightness from Fluorescence Fluctuation Data with an Electron Multiplied CCD Camera

Journal: Biophysical Journal

doi: 10.1529/biophysj.108.130310

B / S versus 〈 I 〉 − offset / S plots for photon-counting simulations ( A ) and simulations with the same analog noise as the Cascade camera ( B – D ). ( C and D ) G 1 and G 1 y analyses, respectively, of the simulation. All simulations used 2D diffusion with 1250 molecules in a 3.5 × 3.5- μ m box, except for the high-intensity, low-brightness point in B , which used 25,000 molecules in the same box size. The diffusion coefficient was 2 μ m 2 /s, with a frame rate of 200 frames/s. Each simulation was run for 100 frames with a frame resolution of 128 × 128 pixels. Brightness values were 0.5, 2, 5, and 10 cpfm.
Figure Legend Snippet: B / S versus 〈 I 〉 − offset / S plots for photon-counting simulations ( A ) and simulations with the same analog noise as the Cascade camera ( B – D ). ( C and D ) G 1 and G 1 y analyses, respectively, of the simulation. All simulations used 2D diffusion with 1250 molecules in a 3.5 × 3.5- μ m box, except for the high-intensity, low-brightness point in B , which used 25,000 molecules in the same box size. The diffusion coefficient was 2 μ m 2 /s, with a frame rate of 200 frames/s. Each simulation was run for 100 frames with a frame resolution of 128 × 128 pixels. Brightness values were 0.5, 2, 5, and 10 cpfm.

Techniques Used: Diffusion-based Assay

3) Product Images from "The crossover conformational shift of the GTPase atlastin provides the energy driving ER fusion"

Article Title: The crossover conformational shift of the GTPase atlastin provides the energy driving ER fusion

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201609071

Mutant variants show no defects in GTP-catalyzed crossover formation. (A and B, left) The positions of K320 and P317 mutations made to target DATL crossover superimposed onto PyMOL renderings of (A) the hATL1 form2 extended dimer PDB 3QOF and (B) the hATL1 form3 crossover dimer PDB 4IDP . The position of the Cy3 dye in each structure is indicated with a red circle. (A and B, right) Enlargement of the boxed regions in A and B showing the K320 and P317 side chains highlighted in cyan. (C) Normalized PIFE over time when each of the indicated Cy3-cytoDATL mutant variants is mixed with GTP. (C, right) Zoomed-in view of the first 100 ms of the trace in C. The mean of seven runs is shown for each trace (SEM
Figure Legend Snippet: Mutant variants show no defects in GTP-catalyzed crossover formation. (A and B, left) The positions of K320 and P317 mutations made to target DATL crossover superimposed onto PyMOL renderings of (A) the hATL1 form2 extended dimer PDB 3QOF and (B) the hATL1 form3 crossover dimer PDB 4IDP . The position of the Cy3 dye in each structure is indicated with a red circle. (A and B, right) Enlargement of the boxed regions in A and B showing the K320 and P317 side chains highlighted in cyan. (C) Normalized PIFE over time when each of the indicated Cy3-cytoDATL mutant variants is mixed with GTP. (C, right) Zoomed-in view of the first 100 ms of the trace in C. The mean of seven runs is shown for each trace (SEM

Techniques Used: Mutagenesis, Mass Spectrometry

A subset of mutant variants causes abnormal ER network structure. (A) COS-7 cells transfected with each indicated variant of full-length Venus-tagged DATL were fixed and imaged 48 h later by confocal microscopy. Bar, 10 µm. (B) Quantification of the percentage of expressing cells displaying a normal branched ER ( > 100 cells per measurement; data represent mean of three independent measurements ± SD); *, P
Figure Legend Snippet: A subset of mutant variants causes abnormal ER network structure. (A) COS-7 cells transfected with each indicated variant of full-length Venus-tagged DATL were fixed and imaged 48 h later by confocal microscopy. Bar, 10 µm. (B) Quantification of the percentage of expressing cells displaying a normal branched ER ( > 100 cells per measurement; data represent mean of three independent measurements ± SD); *, P

Techniques Used: Mutagenesis, Transfection, Variant Assay, Confocal Microscopy, Expressing

4) Product Images from "Biochemical Reconstitution of Hemorrhagic-Fever Arenavirus Envelope Glycoprotein-Mediated Membrane Fusion"

Article Title: Biochemical Reconstitution of Hemorrhagic-Fever Arenavirus Envelope Glycoprotein-Mediated Membrane Fusion

Journal: PLoS ONE

doi: 10.1371/journal.pone.0051114

pH-induced membrane fusion by rGPC fur proteoliposomes: content mixing. A. rGPC fur was incorporated into POPG-POPC-CHS liposomes containing 50 µM Zn ++ and these proteoliposomes were mixed with target POPG-POPC-CHS liposomes containing 1 µM FluoZin-1. The experimental treatments, labels and colors are as described in the legend of Fig. 6 . Content mixing was determined by Zn ++ -induced FluoZin-1 fluorescence measured at 485 nm (excitation 370 nm). Complete mixing was determined by solubilization with Triton X-100 in buffer containing 50 µM Zn ++ . B. pH-induced content mixing was determined in the continued presence of MAbs F100G5, BF11 or an irrelevant IgG (5 µM, 25 µM and 25 µM, respectively). In this experiment, content mixing in the absence of MAb (pH 5.0) was ∼50%.
Figure Legend Snippet: pH-induced membrane fusion by rGPC fur proteoliposomes: content mixing. A. rGPC fur was incorporated into POPG-POPC-CHS liposomes containing 50 µM Zn ++ and these proteoliposomes were mixed with target POPG-POPC-CHS liposomes containing 1 µM FluoZin-1. The experimental treatments, labels and colors are as described in the legend of Fig. 6 . Content mixing was determined by Zn ++ -induced FluoZin-1 fluorescence measured at 485 nm (excitation 370 nm). Complete mixing was determined by solubilization with Triton X-100 in buffer containing 50 µM Zn ++ . B. pH-induced content mixing was determined in the continued presence of MAbs F100G5, BF11 or an irrelevant IgG (5 µM, 25 µM and 25 µM, respectively). In this experiment, content mixing in the absence of MAb (pH 5.0) was ∼50%.

Techniques Used: Fluorescence

pH-induced membrane fusion by rGPC fur proteoliposomes: lipid mixing. rGPC fur was incorporated into POPG-POPC liposomes and mixed with target POPG-POPC liposomes doped with 1% Rhodamine-PE. In most cases, the rGPC fur proteoliposomes were first treated with sFurin (as indicated by+in the first position of the labels, at right). Exposure to acidic pH at the start of the experiment (time = 0) is indicated by+in the second position of the labels. 15 µM of ST-294 or ST-161 was present prior to and during exposure to acidic pH, where indicated. Lipid mixing and the resulting dequenching of the rhodamine fluorophore were measured at 600 nm (excitation 508 nm). Complete dequenching (100%) was determined by subsequent solubilization in Triton X-100 nonionic detergent.
Figure Legend Snippet: pH-induced membrane fusion by rGPC fur proteoliposomes: lipid mixing. rGPC fur was incorporated into POPG-POPC liposomes and mixed with target POPG-POPC liposomes doped with 1% Rhodamine-PE. In most cases, the rGPC fur proteoliposomes were first treated with sFurin (as indicated by+in the first position of the labels, at right). Exposure to acidic pH at the start of the experiment (time = 0) is indicated by+in the second position of the labels. 15 µM of ST-294 or ST-161 was present prior to and during exposure to acidic pH, where indicated. Lipid mixing and the resulting dequenching of the rhodamine fluorophore were measured at 600 nm (excitation 508 nm). Complete dequenching (100%) was determined by subsequent solubilization in Triton X-100 nonionic detergent.

Techniques Used:

SPR studies of interactions with lipid-reconstituted rGPC fur . The rGPC fur precursor was immobilized onto a Biacore L1 chip and reconstituted in a lipid bilayer as described in Material and Methods . Two or three concentration-dependent data sets were analyzed and sensorgram figures were generated using a five-point smoothing procedure and ORIGIN graphing software. Labels to the right are drawn to coincide with the maximum RUs achieved by the respective ligand. A. Binding of G1-directed MAbs (BF11, BF09, BE08, and AG02; 0.5 µM), sTfR (1.5 µM), and a nucleoprotein-directed MAb (aN; BG12). B. Binding of SIGA (ST-294, ST-375, ST-193, and ST-761; 150 µM) and TSRI (17C8; 100 µM) small-molecule fusion inhibitors. ST-161 and TSRI 8C1 are specific to the OW LASV and do not inhibit the NW arenavirus JUNV.
Figure Legend Snippet: SPR studies of interactions with lipid-reconstituted rGPC fur . The rGPC fur precursor was immobilized onto a Biacore L1 chip and reconstituted in a lipid bilayer as described in Material and Methods . Two or three concentration-dependent data sets were analyzed and sensorgram figures were generated using a five-point smoothing procedure and ORIGIN graphing software. Labels to the right are drawn to coincide with the maximum RUs achieved by the respective ligand. A. Binding of G1-directed MAbs (BF11, BF09, BE08, and AG02; 0.5 µM), sTfR (1.5 µM), and a nucleoprotein-directed MAb (aN; BG12). B. Binding of SIGA (ST-294, ST-375, ST-193, and ST-761; 150 µM) and TSRI (17C8; 100 µM) small-molecule fusion inhibitors. ST-161 and TSRI 8C1 are specific to the OW LASV and do not inhibit the NW arenavirus JUNV.

Techniques Used: SPR Assay, Chromatin Immunoprecipitation, Concentration Assay, Generated, Software, Binding Assay

5) Product Images from "Charge-Dependent Translocation of the Trojan Peptide Penetratin across Lipid Membranes"

Article Title: Charge-Dependent Translocation of the Trojan Peptide Penetratin across Lipid Membranes

Journal: Biophysical Journal

doi:

Lipid-to-peptide experiments for mixed DOPC/DOPG SUV of different composition. The molar fraction of DOPG, X PG , is given in the figure. Calorimeter tracings are shown in the left panel ( δV syr = 7 μ l, SUV in TRIS + 100 mM NaCl; L syr = 15 mM, P 0 = 0.0125 mM). The right panel displays the differential heats, q L , as a function of the molar ratio lipid-to-peptide in the sample cell.
Figure Legend Snippet: Lipid-to-peptide experiments for mixed DOPC/DOPG SUV of different composition. The molar fraction of DOPG, X PG , is given in the figure. Calorimeter tracings are shown in the left panel ( δV syr = 7 μ l, SUV in TRIS + 100 mM NaCl; L syr = 15 mM, P 0 = 0.0125 mM). The right panel displays the differential heats, q L , as a function of the molar ratio lipid-to-peptide in the sample cell.

Techniques Used:

6) Product Images from "Membrane tethering by the atlastin GTPase depends on GTP hydrolysis but not on forming the cross-over configuration"

Article Title: Membrane tethering by the atlastin GTPase depends on GTP hydrolysis but not on forming the cross-over configuration

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E14-08-1284

Tethering does not require cross-over. (A) The K320E mutation partially inhibits cross-over, but the double mutation P319G K320E abolishes it. The soluble domain of wild-type, K320E, P317G, P319G, or P319G K320E D-ATL was incubated at RT for 20 min in the presence or absence of the indicated nucleotides and then subjected to BMOE cross-linking. The single asterisk marks the soluble-domain monomer and the double asterisk marks the cross-linked dimer. All variants had the G343C substitution. (B) The positions of linker mutations made to block D-ATL cross-over superimposed onto a PyMOL rendering of the ATL1 cross-over dimer PDB 3QNU. (C) Expression of linker mutant variants in Cos-7 cells perturbs ER network morphology. Cells transfected with the indicated FL eYFP-tagged D-ATL mutant variants were visualized 48 h later by confocal microscopy. Scale bar: 10 μm. (D) The percent of cells expressing each variant and showing a normal branched ER, > 100 cells per measurement, average of three independent measurements ± SD are quantified. The dimerization- and hydrolysis-defective R48E D-ATL is also shown for comparison. (E) Mutations that block cross-over do not impair GTPase activity. The soluble domain of each cross-over–defective variant was assayed for GTPase activity. The average of three independent measurements ± SD is shown. R48E is shown for comparison. (F) Cross-over–defective P319G K320E D-ATL can tether. Vesicles containing P319G K320E FL D-ATL were assessed for tethering by DLS after incubation at 37°C for 10 min in the presence or absence of the indicated nucleotides. The average of three independent measurements ± SD is shown for each condition. (G) The P319G K320E mutations block fusion. Wild-type or P319G K320E D-ATL was reconstituted into donor and acceptor vesicles. Fusion was monitored by the dequenching of NBD-labeled lipid present in the donor vesicles in the presence or absence of GTP.
Figure Legend Snippet: Tethering does not require cross-over. (A) The K320E mutation partially inhibits cross-over, but the double mutation P319G K320E abolishes it. The soluble domain of wild-type, K320E, P317G, P319G, or P319G K320E D-ATL was incubated at RT for 20 min in the presence or absence of the indicated nucleotides and then subjected to BMOE cross-linking. The single asterisk marks the soluble-domain monomer and the double asterisk marks the cross-linked dimer. All variants had the G343C substitution. (B) The positions of linker mutations made to block D-ATL cross-over superimposed onto a PyMOL rendering of the ATL1 cross-over dimer PDB 3QNU. (C) Expression of linker mutant variants in Cos-7 cells perturbs ER network morphology. Cells transfected with the indicated FL eYFP-tagged D-ATL mutant variants were visualized 48 h later by confocal microscopy. Scale bar: 10 μm. (D) The percent of cells expressing each variant and showing a normal branched ER, > 100 cells per measurement, average of three independent measurements ± SD are quantified. The dimerization- and hydrolysis-defective R48E D-ATL is also shown for comparison. (E) Mutations that block cross-over do not impair GTPase activity. The soluble domain of each cross-over–defective variant was assayed for GTPase activity. The average of three independent measurements ± SD is shown. R48E is shown for comparison. (F) Cross-over–defective P319G K320E D-ATL can tether. Vesicles containing P319G K320E FL D-ATL were assessed for tethering by DLS after incubation at 37°C for 10 min in the presence or absence of the indicated nucleotides. The average of three independent measurements ± SD is shown for each condition. (G) The P319G K320E mutations block fusion. Wild-type or P319G K320E D-ATL was reconstituted into donor and acceptor vesicles. Fusion was monitored by the dequenching of NBD-labeled lipid present in the donor vesicles in the presence or absence of GTP.

Techniques Used: Mutagenesis, Incubation, Blocking Assay, Expressing, Transfection, Confocal Microscopy, Variant Assay, Activity Assay, Labeling

Cryo-EM visualization of vesicle tethering by D-ATL. (A and B) Single TM D-ATL reconstituted into vesicles was incubated at RT for 20 min in the absence or presence of GTP and imaged with cryo-EM at low (A) and high (B) magnification. Scale bars: (A) 1 μm; (B) 100 nm. Arrowheads (B) mark zipper-like structures formed between opposing membranes. (C) Enlarged views of the zipper-like structures from wild-type single TM (left), P319G K320E FL (middle), and K320E FL (right) D-ATL. Arrows indicate where two membranes are “zippered” together. Scale bar: 20 nm. (D) Comparison of the average number of tethered structures per micrograph, in the absence (black) and presence (gray) of GTP. Data shown are from 70–100 micrographs per sample. Also shown is a comparison of the average width of the “zippers” (green) in the presence of GTP from wild-type single TM, P319G K320E FL and K320E FL D-ATL samples. Measurements are derived from > 10 “zippers” for each, ± SD The indicated p values were determined using an unpaired Student's t test.
Figure Legend Snippet: Cryo-EM visualization of vesicle tethering by D-ATL. (A and B) Single TM D-ATL reconstituted into vesicles was incubated at RT for 20 min in the absence or presence of GTP and imaged with cryo-EM at low (A) and high (B) magnification. Scale bars: (A) 1 μm; (B) 100 nm. Arrowheads (B) mark zipper-like structures formed between opposing membranes. (C) Enlarged views of the zipper-like structures from wild-type single TM (left), P319G K320E FL (middle), and K320E FL (right) D-ATL. Arrows indicate where two membranes are “zippered” together. Scale bar: 20 nm. (D) Comparison of the average number of tethered structures per micrograph, in the absence (black) and presence (gray) of GTP. Data shown are from 70–100 micrographs per sample. Also shown is a comparison of the average width of the “zippers” (green) in the presence of GTP from wild-type single TM, P319G K320E FL and K320E FL D-ATL samples. Measurements are derived from > 10 “zippers” for each, ± SD The indicated p values were determined using an unpaired Student's t test.

Techniques Used: Incubation, Derivative Assay

Fusion but not tethering depends on a K320-E328 salt bridge. (A) The position of the K320E-E328 salt bridge is superimposed onto a PyMOL rendering of the ATL1 cross-over dimer PDB 3QNU. (B) Expression of the individual K320E and E328R FL D-ATL variants but not the compensatory double-mutant (K320E,E328R) variant leads to abnormal ER network structure in Cos-7 cells. Cells transfected with the indicated eYFP-tagged variants were imaged 48 h later by confocal microscopy. Scale bar: 10 μm. (C) Quantification of the percent of expressing cells displaying a normal branched ER, > 100 cells per measurement, average of three independent measurements ± SD. (D) The K320-E328 salt bridge is required for fusion. Wild-type, K320E, E328R, or the double-mutant (K320E,E328R) variant of FL D-ATL were reconstituted into donor and acceptor vesicles. Fusion was monitored by the dequenching of NBD-labeled lipid present in the donor vesicles in the presence or absence of GTP. (E) The K320-E328 salt bridge is not required for tethering. Either single TM or FL versions of either K320E or E328R D-ATL were incorporated into vesicles and incubated at 37°C for 5 min with the indicated nucleotides. Thereafter samples were subjected to DLS. For comparison, tethering by the single TM R48E D-ATL variant is also shown. The average of three independent measurements ± SD is shown for each condition.
Figure Legend Snippet: Fusion but not tethering depends on a K320-E328 salt bridge. (A) The position of the K320E-E328 salt bridge is superimposed onto a PyMOL rendering of the ATL1 cross-over dimer PDB 3QNU. (B) Expression of the individual K320E and E328R FL D-ATL variants but not the compensatory double-mutant (K320E,E328R) variant leads to abnormal ER network structure in Cos-7 cells. Cells transfected with the indicated eYFP-tagged variants were imaged 48 h later by confocal microscopy. Scale bar: 10 μm. (C) Quantification of the percent of expressing cells displaying a normal branched ER, > 100 cells per measurement, average of three independent measurements ± SD. (D) The K320-E328 salt bridge is required for fusion. Wild-type, K320E, E328R, or the double-mutant (K320E,E328R) variant of FL D-ATL were reconstituted into donor and acceptor vesicles. Fusion was monitored by the dequenching of NBD-labeled lipid present in the donor vesicles in the presence or absence of GTP. (E) The K320-E328 salt bridge is not required for tethering. Either single TM or FL versions of either K320E or E328R D-ATL were incorporated into vesicles and incubated at 37°C for 5 min with the indicated nucleotides. Thereafter samples were subjected to DLS. For comparison, tethering by the single TM R48E D-ATL variant is also shown. The average of three independent measurements ± SD is shown for each condition.

Techniques Used: Expressing, Mutagenesis, Variant Assay, Transfection, Confocal Microscopy, Labeling, Incubation

7) Product Images from "Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of ?-Synuclein"

Article Title: Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of ?-Synuclein

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M809067200

tTG cross-linking does not interfere with the binding of α -syn to lipid vesicle membranes. A, CD spectra of cross-linked ( cl )α-syn in the presence of vesicles. In the presence of POPG vesicles, cross-linked A30P α-syn undergoes a change in secondary structure from random-coil (—) to α-helices (- - -). B, SDS-PAGE analysis of membrane-bound native ( lane 5 ) and cross-linked ( lane 6 ) monomeric α-syn from A after passage through a filter with a molecular mass cutoff of 100 kDa. Both retentate ( R ) and flow-through ( F ) were separated onto a 12% SDS-PAGE ( lanes 5-8 ; M, monomeric; m, cross-linked monomeric; L, lipids). C and D, CD spectra of membrane-bound WT and Q mutant α-syn before ( C ) and after ( D ) cross-linking.
Figure Legend Snippet: tTG cross-linking does not interfere with the binding of α -syn to lipid vesicle membranes. A, CD spectra of cross-linked ( cl )α-syn in the presence of vesicles. In the presence of POPG vesicles, cross-linked A30P α-syn undergoes a change in secondary structure from random-coil (—) to α-helices (- - -). B, SDS-PAGE analysis of membrane-bound native ( lane 5 ) and cross-linked ( lane 6 ) monomeric α-syn from A after passage through a filter with a molecular mass cutoff of 100 kDa. Both retentate ( R ) and flow-through ( F ) were separated onto a 12% SDS-PAGE ( lanes 5-8 ; M, monomeric; m, cross-linked monomeric; L, lipids). C and D, CD spectra of membrane-bound WT and Q mutant α-syn before ( C ) and after ( D ) cross-linking.

Techniques Used: Binding Assay, SDS Page, Flow Cytometry, Mutagenesis

8) Product Images from "ER network formation and membrane fusion by atlastin1/SPG3A disease variants"

Article Title: ER network formation and membrane fusion by atlastin1/SPG3A disease variants

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E14-10-1447

Some but not all atlastin1/SPG3A variants are defective in forming a branched ER network. (A) At 48 h after transfection with the indicated HA-tagged atlastin1 variant constructs, HeLa cells were further transfected using siRNAs targeting endogenous atlastin2/3. At 72 h after knockdown, cells were fixed and stained using an antibody against the HA epitope and viewed by confocal microscopy. The negative control was Myc-tagged REEP5/DP1/TB2 stained with an antibody against the Myc epitope. Scale bar, 10 μm. (B) Quantification of the percentage of cells expressing the HA- or Myc-tagged construct and showing an abnormal unbranched ER morphology. Values represent the means of three independent experiments ( > 100 cells each) ± SD. * p
Figure Legend Snippet: Some but not all atlastin1/SPG3A variants are defective in forming a branched ER network. (A) At 48 h after transfection with the indicated HA-tagged atlastin1 variant constructs, HeLa cells were further transfected using siRNAs targeting endogenous atlastin2/3. At 72 h after knockdown, cells were fixed and stained using an antibody against the HA epitope and viewed by confocal microscopy. The negative control was Myc-tagged REEP5/DP1/TB2 stained with an antibody against the Myc epitope. Scale bar, 10 μm. (B) Quantification of the percentage of cells expressing the HA- or Myc-tagged construct and showing an abnormal unbranched ER morphology. Values represent the means of three independent experiments ( > 100 cells each) ± SD. * p

Techniques Used: Transfection, Variant Assay, Construct, Staining, Confocal Microscopy, Negative Control, Expressing

Some but not all SPG3A mutations impair ER network formation when expressed in Drosophila atlastin. (A) At 48 h after transfection with the indicated eYFP-tagged Drosophila atlastin variant constructs, HeLa cells were further transfected using siRNAs targeting endogenous atlastin2 and atlastin3. At 72 h after knockdown, cells were fixed and viewed by confocal microscopy. The negative control Myc-tagged REEP5/DP1/TB2 was stained with an antibody against the Myc epitope. Scale bar, 10 μm. (B) Quantification of the percentage of cells expressing either the Myc-tagged or eYFP-tagged construct and showing an abnormal unbranched ER morphology. Values represent the means of three independent experiments ( > 100 cells each) ± SD. * p
Figure Legend Snippet: Some but not all SPG3A mutations impair ER network formation when expressed in Drosophila atlastin. (A) At 48 h after transfection with the indicated eYFP-tagged Drosophila atlastin variant constructs, HeLa cells were further transfected using siRNAs targeting endogenous atlastin2 and atlastin3. At 72 h after knockdown, cells were fixed and viewed by confocal microscopy. The negative control Myc-tagged REEP5/DP1/TB2 was stained with an antibody against the Myc epitope. Scale bar, 10 μm. (B) Quantification of the percentage of cells expressing either the Myc-tagged or eYFP-tagged construct and showing an abnormal unbranched ER morphology. Values represent the means of three independent experiments ( > 100 cells each) ± SD. * p

Techniques Used: Transfection, Variant Assay, Construct, Confocal Microscopy, Negative Control, Staining, Expressing

Atlastin1 and Drosophila atlastin maintain a normal branched ER network in HeLa cells in the absence of atlastin2/3. At 48 h after transfection with Myc-tagged REEP5/DP1/TB2 (neg con; A, B), eYFP-tagged Drosophila atlastin (C), or HA-tagged atlastin1 (D), cells were either left untreated (A) or further transfected using siRNAs targeting atlastin2/3 (B–D). At 72 h after knockdown, cells were fixed and stained using an antibody against the Myc or HA epitope and viewed by confocal microscopy. Scale bars, 10 μm. The insets in A–D show magnified views of a boxed region of the peripheral ER. (E) Quantification of the average number of network branch points (±SD) in five representative 100-μm 2 boxed peripheral regions of the ER under each condition. (F) Quantification of the percentage of cells expressing Myc-REEP5/DP1/TB2 (with or without RNAi), eYFP– Drosophila atlastin (with RNAi), or HA-atlastin1 (with RNAi) that displayed an abnormal unbranched ER morphology. Values represent the means of three independent experiments ( > 100 cells each) ± SD. * p
Figure Legend Snippet: Atlastin1 and Drosophila atlastin maintain a normal branched ER network in HeLa cells in the absence of atlastin2/3. At 48 h after transfection with Myc-tagged REEP5/DP1/TB2 (neg con; A, B), eYFP-tagged Drosophila atlastin (C), or HA-tagged atlastin1 (D), cells were either left untreated (A) or further transfected using siRNAs targeting atlastin2/3 (B–D). At 72 h after knockdown, cells were fixed and stained using an antibody against the Myc or HA epitope and viewed by confocal microscopy. Scale bars, 10 μm. The insets in A–D show magnified views of a boxed region of the peripheral ER. (E) Quantification of the average number of network branch points (±SD) in five representative 100-μm 2 boxed peripheral regions of the ER under each condition. (F) Quantification of the percentage of cells expressing Myc-REEP5/DP1/TB2 (with or without RNAi), eYFP– Drosophila atlastin (with RNAi), or HA-atlastin1 (with RNAi) that displayed an abnormal unbranched ER morphology. Values represent the means of three independent experiments ( > 100 cells each) ± SD. * p

Techniques Used: Transfection, Staining, Confocal Microscopy, Expressing

9) Product Images from "Raft Composition at Physiological Temperature and pH in the Absence of Detergents"

Article Title: Raft Composition at Physiological Temperature and pH in the Absence of Detergents

Journal:

doi: 10.1529/biophysj.107.118596

( A ) High SM content retards liposome flotation in sucrose gradients. DOPC/cholesterol liposomes 100 nm in diameter labeled by Rho-DOPE and SM/cholesterol liposomes labeled with NBD-DPPE were placed at the bottom of a sucrose gradient and allowed to come
Figure Legend Snippet: ( A ) High SM content retards liposome flotation in sucrose gradients. DOPC/cholesterol liposomes 100 nm in diameter labeled by Rho-DOPE and SM/cholesterol liposomes labeled with NBD-DPPE were placed at the bottom of a sucrose gradient and allowed to come

Techniques Used: Labeling

10) Product Images from "FUNCTIONAL RECONSTITUTION OF INFLUENZA A M2(22-62)"

Article Title: FUNCTIONAL RECONSTITUTION OF INFLUENZA A M2(22-62)

Journal: Biochimica et biophysica acta

doi: 10.1016/j.bbamem.2010.10.010

Valinomycin-induced uptake as inhibited by 100 μM amantadine, showing the small change in external pH under consideration. Valinomycin was added at t = 0 seconds. A linear baseline drift has been subtracted. Black dots: M2(22-62) proteoliposomes.
Figure Legend Snippet: Valinomycin-induced uptake as inhibited by 100 μM amantadine, showing the small change in external pH under consideration. Valinomycin was added at t = 0 seconds. A linear baseline drift has been subtracted. Black dots: M2(22-62) proteoliposomes.

Techniques Used:

11) Product Images from "Domain Formation in Phosphatidylinositol Monophosphate/Phosphatidylcholine Mixed Vesicles"

Article Title: Domain Formation in Phosphatidylinositol Monophosphate/Phosphatidylcholine Mixed Vesicles

Journal: Biophysical Journal

doi:

DSC thermograms of DPPC, DPPI, and DPPC/DPPI (1:1) multilamellar vesicles (0.3 mM total lipid concentration, third heating scan, heating rate = 1°C/min, pH 7.4, 100 mM NaCl, 10 mM HEPES, 0.1 mM EDTA).
Figure Legend Snippet: DSC thermograms of DPPC, DPPI, and DPPC/DPPI (1:1) multilamellar vesicles (0.3 mM total lipid concentration, third heating scan, heating rate = 1°C/min, pH 7.4, 100 mM NaCl, 10 mM HEPES, 0.1 mM EDTA).

Techniques Used: Concentration Assay

DSC thermograms of DPPC/DPPI-xP (85:15) mixed multilamellar vesicles as a function of pH value (0.3 mM total lipid concentration, third heating scan, heating rate = 1°C/min, 100 mM NaCl, 10 mM buffer, 0.1 mM EDTA, see Materials).
Figure Legend Snippet: DSC thermograms of DPPC/DPPI-xP (85:15) mixed multilamellar vesicles as a function of pH value (0.3 mM total lipid concentration, third heating scan, heating rate = 1°C/min, 100 mM NaCl, 10 mM buffer, 0.1 mM EDTA, see Materials).

Techniques Used: Concentration Assay

12) Product Images from "Biophysical Parameters of the Sec14 Phospholipid Exchange Cycle"

Article Title: Biophysical Parameters of the Sec14 Phospholipid Exchange Cycle

Journal: Biophysical Journal

doi: 10.1016/j.bpj.2018.11.3131

SANS-based lipid transfer assay and floating assay. ( a ) Principle of the detection of lipid transfer by SANS. ( b ) Temporal changes in the normalized contrasts of PC, PC/PI (90:10), and PC/PS (90:10) LUVs ( closed circles ). Sec14 (50 μ g/mL) was injected into mixtures of D- and H-vesicles (15 mM each) at t = 0. Solid lines represent fitting curves with double exponential function. Experiments were also conducted for mixtures of D-vesicles consisting of 100% d -PC and H-vesicles consisting of 90% h -PC and 10% PI or PS ( open symbols ). ( c ) Temporal changes in the normalized contrasts of PC LUVs and PC SUVs ( closed circles ). Protein and lipid concentrations were the same as those in ( b ). Experiments were also conducted for mixtures of LUVs consisting of 100% d -PC and SUVs consisting of 100% h -PC ( open circles ). Data in ( b ) and ( c ) were collected at SANS-U. ( d ) A schematic representation of the Sec14-mediated lipid exchange in the presence of LUVs and SUVs. Sec14 preferentially associates to SUVs and transfers PC between SUVs, so that the transfer between LUV and SUV rarely occurs. ( e ) Vesicle flotation assay. Sec14 (175 μ g/mL) was incubated with PC LUVs or SUVs (2 mM PC) and separated to top (T) and bottom (B) fractions, which were analyzed by SDS-PAGE. ( f ) Quantification of the binding fraction of Sec14 from SDS-PAGE images. Each bar represents the percentage of top fraction against total Sec14 amount (i.e., top fraction + bottom fraction ). Bars show the mean ± SD of four independent experiments. To see this figure in color, go online.
Figure Legend Snippet: SANS-based lipid transfer assay and floating assay. ( a ) Principle of the detection of lipid transfer by SANS. ( b ) Temporal changes in the normalized contrasts of PC, PC/PI (90:10), and PC/PS (90:10) LUVs ( closed circles ). Sec14 (50 μ g/mL) was injected into mixtures of D- and H-vesicles (15 mM each) at t = 0. Solid lines represent fitting curves with double exponential function. Experiments were also conducted for mixtures of D-vesicles consisting of 100% d -PC and H-vesicles consisting of 90% h -PC and 10% PI or PS ( open symbols ). ( c ) Temporal changes in the normalized contrasts of PC LUVs and PC SUVs ( closed circles ). Protein and lipid concentrations were the same as those in ( b ). Experiments were also conducted for mixtures of LUVs consisting of 100% d -PC and SUVs consisting of 100% h -PC ( open circles ). Data in ( b ) and ( c ) were collected at SANS-U. ( d ) A schematic representation of the Sec14-mediated lipid exchange in the presence of LUVs and SUVs. Sec14 preferentially associates to SUVs and transfers PC between SUVs, so that the transfer between LUV and SUV rarely occurs. ( e ) Vesicle flotation assay. Sec14 (175 μ g/mL) was incubated with PC LUVs or SUVs (2 mM PC) and separated to top (T) and bottom (B) fractions, which were analyzed by SDS-PAGE. ( f ) Quantification of the binding fraction of Sec14 from SDS-PAGE images. Each bar represents the percentage of top fraction against total Sec14 amount (i.e., top fraction + bottom fraction ). Bars show the mean ± SD of four independent experiments. To see this figure in color, go online.

Techniques Used: Injection, Incubation, SDS Page, Binding Assay

13) Product Images from "Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin"

Article Title: Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Inhibition of SNARE complex assembly by calcium/calmodulin. ( A ) In vitro translated 35 S-SNAP-25 was incubated in the presence or absence of GST, GST-VAMP 1–96 , and untagged syntaxin 1A 1–261 for 3 h at 4°C. Protein complexes were recovered on glutathione beads and, after washing, bound 35 S-SNAP-25 was measured by β counting. Results are means ± SD, n = 3. ( B ) Samples prepared as in A were analyzed by SDS/PAGE and autoradiography after denaturation in SDS at 37°C or 100°C. Arrows indicate the migration of the trimeric core complex (upper arrow) and 35 S-SNAP-25 (lower arrow). The radioactive band at about 40 kDa in the first two lanes is an unidentified translation product that did not interact with VAMP 2 or syntaxin 1A. ( C ) 35 S-SNAP-25, GST-VAMP 1–96 , and syntaxin 1A 1–261 were incubated at 4°C in a buffer containing 1 mM CaCl 2 , in the presence or absence of calmodulin (10 μM). At the indicated times, samples were removed, and bound 35 S-SNAP-25 was evaluated by β counting. The curve is representative of three independent experiments. ( D ) SNARE assembly was performed as in A , except that complexes were recovered on calmodulin–agarose beads in the presence or absence of 1 mM CaCl 2 and after pretreatment of GST-VAMP 1–96 with TeTx light chain. 35 S-SNAP-25 retained by calmodulin–agarose is shown as a percentage, taking the radioactivity recovered on glutathione beads as 100%, means + SD, n = 3.
Figure Legend Snippet: Inhibition of SNARE complex assembly by calcium/calmodulin. ( A ) In vitro translated 35 S-SNAP-25 was incubated in the presence or absence of GST, GST-VAMP 1–96 , and untagged syntaxin 1A 1–261 for 3 h at 4°C. Protein complexes were recovered on glutathione beads and, after washing, bound 35 S-SNAP-25 was measured by β counting. Results are means ± SD, n = 3. ( B ) Samples prepared as in A were analyzed by SDS/PAGE and autoradiography after denaturation in SDS at 37°C or 100°C. Arrows indicate the migration of the trimeric core complex (upper arrow) and 35 S-SNAP-25 (lower arrow). The radioactive band at about 40 kDa in the first two lanes is an unidentified translation product that did not interact with VAMP 2 or syntaxin 1A. ( C ) 35 S-SNAP-25, GST-VAMP 1–96 , and syntaxin 1A 1–261 were incubated at 4°C in a buffer containing 1 mM CaCl 2 , in the presence or absence of calmodulin (10 μM). At the indicated times, samples were removed, and bound 35 S-SNAP-25 was evaluated by β counting. The curve is representative of three independent experiments. ( D ) SNARE assembly was performed as in A , except that complexes were recovered on calmodulin–agarose beads in the presence or absence of 1 mM CaCl 2 and after pretreatment of GST-VAMP 1–96 with TeTx light chain. 35 S-SNAP-25 retained by calmodulin–agarose is shown as a percentage, taking the radioactivity recovered on glutathione beads as 100%, means + SD, n = 3.

Techniques Used: Inhibition, In Vitro, Incubation, SDS Page, Autoradiography, Migration, Radioactivity

Related Articles

Centrifugation:

Article Title: GTP hydrolysis promotes disassembly of the atlastin crossover dimer during ER fusion
Article Snippet: Liposomes (100–300-nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin) and checked for size by dynamic light scattering (Zen3600). .. Insoluble protein aggregates were pelleted by centrifugation in a microcentrifuge for 10 min at 16,000 g .

Article Title: The crossover conformational shift of the GTPase atlastin provides the energy driving ER fusion
Article Snippet: Liposomes (100–300 nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin) and checked for size by dynamic light scattering (Zen3600; Malvern Instruments). .. Insoluble protein aggregates were pelleted by centrifugation of the samples in a microcentrifuge for 10 min at 16,000g .

Article Title: Raft Composition at Physiological Temperature and pH in the Absence of Detergents
Article Snippet: SUVs were prepared at 60°C in 0.5 M sucrose solution containing 10 mM Tris-HCl, pH 7.3, by extrusion through a 100-nm-pore polycarbonate filters (LiposoFast extruder, Avestin, Ottawa, Canada). .. After a 33,000 rpm centrifugation in an SW 41 Ti rotor for 2.5 h, 10 1-ml fractions were collected.

Blocking Assay:

Article Title: FUNCTIONAL RECONSTITUTION OF INFLUENZA A M2(22-62)
Article Snippet: Internal buffer (50 mM KCl, 50 mM K2 HPO4 , 50 mM KH2 PO4 , pH 8.0, 320 mOsm) was added to the thin film, vortexed to form nonuniform liposomes, then extruded through a 100-nm pore-size polycarbonate filter (Liposofast membrane extruder, Avestin, Ottawa, Canada) at 50-60°C. .. After extrusion, samples were divided for matched-pair drug block assessment.

Incubation:

Article Title: Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of ?-Synuclein
Article Snippet: Prior to gel loading, aggregates of very high molecular weight that formed during the incubation were removed using microcon filters with a molecular mass cutoff of 100 kDa (Millipore Corp., Bedford, MA). .. Small unilamellar vesicles were prepared by extrusion through a 100-nm polycarbonate membrane (Avestin, Mannheim, Germany) according to the manufacturer's instructions.

Article Title: Filter gate closure inhibits ion but not water transport through potassium channels
Article Snippet: Proteoliposomes or control vesicles were diluted 10-fold in buffer (150 mM choline chloride, 25 mM Tris, 25 mM Hepes, pH 7.50) and extruded through two stacked 100-nm polycarbonate filters (Avestin). .. After 30 min of incubation with 8 µM valinomycin and 5 µM CCCP (both from Sigma), 75 µL of the suspension was mixed with the same volume of buffer containing 600 mM sucrose in a stopped-flow device (model SFM-300; Bio-Logic).

Mass Spectrometry:

Article Title: Filter gate closure inhibits ion but not water transport through potassium channels
Article Snippet: Proteoliposomes or control vesicles were diluted 10-fold in buffer (150 mM choline chloride, 25 mM Tris, 25 mM Hepes, pH 7.50) and extruded through two stacked 100-nm polycarbonate filters (Avestin). .. Recording settings were 7,000 data points at an interval of 100 µs and 1,000 data points at an interval of 2 ms with a filter of 300 µs according to the manufacturer’s recommendations.

Modification:

Article Title: THE PDZ2 DOMAIN OF ZONULA OCCLUDENS -1 AND -2 IS A PHOSPHOINOSITIDE BINDING DOMAIN
Article Snippet: Phospholipid concentrations were determined by a modified Bartlett analysis [ ]. .. The Liposofast microextruder and 100-nm polycarbonate filters were from Avestin (Ottawa, Ontario).

Flow Cytometry:

Article Title: Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of ?-Synuclein
Article Snippet: The retentate (high molecular weight aggregates) and the flow-through solution (monomeric α-syn) were analyzed by 12% native PAGE or SDS-PAGE. .. Small unilamellar vesicles were prepared by extrusion through a 100-nm polycarbonate membrane (Avestin, Mannheim, Germany) according to the manufacturer's instructions.

Sublimation:

Article Title: Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin
Article Snippet: Dry lipid films were hydrated in 150 mM NaCl/1 mM EDTA/25 mM Tris⋅HCl/ 25 mM sodium acetate, adjusted to pH 7.2, and allowed to swell at a temperature above the phase-transition temperature of the phospholipids (typically > 45°C). .. Liposomes were formed by 19 passes through 100-nm polycarbonate filters by using a Lipofast basic apparatus (Avestin, Ottawa) and stored at 4°C under nitrogen.

Solubility:

Article Title: Domain Formation in Phosphatidylinositol Monophosphate/Phosphatidylcholine Mixed Vesicles
Article Snippet: To avoid lipid demixing due to solubility differences among the components of the lipid mixture (which has been shown in some instances to affect the mixing properties in the later formed vesicles), this drying process was carried out as quickly as possible at slightly elevated temperatures (≈50°C). .. For the fluorescence measurements, unilamellar vesicles were obtained by extruding the multilamellar vesicles at ≈60°C through a 100-nm pore size membrane (Avestin, Ottawa, ON).

SPR Assay:

Article Title: Biochemical Reconstitution of Hemorrhagic-Fever Arenavirus Envelope Glycoprotein-Mediated Membrane Fusion
Article Snippet: Preparation of Liposomes and Proteoliposomes DMPC:PC liposomes used to form lipid bilayers in SPR studies were prepared as previously described . .. LUVs were produced by extrusion through 100-nm pore size polycarbonate membranes (Avestin).

Generated:

Article Title: Phospholipid-esterified Eicosanoids Are Generated in Agonist-activated Human Platelets and Enhance Tissue Factor-dependent Thrombin Generation *
Article Snippet: Varying amounts of 16:0/20:4-PC or 16:0/HETE-PC generated by air oxidation and purified as described above were added to the lipid extract from unactivated control platelets (human platelets were isolated as above and immediately extracted) in a glass vial, and then the solvent was evaporated under N2 . .. Liposomes were then prepared by 10 freeze-thaw cycles followed by passing through Liposofast miniextruder using 100-nm pore membranes (Avestin, Ottawa, ON, Canada) 29 times.

Sonication:

Article Title: Charge-Dependent Translocation of the Trojan Peptide Penetratin across Lipid Membranes
Article Snippet: .. After five freeze-thaw cycles, the lipid suspension was either passed at least 15 times through two stacked Osmonics polycarbonate membranes of 100-nm pore size in a 1-ml mini-Liposo-Fast-Pneumatic extruder (Avestin, Canada), or it was sonicated in cold water for 20 min using a titanium tip ultrasonicator followed by 10-min ultracentrifugation (3000 g) to prepare large and small unilamellar vesicles, LUV and SUV, respectively. .. The measurements were performed using a VP isothermal titration calorimeter produced by MicroCal Inc. (Northampton, MA).

Article Title: Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin
Article Snippet: The dispersed lipid (1 ml) was then sonicated for 2 min by using an Ultrasons apparatus (Annemasse, France) with a 2-mm microtip. .. Liposomes were formed by 19 passes through 100-nm polycarbonate filters by using a Lipofast basic apparatus (Avestin, Ottawa) and stored at 4°C under nitrogen.

Molecular Weight:

Article Title: Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of ?-Synuclein
Article Snippet: The retentate (high molecular weight aggregates) and the flow-through solution (monomeric α-syn) were analyzed by 12% native PAGE or SDS-PAGE. .. Small unilamellar vesicles were prepared by extrusion through a 100-nm polycarbonate membrane (Avestin, Mannheim, Germany) according to the manufacturer's instructions.

Article Title: FUNCTIONAL RECONSTITUTION OF INFLUENZA A M2(22-62)
Article Snippet: Liposomes were prepared by mixing chloroform-suspended E. coli polar lipid extract (67% phosphatidylethanolamine, 23.2% phosphatidylglycerol, 9.8% cardiolipin, average molecular weight: 798 Da; Avanti Polar Lipids, Alabaster, AL, USA) with the methanol-suspended M2(22-62) peptide. .. Internal buffer (50 mM KCl, 50 mM K2 HPO4 , 50 mM KH2 PO4 , pH 8.0, 320 mOsm) was added to the thin film, vortexed to form nonuniform liposomes, then extruded through a 100-nm pore-size polycarbonate filter (Liposofast membrane extruder, Avestin, Ottawa, Canada) at 50-60°C.

Fluorescence:

Article Title: Domain Formation in Phosphatidylinositol Monophosphate/Phosphatidylcholine Mixed Vesicles
Article Snippet: .. For the fluorescence measurements, unilamellar vesicles were obtained by extruding the multilamellar vesicles at ≈60°C through a 100-nm pore size membrane (Avestin, Ottawa, ON). .. The quality of the extrusion was checked regularly by Dynamic Light Scattering (HPPS, Malvern Instruments, Southborough, MA).

Isolation:

Article Title: Phospholipid-esterified Eicosanoids Are Generated in Agonist-activated Human Platelets and Enhance Tissue Factor-dependent Thrombin Generation *
Article Snippet: Varying amounts of 16:0/20:4-PC or 16:0/HETE-PC generated by air oxidation and purified as described above were added to the lipid extract from unactivated control platelets (human platelets were isolated as above and immediately extracted) in a glass vial, and then the solvent was evaporated under N2 . .. Liposomes were then prepared by 10 freeze-thaw cycles followed by passing through Liposofast miniextruder using 100-nm pore membranes (Avestin, Ottawa, ON, Canada) 29 times.

Labeling:

Article Title: GTP hydrolysis promotes disassembly of the atlastin crossover dimer during ER fusion
Article Snippet: Liposomes (100–300-nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin) and checked for size by dynamic light scattering (Zen3600). .. Purified full-length D -ATL was incorporated at a 1:1,000 protein/lipid ratio into labeled and unlabeled liposome populations at an effective detergent/lipid ratio of ∼0.7 by incubating protein and lipid at 4°C for 1 h followed by four detergent-removal incubations by SM-2 Bio-Beads (Bio-Rad) at 1 g beads per 70 mg Anapoe X-100.

Article Title: Membrane tethering by the atlastin GTPase depends on GTP hydrolysis but not on forming the cross-over configuration
Article Snippet: Liposomes (100- to 300-nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin, Ottawa, ON, Canada) and checked for size by DLS (as described below and in Supplemental Figure S1). .. Un­labeled liposomes consisted of 1-palmitoyl-2-oleoyl-sn -glycero-3-phosphocholine:1,2-dioleoyl-sn -glycero-3-phospho-l -serine (85:15), and labeled liposomes had 1-palmitoyl-2-oleoyl-sn -glycero-3-phosphocholine:1,2-dioleoyl-sn -glycero-3-phospho-l -serine:1,3-dipalmitoyl-sn -glycero-3-phosphoethanolamine-N -(7-nitro-2-1,3-benzoxadiazol-4-yl:1,2-dipalmitoyl-sn -glycero-3-phosphoethanolamine-N -(lissamine rhodamine B sulfonyl) (82:15: 1.5:1.5).

Article Title: The crossover conformational shift of the GTPase atlastin provides the energy driving ER fusion
Article Snippet: Liposomes (100–300 nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin) and checked for size by dynamic light scattering (Zen3600; Malvern Instruments). .. Full-length DATL was inserted at a 1:1,000 ratio of proteins to lipids into labeled and unlabeled populations of liposomes at an effective detergent-to-lipid ratio of ∼0.7 by incubating protein and lipid at 4°C for 1 h followed by detergent removal by SM-2 Bio-Beads (Bio-Rad) at 1 g beads per 70 mg Anapoe X-100.

Article Title: ER network formation and membrane fusion by atlastin1/SPG3A disease variants
Article Snippet: Liposomes of diameter 100–300 nm were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin, Ottawa, ON, Canada). .. Unlabeled liposomes consisted of palmitoyloleoylphosphatidylcholine (POPC):dioleoyl phosphatidylserine (DOPS; 85:15), and labeled liposomes had POPC:DOPS:1,2-dipalmitoyl-sn -glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl (DPPE-NBD):rhodamine-DPPE (82:15:1.5:1.5).

Purification:

Article Title: GTP hydrolysis promotes disassembly of the atlastin crossover dimer during ER fusion
Article Snippet: Liposomes (100–300-nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin) and checked for size by dynamic light scattering (Zen3600). .. Purified full-length D -ATL was incorporated at a 1:1,000 protein/lipid ratio into labeled and unlabeled liposome populations at an effective detergent/lipid ratio of ∼0.7 by incubating protein and lipid at 4°C for 1 h followed by four detergent-removal incubations by SM-2 Bio-Beads (Bio-Rad) at 1 g beads per 70 mg Anapoe X-100.

Article Title: Phospholipid-esterified Eicosanoids Are Generated in Agonist-activated Human Platelets and Enhance Tissue Factor-dependent Thrombin Generation *
Article Snippet: Varying amounts of 16:0/20:4-PC or 16:0/HETE-PC generated by air oxidation and purified as described above were added to the lipid extract from unactivated control platelets (human platelets were isolated as above and immediately extracted) in a glass vial, and then the solvent was evaporated under N2 . .. Liposomes were then prepared by 10 freeze-thaw cycles followed by passing through Liposofast miniextruder using 100-nm pore membranes (Avestin, Ottawa, ON, Canada) 29 times.

Spectroscopy:

Article Title: Charge-Dependent Translocation of the Trojan Peptide Penetratin across Lipid Membranes
Article Snippet: The final concentrations of penetratin in the samples were determined by ultraviolet absorption spectroscopy ( ). .. After five freeze-thaw cycles, the lipid suspension was either passed at least 15 times through two stacked Osmonics polycarbonate membranes of 100-nm pore size in a 1-ml mini-Liposo-Fast-Pneumatic extruder (Avestin, Canada), or it was sonicated in cold water for 20 min using a titanium tip ultrasonicator followed by 10-min ultracentrifugation (3000 g) to prepare large and small unilamellar vesicles, LUV and SUV, respectively.

Chromatin Immunoprecipitation:

Article Title: THE PDZ2 DOMAIN OF ZONULA OCCLUDENS -1 AND -2 IS A PHOSPHOINOSITIDE BINDING DOMAIN
Article Snippet: The Liposofast microextruder and 100-nm polycarbonate filters were from Avestin (Ottawa, Ontario). .. Pioneer L1 sensor chip was from Biacore AB (Piscataway, NJ).

Article Title: Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin
Article Snippet: Liposomes were formed by 19 passes through 100-nm polycarbonate filters by using a Lipofast basic apparatus (Avestin, Ottawa) and stored at 4°C under nitrogen. .. Anti-GST antibodies were covalently coupled to a CM5 sensor chip by standard amine-coupling procedure.

SDS Page:

Article Title: Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of ?-Synuclein
Article Snippet: The retentate (high molecular weight aggregates) and the flow-through solution (monomeric α-syn) were analyzed by 12% native PAGE or SDS-PAGE. .. Small unilamellar vesicles were prepared by extrusion through a 100-nm polycarbonate membrane (Avestin, Mannheim, Germany) according to the manufacturer's instructions.

Sample Prep:

Article Title: Domain Formation in Phosphatidylinositol Monophosphate/Phosphatidylcholine Mixed Vesicles
Article Snippet: Paragraph title: Sample preparation ... For the fluorescence measurements, unilamellar vesicles were obtained by extruding the multilamellar vesicles at ≈60°C through a 100-nm pore size membrane (Avestin, Ottawa, ON).

Article Title: Charge-Dependent Translocation of the Trojan Peptide Penetratin across Lipid Membranes
Article Snippet: No substantial loss of penetratin during sample preparation was established. .. After five freeze-thaw cycles, the lipid suspension was either passed at least 15 times through two stacked Osmonics polycarbonate membranes of 100-nm pore size in a 1-ml mini-Liposo-Fast-Pneumatic extruder (Avestin, Canada), or it was sonicated in cold water for 20 min using a titanium tip ultrasonicator followed by 10-min ultracentrifugation (3000 g) to prepare large and small unilamellar vesicles, LUV and SUV, respectively.

Evaporation:

Article Title: Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of ?-Synuclein
Article Snippet: Preparation of Large Unilamellar Vesicles, Small Unilamellar Vesicles, and α -Syn-Liposome Complexes —1-Palmitoyl-2-oleoyl- sn -glycero-3-[phospho- rac -(1-glycerol)] sodium salt (POPG, Avanti Polar Lipids Inc., Pelham, AL) was purchased as a chloroform solution, and the solvent was removed by evaporation and lyophilization. .. Small unilamellar vesicles were prepared by extrusion through a 100-nm polycarbonate membrane (Avestin, Mannheim, Germany) according to the manufacturer's instructions.

Article Title: GTP hydrolysis promotes disassembly of the atlastin crossover dimer during ER fusion
Article Snippet: Preparation of liposomes and lipid-mixing fusion assay Lipids in chloroform dried down by rotary evaporation were hydrated by resuspension in A100 buffer (25 mM Hepes, pH 7.4, 100 mM KCl, 10% glycerol, 1 mM EDTA, and 2 mM 2-ME) at a final 10-mM lipid concentration and subjected to 11 freeze–thaw cycles in liquid N2 and 42°C water bath. .. Liposomes (100–300-nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin) and checked for size by dynamic light scattering (Zen3600).

Article Title: Membrane tethering by the atlastin GTPase depends on GTP hydrolysis but not on forming the cross-over configuration
Article Snippet: Proteoliposome production Lipids in chloroform dried down by rotary evaporation were hydrated by resuspension in A100 buffer (25 mM HEPES, pH 7.4, 100 mM KCl, 10% glycerol, 2 mM β-mercaptoethanol, 1 mM EDTA; ), final lipid concentration ∼10 mM, and subjected to 12 freeze–thaw cycles in liquid N2 and room temperature (RT) water. .. Liposomes (100- to 300-nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin, Ottawa, ON, Canada) and checked for size by DLS (as described below and in Supplemental Figure S1).

Article Title: The crossover conformational shift of the GTPase atlastin provides the energy driving ER fusion
Article Snippet: Proteoliposome production and fusion assay Lipids in chloroform dried down by rotary evaporation were hydrated by resuspension in A100 buffer (25 mM Hepes, pH 7.4, 100 mM KCl, 10% glycerol, 2 mM 2-mercaptoethanol, and 1 mM EDTA containing 5 mM MgCl2 ) at a final lipid concentration of ∼10 mM and subjected to 12 freeze–thaw cycles in liquid N2 and RT water. .. Liposomes (100–300 nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin) and checked for size by dynamic light scattering (Zen3600; Malvern Instruments).

Article Title: ER network formation and membrane fusion by atlastin1/SPG3A disease variants
Article Snippet: Proteoliposome production Lipids in chloroform dried down by rotary evaporation were hydrated by resuspension in A100 buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 100 mM KCl, 10% glycerol, 2 mM β-mercaptoethanol, 1 mM EDTA; ), final lipid concentration ∼10 mM, and subjected to 12 freeze–thaw cycles in liquid N2 and room temperature water. .. Liposomes of diameter 100–300 nm were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin, Ottawa, ON, Canada).

Produced:

Article Title: Biochemical Reconstitution of Hemorrhagic-Fever Arenavirus Envelope Glycoprotein-Mediated Membrane Fusion
Article Snippet: .. LUVs were produced by extrusion through 100-nm pore size polycarbonate membranes (Avestin). .. To generate target liposomes bearing a self-quenching concentration of the fluorescent lipid rhodamine-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl); Avanti Polar Lipids), the lipid mix was doped with 1% of the compound.

Concentration Assay:

Article Title: Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of ?-Synuclein
Article Snippet: The phospholipid was resuspended to a final concentration of 10 mg/ml in a solution of 50 m m HEPES (pH 7.5), 150 m m NaCl. .. Small unilamellar vesicles were prepared by extrusion through a 100-nm polycarbonate membrane (Avestin, Mannheim, Germany) according to the manufacturer's instructions.

Article Title: Charge-Dependent Translocation of the Trojan Peptide Penetratin across Lipid Membranes
Article Snippet: The dried lipid films were resuspended in defined buffer volumes by vortexing to give the final lipid concentration, L = C DOPC + C DOPG . .. After five freeze-thaw cycles, the lipid suspension was either passed at least 15 times through two stacked Osmonics polycarbonate membranes of 100-nm pore size in a 1-ml mini-Liposo-Fast-Pneumatic extruder (Avestin, Canada), or it was sonicated in cold water for 20 min using a titanium tip ultrasonicator followed by 10-min ultracentrifugation (3000 g) to prepare large and small unilamellar vesicles, LUV and SUV, respectively.

Article Title: Interaction of human peripheral blood monocytes with apoptotic polymorphonuclear cells
Article Snippet: The final concentration of lipids in PBS was 1 m m . .. The multilamellar liposome suspension was freeze–thawed several times and then extruded 13 or 15 times through a LiposoFast extruder equipped with polycarbon filters of 100-nm diameter (both from Avestin, Inc., Ottawa, ON, Canada).

Article Title: GTP hydrolysis promotes disassembly of the atlastin crossover dimer during ER fusion
Article Snippet: Preparation of liposomes and lipid-mixing fusion assay Lipids in chloroform dried down by rotary evaporation were hydrated by resuspension in A100 buffer (25 mM Hepes, pH 7.4, 100 mM KCl, 10% glycerol, 1 mM EDTA, and 2 mM 2-ME) at a final 10-mM lipid concentration and subjected to 11 freeze–thaw cycles in liquid N2 and 42°C water bath. .. Liposomes (100–300-nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin) and checked for size by dynamic light scattering (Zen3600).

Article Title: Membrane tethering by the atlastin GTPase depends on GTP hydrolysis but not on forming the cross-over configuration
Article Snippet: Proteoliposome production Lipids in chloroform dried down by rotary evaporation were hydrated by resuspension in A100 buffer (25 mM HEPES, pH 7.4, 100 mM KCl, 10% glycerol, 2 mM β-mercaptoethanol, 1 mM EDTA; ), final lipid concentration ∼10 mM, and subjected to 12 freeze–thaw cycles in liquid N2 and room temperature (RT) water. .. Liposomes (100- to 300-nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin, Ottawa, ON, Canada) and checked for size by DLS (as described below and in Supplemental Figure S1).

Article Title: The crossover conformational shift of the GTPase atlastin provides the energy driving ER fusion
Article Snippet: Proteoliposome production and fusion assay Lipids in chloroform dried down by rotary evaporation were hydrated by resuspension in A100 buffer (25 mM Hepes, pH 7.4, 100 mM KCl, 10% glycerol, 2 mM 2-mercaptoethanol, and 1 mM EDTA containing 5 mM MgCl2 ) at a final lipid concentration of ∼10 mM and subjected to 12 freeze–thaw cycles in liquid N2 and RT water. .. Liposomes (100–300 nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin) and checked for size by dynamic light scattering (Zen3600; Malvern Instruments).

Article Title: ER network formation and membrane fusion by atlastin1/SPG3A disease variants
Article Snippet: Proteoliposome production Lipids in chloroform dried down by rotary evaporation were hydrated by resuspension in A100 buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 100 mM KCl, 10% glycerol, 2 mM β-mercaptoethanol, 1 mM EDTA; ), final lipid concentration ∼10 mM, and subjected to 12 freeze–thaw cycles in liquid N2 and room temperature water. .. Liposomes of diameter 100–300 nm were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin, Ottawa, ON, Canada).

Article Title: FUNCTIONAL RECONSTITUTION OF INFLUENZA A M2(22-62)
Article Snippet: Internal buffer (50 mM KCl, 50 mM K2 HPO4 , 50 mM KH2 PO4 , pH 8.0, 320 mOsm) was added to the thin film, vortexed to form nonuniform liposomes, then extruded through a 100-nm pore-size polycarbonate filter (Liposofast membrane extruder, Avestin, Ottawa, Canada) at 50-60°C. .. Amantadine or cycloctylamine were added to liposome and external buffers to a nominal concentration of 100 μM.

Article Title: Raft Composition at Physiological Temperature and pH in the Absence of Detergents
Article Snippet: SUVs were prepared at 60°C in 0.5 M sucrose solution containing 10 mM Tris-HCl, pH 7.3, by extrusion through a 100-nm-pore polycarbonate filters (LiposoFast extruder, Avestin, Ottawa, Canada). .. After adjusting the external sucrose concentration to 1.7 M, we layered the liposomes at the bottom of a step gradient composed of 0.125, 0.25, 0.4, 0.8, and 1.2 M sucrose steps (10.5 ml total volume).

Article Title: Biochemical Reconstitution of Hemorrhagic-Fever Arenavirus Envelope Glycoprotein-Mediated Membrane Fusion
Article Snippet: LUVs were produced by extrusion through 100-nm pore size polycarbonate membranes (Avestin). .. To generate target liposomes bearing a self-quenching concentration of the fluorescent lipid rhodamine-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl); Avanti Polar Lipids), the lipid mix was doped with 1% of the compound.

Article Title: Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin
Article Snippet: Unilamellar vesicles of PC or PC/PS, 7.5/2.5, wt/wt, were prepared at a phospholipid concentration of 1 mg/ml. .. Liposomes were formed by 19 passes through 100-nm polycarbonate filters by using a Lipofast basic apparatus (Avestin, Ottawa) and stored at 4°C under nitrogen.

Single Vesicle Fusion Assay:

Article Title: GTP hydrolysis promotes disassembly of the atlastin crossover dimer during ER fusion
Article Snippet: Paragraph title: Preparation of liposomes and lipid-mixing fusion assay ... Liposomes (100–300-nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin) and checked for size by dynamic light scattering (Zen3600).

Article Title: The crossover conformational shift of the GTPase atlastin provides the energy driving ER fusion
Article Snippet: Paragraph title: Proteoliposome production and fusion assay ... Liposomes (100–300 nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin) and checked for size by dynamic light scattering (Zen3600; Malvern Instruments).

Clear Native PAGE:

Article Title: Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of ?-Synuclein
Article Snippet: The retentate (high molecular weight aggregates) and the flow-through solution (monomeric α-syn) were analyzed by 12% native PAGE or SDS-PAGE. .. Small unilamellar vesicles were prepared by extrusion through a 100-nm polycarbonate membrane (Avestin, Mannheim, Germany) according to the manufacturer's instructions.

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  • 86
    AVESTIN 100 nm pore diameter
    , with the following parameters: Δφ m = 14.2 ± 1.3 mV, K 1 = (2.6 ± 0.4) × 10 7 M −1 , K 2 = (3.7 ± 0.3) × 10 6 M −1 , and R 2 = 0.998. The inset shows the kinetics of the PD change from <t>100</t> nM protein added in one (red) or two (blue) steps. The arrow indicates the moment of the second addition of protein.
    100 Nm Pore Diameter, supplied by AVESTIN, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AVESTIN 100 nm polycarbonate filters
    Mutant variants show no defects in GTP-catalyzed crossover formation. (A and B, left) The positions of K320 and P317 mutations made to target DATL crossover superimposed onto PyMOL renderings of (A) the hATL1 form2 extended dimer PDB 3QOF and (B) the hATL1 form3 crossover dimer PDB 4IDP . The position of the Cy3 dye in each structure is indicated with a red circle. (A and B, right) Enlargement of the boxed regions in A and B showing the K320 and P317 side chains highlighted in cyan. (C) Normalized PIFE over time when each of the indicated Cy3-cytoDATL mutant variants is mixed with GTP. (C, right) Zoomed-in view of the first <t>100</t> ms of the trace in C. The mean of seven runs is shown for each trace (SEM
    100 Nm Polycarbonate Filters, supplied by AVESTIN, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    AVESTIN 100 nm pore size polycarbonate membranes
    pH-induced membrane fusion by rGPC fur proteoliposomes: content mixing. A. rGPC fur was incorporated into POPG-POPC-CHS liposomes containing 50 µM Zn ++ and these proteoliposomes were mixed with target POPG-POPC-CHS liposomes containing 1 µM FluoZin-1. The experimental treatments, labels and colors are as described in the legend of Fig. 6 . Content mixing was determined by Zn ++ -induced FluoZin-1 fluorescence measured at 485 nm (excitation 370 nm). Complete mixing was determined by solubilization with Triton <t>X-100</t> in buffer containing 50 µM Zn ++ . B. pH-induced content mixing was determined in the continued presence of MAbs F100G5, BF11 or an irrelevant IgG (5 µM, 25 µM and 25 µM, respectively). In this experiment, content mixing in the absence of MAb (pH 5.0) was ∼50%.
    100 Nm Pore Size Polycarbonate Membranes, supplied by AVESTIN, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    , with the following parameters: Δφ m = 14.2 ± 1.3 mV, K 1 = (2.6 ± 0.4) × 10 7 M −1 , K 2 = (3.7 ± 0.3) × 10 6 M −1 , and R 2 = 0.998. The inset shows the kinetics of the PD change from 100 nM protein added in one (red) or two (blue) steps. The arrow indicates the moment of the second addition of protein.

    Journal: Journal of Virology

    Article Title: pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion

    doi: 10.1128/JVI.01539-15

    Figure Lengend Snippet: , with the following parameters: Δφ m = 14.2 ± 1.3 mV, K 1 = (2.6 ± 0.4) × 10 7 M −1 , K 2 = (3.7 ± 0.3) × 10 6 M −1 , and R 2 = 0.998. The inset shows the kinetics of the PD change from 100 nM protein added in one (red) or two (blue) steps. The arrow indicates the moment of the second addition of protein.

    Article Snippet: Large unilamellar vesicles (LUVs) containing 30% DOPS and 70% DOPC were prepared by extrusion through filters with a 100-nm pore diameter (Avestin) in buffer containing 4 mM TbCl3 , 40 mM dipicolinic acid (DPA), 30 mM sodium citrate, 20 mM HEPES, and 70 mM NaCl (pH 7.0) with the final liposome concentration of 15 g/liter ( ).

    Techniques:

    AFM topography image of the protein layer adsorbed at an M1 bulk concentration of 100 nM with a removed square patch of the protein layer with dimensions of 0.7 by 0.7 μm 2 . The full z -axis scale is 10 nm.

    Journal: Journal of Virology

    Article Title: pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion

    doi: 10.1128/JVI.01539-15

    Figure Lengend Snippet: AFM topography image of the protein layer adsorbed at an M1 bulk concentration of 100 nM with a removed square patch of the protein layer with dimensions of 0.7 by 0.7 μm 2 . The full z -axis scale is 10 nm.

    Article Snippet: Large unilamellar vesicles (LUVs) containing 30% DOPS and 70% DOPC were prepared by extrusion through filters with a 100-nm pore diameter (Avestin) in buffer containing 4 mM TbCl3 , 40 mM dipicolinic acid (DPA), 30 mM sodium citrate, 20 mM HEPES, and 70 mM NaCl (pH 7.0) with the final liposome concentration of 15 g/liter ( ).

    Techniques: Concentration Assay

    AFM topography images of M1 protein layer structures on supported BLMs (A to C) and mica (D to F). (A, D) Changes in M1 layer structure upon a pH drop from 7.1 to 5.0 or pH transitions from 7.1 to 6.0 to 5.0. The bright spots are protein aggregates. (B, E) M1 initially adsorbed at pH 6.0. (C, F) M1 initially adsorbed at pH 5.0. The protein bulk concentration was 70 nM. The working buffer was 100 mM KCl–50 mM MES. The full z -axis scale is 10 nm in panels A to D and 5 nm in panels E and F.

    Journal: Journal of Virology

    Article Title: pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion

    doi: 10.1128/JVI.01539-15

    Figure Lengend Snippet: AFM topography images of M1 protein layer structures on supported BLMs (A to C) and mica (D to F). (A, D) Changes in M1 layer structure upon a pH drop from 7.1 to 5.0 or pH transitions from 7.1 to 6.0 to 5.0. The bright spots are protein aggregates. (B, E) M1 initially adsorbed at pH 6.0. (C, F) M1 initially adsorbed at pH 5.0. The protein bulk concentration was 70 nM. The working buffer was 100 mM KCl–50 mM MES. The full z -axis scale is 10 nm in panels A to D and 5 nm in panels E and F.

    Article Snippet: Large unilamellar vesicles (LUVs) containing 30% DOPS and 70% DOPC were prepared by extrusion through filters with a 100-nm pore diameter (Avestin) in buffer containing 4 mM TbCl3 , 40 mM dipicolinic acid (DPA), 30 mM sodium citrate, 20 mM HEPES, and 70 mM NaCl (pH 7.0) with the final liposome concentration of 15 g/liter ( ).

    Techniques: Concentration Assay

    AFM topography images of M1 adsorbed at pH 7.1 to supported BLMs (A to C) and mica (D to F) at the following bulk concentrations: A and D, 10 nM; B and E, 50 nM; C and F, 70 nM. The full z -axis scale is 5 nm for panels A and D to F and 10 nm for panels B and C. The bright spots in panels B and C are protein aggregates. The working buffer was 100 mM KCl–50 mM MES. The inset in panel C is a 300-by-300-nm 2 enlargement of the image. The full z -axis scale of the inset is 5 nm.

    Journal: Journal of Virology

    Article Title: pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion

    doi: 10.1128/JVI.01539-15

    Figure Lengend Snippet: AFM topography images of M1 adsorbed at pH 7.1 to supported BLMs (A to C) and mica (D to F) at the following bulk concentrations: A and D, 10 nM; B and E, 50 nM; C and F, 70 nM. The full z -axis scale is 5 nm for panels A and D to F and 10 nm for panels B and C. The bright spots in panels B and C are protein aggregates. The working buffer was 100 mM KCl–50 mM MES. The inset in panel C is a 300-by-300-nm 2 enlargement of the image. The full z -axis scale of the inset is 5 nm.

    Article Snippet: Large unilamellar vesicles (LUVs) containing 30% DOPS and 70% DOPC were prepared by extrusion through filters with a 100-nm pore diameter (Avestin) in buffer containing 4 mM TbCl3 , 40 mM dipicolinic acid (DPA), 30 mM sodium citrate, 20 mM HEPES, and 70 mM NaCl (pH 7.0) with the final liposome concentration of 15 g/liter ( ).

    Techniques:

    Mutant variants show no defects in GTP-catalyzed crossover formation. (A and B, left) The positions of K320 and P317 mutations made to target DATL crossover superimposed onto PyMOL renderings of (A) the hATL1 form2 extended dimer PDB 3QOF and (B) the hATL1 form3 crossover dimer PDB 4IDP . The position of the Cy3 dye in each structure is indicated with a red circle. (A and B, right) Enlargement of the boxed regions in A and B showing the K320 and P317 side chains highlighted in cyan. (C) Normalized PIFE over time when each of the indicated Cy3-cytoDATL mutant variants is mixed with GTP. (C, right) Zoomed-in view of the first 100 ms of the trace in C. The mean of seven runs is shown for each trace (SEM

    Journal: The Journal of Cell Biology

    Article Title: The crossover conformational shift of the GTPase atlastin provides the energy driving ER fusion

    doi: 10.1083/jcb.201609071

    Figure Lengend Snippet: Mutant variants show no defects in GTP-catalyzed crossover formation. (A and B, left) The positions of K320 and P317 mutations made to target DATL crossover superimposed onto PyMOL renderings of (A) the hATL1 form2 extended dimer PDB 3QOF and (B) the hATL1 form3 crossover dimer PDB 4IDP . The position of the Cy3 dye in each structure is indicated with a red circle. (A and B, right) Enlargement of the boxed regions in A and B showing the K320 and P317 side chains highlighted in cyan. (C) Normalized PIFE over time when each of the indicated Cy3-cytoDATL mutant variants is mixed with GTP. (C, right) Zoomed-in view of the first 100 ms of the trace in C. The mean of seven runs is shown for each trace (SEM

    Article Snippet: Liposomes (100–300 nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin) and checked for size by dynamic light scattering (Zen3600; Malvern Instruments).

    Techniques: Mutagenesis, Mass Spectrometry

    A subset of mutant variants causes abnormal ER network structure. (A) COS-7 cells transfected with each indicated variant of full-length Venus-tagged DATL were fixed and imaged 48 h later by confocal microscopy. Bar, 10 µm. (B) Quantification of the percentage of expressing cells displaying a normal branched ER ( > 100 cells per measurement; data represent mean of three independent measurements ± SD); *, P

    Journal: The Journal of Cell Biology

    Article Title: The crossover conformational shift of the GTPase atlastin provides the energy driving ER fusion

    doi: 10.1083/jcb.201609071

    Figure Lengend Snippet: A subset of mutant variants causes abnormal ER network structure. (A) COS-7 cells transfected with each indicated variant of full-length Venus-tagged DATL were fixed and imaged 48 h later by confocal microscopy. Bar, 10 µm. (B) Quantification of the percentage of expressing cells displaying a normal branched ER ( > 100 cells per measurement; data represent mean of three independent measurements ± SD); *, P

    Article Snippet: Liposomes (100–300 nm diameter) were formed by extrusion through 100-nm polycarbonate filters using the LipoFast LF-50 extruder (Avestin) and checked for size by dynamic light scattering (Zen3600; Malvern Instruments).

    Techniques: Mutagenesis, Transfection, Variant Assay, Confocal Microscopy, Expressing

    B / S versus 〈 I 〉 − offset / S plots for photon-counting simulations ( A ) and simulations with the same analog noise as the Cascade camera ( B – D ). ( C and D ) G 1 and G 1 y analyses, respectively, of the simulation. All simulations used 2D diffusion with 1250 molecules in a 3.5 × 3.5- μ m box, except for the high-intensity, low-brightness point in B , which used 25,000 molecules in the same box size. The diffusion coefficient was 2 μ m 2 /s, with a frame rate of 200 frames/s. Each simulation was run for 100 frames with a frame resolution of 128 × 128 pixels. Brightness values were 0.5, 2, 5, and 10 cpfm.

    Journal: Biophysical Journal

    Article Title: Analysis of Molecular Concentration and Brightness from Fluorescence Fluctuation Data with an Electron Multiplied CCD Camera

    doi: 10.1529/biophysj.108.130310

    Figure Lengend Snippet: B / S versus 〈 I 〉 − offset / S plots for photon-counting simulations ( A ) and simulations with the same analog noise as the Cascade camera ( B – D ). ( C and D ) G 1 and G 1 y analyses, respectively, of the simulation. All simulations used 2D diffusion with 1250 molecules in a 3.5 × 3.5- μ m box, except for the high-intensity, low-brightness point in B , which used 25,000 molecules in the same box size. The diffusion coefficient was 2 μ m 2 /s, with a frame rate of 200 frames/s. Each simulation was run for 100 frames with a frame resolution of 128 × 128 pixels. Brightness values were 0.5, 2, 5, and 10 cpfm.

    Article Snippet: The sample was then extruded through two stacked 100-nm polycarbonate filters using the Avestin extruder.

    Techniques: Diffusion-based Assay

    pH-induced membrane fusion by rGPC fur proteoliposomes: content mixing. A. rGPC fur was incorporated into POPG-POPC-CHS liposomes containing 50 µM Zn ++ and these proteoliposomes were mixed with target POPG-POPC-CHS liposomes containing 1 µM FluoZin-1. The experimental treatments, labels and colors are as described in the legend of Fig. 6 . Content mixing was determined by Zn ++ -induced FluoZin-1 fluorescence measured at 485 nm (excitation 370 nm). Complete mixing was determined by solubilization with Triton X-100 in buffer containing 50 µM Zn ++ . B. pH-induced content mixing was determined in the continued presence of MAbs F100G5, BF11 or an irrelevant IgG (5 µM, 25 µM and 25 µM, respectively). In this experiment, content mixing in the absence of MAb (pH 5.0) was ∼50%.

    Journal: PLoS ONE

    Article Title: Biochemical Reconstitution of Hemorrhagic-Fever Arenavirus Envelope Glycoprotein-Mediated Membrane Fusion

    doi: 10.1371/journal.pone.0051114

    Figure Lengend Snippet: pH-induced membrane fusion by rGPC fur proteoliposomes: content mixing. A. rGPC fur was incorporated into POPG-POPC-CHS liposomes containing 50 µM Zn ++ and these proteoliposomes were mixed with target POPG-POPC-CHS liposomes containing 1 µM FluoZin-1. The experimental treatments, labels and colors are as described in the legend of Fig. 6 . Content mixing was determined by Zn ++ -induced FluoZin-1 fluorescence measured at 485 nm (excitation 370 nm). Complete mixing was determined by solubilization with Triton X-100 in buffer containing 50 µM Zn ++ . B. pH-induced content mixing was determined in the continued presence of MAbs F100G5, BF11 or an irrelevant IgG (5 µM, 25 µM and 25 µM, respectively). In this experiment, content mixing in the absence of MAb (pH 5.0) was ∼50%.

    Article Snippet: LUVs were produced by extrusion through 100-nm pore size polycarbonate membranes (Avestin).

    Techniques: Fluorescence

    pH-induced membrane fusion by rGPC fur proteoliposomes: lipid mixing. rGPC fur was incorporated into POPG-POPC liposomes and mixed with target POPG-POPC liposomes doped with 1% Rhodamine-PE. In most cases, the rGPC fur proteoliposomes were first treated with sFurin (as indicated by+in the first position of the labels, at right). Exposure to acidic pH at the start of the experiment (time = 0) is indicated by+in the second position of the labels. 15 µM of ST-294 or ST-161 was present prior to and during exposure to acidic pH, where indicated. Lipid mixing and the resulting dequenching of the rhodamine fluorophore were measured at 600 nm (excitation 508 nm). Complete dequenching (100%) was determined by subsequent solubilization in Triton X-100 nonionic detergent.

    Journal: PLoS ONE

    Article Title: Biochemical Reconstitution of Hemorrhagic-Fever Arenavirus Envelope Glycoprotein-Mediated Membrane Fusion

    doi: 10.1371/journal.pone.0051114

    Figure Lengend Snippet: pH-induced membrane fusion by rGPC fur proteoliposomes: lipid mixing. rGPC fur was incorporated into POPG-POPC liposomes and mixed with target POPG-POPC liposomes doped with 1% Rhodamine-PE. In most cases, the rGPC fur proteoliposomes were first treated with sFurin (as indicated by+in the first position of the labels, at right). Exposure to acidic pH at the start of the experiment (time = 0) is indicated by+in the second position of the labels. 15 µM of ST-294 or ST-161 was present prior to and during exposure to acidic pH, where indicated. Lipid mixing and the resulting dequenching of the rhodamine fluorophore were measured at 600 nm (excitation 508 nm). Complete dequenching (100%) was determined by subsequent solubilization in Triton X-100 nonionic detergent.

    Article Snippet: LUVs were produced by extrusion through 100-nm pore size polycarbonate membranes (Avestin).

    Techniques:

    SPR studies of interactions with lipid-reconstituted rGPC fur . The rGPC fur precursor was immobilized onto a Biacore L1 chip and reconstituted in a lipid bilayer as described in Material and Methods . Two or three concentration-dependent data sets were analyzed and sensorgram figures were generated using a five-point smoothing procedure and ORIGIN graphing software. Labels to the right are drawn to coincide with the maximum RUs achieved by the respective ligand. A. Binding of G1-directed MAbs (BF11, BF09, BE08, and AG02; 0.5 µM), sTfR (1.5 µM), and a nucleoprotein-directed MAb (aN; BG12). B. Binding of SIGA (ST-294, ST-375, ST-193, and ST-761; 150 µM) and TSRI (17C8; 100 µM) small-molecule fusion inhibitors. ST-161 and TSRI 8C1 are specific to the OW LASV and do not inhibit the NW arenavirus JUNV.

    Journal: PLoS ONE

    Article Title: Biochemical Reconstitution of Hemorrhagic-Fever Arenavirus Envelope Glycoprotein-Mediated Membrane Fusion

    doi: 10.1371/journal.pone.0051114

    Figure Lengend Snippet: SPR studies of interactions with lipid-reconstituted rGPC fur . The rGPC fur precursor was immobilized onto a Biacore L1 chip and reconstituted in a lipid bilayer as described in Material and Methods . Two or three concentration-dependent data sets were analyzed and sensorgram figures were generated using a five-point smoothing procedure and ORIGIN graphing software. Labels to the right are drawn to coincide with the maximum RUs achieved by the respective ligand. A. Binding of G1-directed MAbs (BF11, BF09, BE08, and AG02; 0.5 µM), sTfR (1.5 µM), and a nucleoprotein-directed MAb (aN; BG12). B. Binding of SIGA (ST-294, ST-375, ST-193, and ST-761; 150 µM) and TSRI (17C8; 100 µM) small-molecule fusion inhibitors. ST-161 and TSRI 8C1 are specific to the OW LASV and do not inhibit the NW arenavirus JUNV.

    Article Snippet: LUVs were produced by extrusion through 100-nm pore size polycarbonate membranes (Avestin).

    Techniques: SPR Assay, Chromatin Immunoprecipitation, Concentration Assay, Generated, Software, Binding Assay