Journal: The Journal of Biological Chemistry

Article Title: A new functional assay reveals that membrane binding is critical for overactivation of the phosphoinositide 3-kinase H1047R mutant

doi: 10.1016/j.jbc.2026.111207

Figure Lengend Snippet: Establishment and titration of the optimal form of the substrate PIP 2 . A , the optimal form of substrate was determined by comparing two different ratios of lPIP 2 to total lipids, 1:10 and 1:2, both at a final lPIP 2 concentration of 7 μM. A water-soluble PIP 2 was also tested, at a concentration of 100 μM. Note that the most advantageous substrate preparation is a 1:2 mixture of lPIP 2 to total lipids. Two replicates from n = 10. B , different concentrations of lPIP 2 (1:2 mixture of lPIP 2 to total lipids) were tested. Note the dose-dependent effect. Two replicates from n = 4. C , schematic diagram of the experimental set up used in ( A ) and ( B ). The individual data points displayed above represent the means of replicates in each experiment. The statistical significance of difference between groups was examined by one-way ANOVA, followed by Tukey’s multiple comparisons test. ns : no significance, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Data were shown as mean ± SD. Specific activity values for PI3Kα, calculated as pmol PIP 3 produced per minute per nanogram of enzyme, are indicated in red fonts in the figure. lPIP 2 , lipid form of PIP 2 ; PIP 2 , phosphatidylinositol-4,5-biphospate; PIP 3 , phosphatidylinositol-3,4,5-triphosphate.

Article Snippet: Lipid PIP 2 [L-α-phosphatidylinositol-4,5 bisphosphate (Brain, Porcine) (ammonium salt)] and soluble PIP 2 [1,2-dioctanoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5-bisphosphate) (ammonium salt)] were from Avanti Polar Lipids.

Techniques: Titration, Concentration Assay, Activity Assay, Produced

Journal: The Journal of Biological Chemistry

Article Title: A new functional assay reveals that membrane binding is critical for overactivation of the phosphoinositide 3-kinase H1047R mutant

doi: 10.1016/j.jbc.2026.111207

Figure Lengend Snippet: Role of membranes in the overactivation of the mutants H1047R and E545K. We compare the PI3Kα enzymatic activity of H1047R, E545K, and WT, using as substrate 7 μM lPIP 2 ( A ), or 100 μM sPIP 2 ( B ). The activities of the mutants were normalized to WT, which was set to value “1.” Note that E545K shows higher activity than WT with either soluble or membranous PIP 2 , whereas H1047R exhibits increased activity only when membranes are used. Two replicates from n = 6 in ( A ) and n = 7 in ( B ). The individual data points displayed above represent the means of replicates in each experiment. The statistical significance of difference between groups in graphs ( A ) and ( B ) was examined by one-way ANOVA, followed by Tukey’s multiple comparisons test. ns : no significance, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Data were shown as mean ± SD. Specific activity values for PI3Kα, calculated as pmol PIP 3 produced per minute per nanogram of enzyme, are indicated in red fonts in the figure. lPIP 2 , lipid form of phosphatidylinositol-4,5-biphospate; SPR, surface plasmon resonance; sPIP 2 , soluble form of PIP 2 ; PIP 3 , phosphatidylinositol-3,4,5-triphosphate.

Article Snippet: Lipid PIP 2 [L-α-phosphatidylinositol-4,5 bisphosphate (Brain, Porcine) (ammonium salt)] and soluble PIP 2 [1,2-dioctanoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5-bisphosphate) (ammonium salt)] were from Avanti Polar Lipids.

Techniques: Activity Assay, Produced, SPR Assay

Journal: PLOS Biology

Article Title: Endocytic protein AP180 assembly domain regulates synaptic vesicle size and release in Caenorhabditis elegans

doi: 10.1371/journal.pbio.3003643

Figure Lengend Snippet: (A) UNC-11 condensates promote the growth of F-actin. Full-length UNC-11 (UNC-11 FL) was labeled with Pacific Blue (cyan), and F-actin was visualized using Alexa Fluor 488–phalloidin (yellow). G-actin (2 µM) and phalloidin (0.2 µM) were added to samples containing UNC-11 droplets, and fluorescence images were collected every 3 min for 48 min. Low-magnification images are shown ( top ), with red rectangles marking regions shown at higher magnification below. Scale bar, 5 µm. Images were acquired on an Olympus FV1000 laser-scanning confocal microscope using a 60×, 1.4 NA objective with 5× zoom. (B) UNC-11 condensates connect F-actin to liposomes containing PIP2. A schematic model is shown ( top ). Representative fluorescence images with two regions of interest (ROIs, red boxes) are shown ( middle ). UNC-11 FL is shown in magenta (Pacific Blue), F-actin in yellow (Alexa Fluor 488–phalloidin), and liposomes in cyan (500 µM total lipids; 2% PI(4,5)P2, 25% PS, 71% PC, and 2% Rhodamine-PE; 50 nm diameter). Zoomed-in views and fluorescence intensity line scans of the ROIs are shown ( bottom ). Images were acquired on an Olympus FV1000 confocal system using a 60×, 1.4 NA objective with 5× zoom. (C) Quantification of UNC-11 condensates associated with both actin filaments and membranes. The fraction of dual association was calculated from total condensates per Z-stack. Data represent three independent experiments. p < 0.01, one-way ANOVA with Dunnett’s multiple comparisons. The data underlying this figure are provided in .

Article Snippet: PI(4,5)P2 (brain L-α-phosphatidylinositol-4,5-bisphosphate, PIP 2 ) and 18:1 Liss Rhodamine PE (Rhod-PE) were obtained from Avanti Polar Lipids.

Techniques: Labeling, Fluorescence, Microscopy, Liposomes

Journal: bioRxiv

Article Title: Supported membrane assay probes PLCγ1 activity in LAT condensates

doi: 10.64898/2026.01.23.701188

Figure Lengend Snippet: (A) A schematic summarizing the lysate assay protocol. PLCγ1-mNG was generated in HEK293T cells, then flowed directly as dilute lysate over pLAT bilayers containing PIP 2 . Active PLCγ1-mNG hydrolyzed PIP 2 to DAG, which was detected using a labeled DAG sensor (PKCθ-C1b-SNAP-Alexa 647). Parts of the figure were drawn using images from Servier Medical Art Commons Attribution 3.0 Unported License ( http://smart.servier.com (accessed on 21 March 2022)). (B) An example TIRF image of the DAG sensor demonstrating the segmentation of 50 x 50 µm images for analysis. TIRF images in a time series were sectioned into 5 x 5 µm patches and analyzed as separate instances. (C) Images of a single 5 x 5 µm patch in the DAG sensor channel (Top) and PLCγ1-mNG channel (Bottom) through time, corresponding to the time axis for the curves below. (D) Curves obtained from processing the raw median fluorescence from membrane patches, representing the amount of DAG in the bilayer (red, left axis, foreground curves) and density of PLCγ1-mNG recruited (green, right axis, background curves) over 20 minutes. PLCγ1-mNG lysate (∼1:1000 dilution) with DAG sensor was injected at t = 0 min. To attain quantitative curves, raw fluorescence was first normalized to the time of injection. DAG was calibrated using the assumption that the average normalized maximum for multiple experiments must be 2% (given 2% PIP 2 bilayers). PLCγ1-mNG was calibrated by matching single-molecule counts (at high laser power) to bulk fluorescence (at low laser power). The two solid, darkly colored curves represent the median of all patches analyzed. Lightly colored curves in the background represent the results from each single patch (n = 100). Lipid composition (mol%): 94:4:2 DOPC:Ni-DGS:PIP 2 . LAT density: ∼1500 µm -2 .

Article Snippet: Small unilamellar vesicles (SUVs) were made using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-[(N-carboxypentyl)iminidiacetic acid)succinyl] (nickel salt) (Ni2+-NTA-DGS), and L-α-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt) (Brain PIP 2 ) (Avanti Polar Lipids).

Techniques: Generated, Labeling, Fluorescence, Membrane, Injection

Journal: bioRxiv

Article Title: Supported membrane assay probes PLCγ1 activity in LAT condensates

doi: 10.64898/2026.01.23.701188

Figure Lengend Snippet: (A) A schematic showing greater detail of the lysate experiment at the membrane. Hck and LAT are functionalized to the SLB via Ni-histidine chelation. Hck phosphorylates LAT allowing for the recruitment of PLCγ1-mNG. PLCγ1-mNG is then subsequently phosphorylated by Hck at Y783 and is active at the membrane after structural rearrangement. (B) Relative observed enzymatic velocities ( v obs ) (black, left axis, defined as the enzymatic rate for the linear portion of the reaction trace) and PLCγ1-mNG densities (green, right axis, defined as density at the midpoint of v obs measurement) for various assay conditions, normalized to the median value for PLCγ1-WT-mNG lysate on a pLAT bilayer. Distributions of 100 membrane patches are shown as half-violin plots, with single observations shown as points and distributions represented by a shaded area. Compact box plots are overlayed over the distributions to represent the median (white circles), quartile regions (box), and extremes with outliers excluded (whiskers). Median values for v obs (m v ) and PLCγ1-mNG density (m ρ ) are expressly denoted for clarity. Lipid composition (mol%): 94:4:2 DOPC:Ni-DGS:PIP 2 . LAT densities: ∼1500 µm -2 .

Article Snippet: Small unilamellar vesicles (SUVs) were made using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-[(N-carboxypentyl)iminidiacetic acid)succinyl] (nickel salt) (Ni2+-NTA-DGS), and L-α-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt) (Brain PIP 2 ) (Avanti Polar Lipids).

Techniques: Membrane

Journal: bioRxiv

Article Title: Supported membrane assay probes PLCγ1 activity in LAT condensates

doi: 10.64898/2026.01.23.701188

Figure Lengend Snippet: (A)-(C) Field-corrected images of LAT-Alexa 555 (Ax555) (gold, top), PLCγ1-mNG (green, middle), and DAG sensor (red, bottom) with 5 µM Grb2 and 2 µM SOS-PR in solution at t = 15 min. The entire FOV is 50 x 50 µm (scale bar = 10 µm) with an inset showing a 4X expansion of the center 5 x 5 µm patch (scale bar = 2.5 µm). D. A binary image mask for LAT segmented into condensed (gold) and disperse (grey) areas obtained from Gaussian filtration (σ=1) and thresholding of the LAT-Ax555 image in (A). Inset and scale bars are equivalent to those in (A). (E)-(F) Images showing the effect of applying the binary LAT mask to PLCγ1-mNG (green) and DAG sensor (red) channels. Only the outline of the applied mask is shown for visibility. Insets and scale bars are equivalent to those in (B-C). (G)-(I) Histograms of relative pixel intensities for LAT-Ax555 (gold, top), PLCγ1-mNG (green, middle), and DAG sensor (red, bottom). Pixel intensities were normalized to the median value for all pixels. Values greater than 1 indicate an increase in brightness from the overall median, while values less than 1 indicate a decrease. Histograms for all pixels are displayed in full color in the background. Histograms for pixels that are in areas of condensed LAT are shown in translucent white in the foreground. Pixel histograms for disperse LAT areas are shown in translucent grey. Dotted lines denoting the median of each histogram are decorated with an icon corresponding to the LAT mask for all pixels (gold and grey), LAT condensed (gold only), or LAT disperse (grey only). Median fold change values for each histogram are displayed in the top right for clarity. Lipid compositions for all experiments (mol%): 94:4:2 DOPC:Ni-DGS:PIP 2 . LAT density: ∼1500 µm -2 (10% labeled).

Article Snippet: Small unilamellar vesicles (SUVs) were made using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-[(N-carboxypentyl)iminidiacetic acid)succinyl] (nickel salt) (Ni2+-NTA-DGS), and L-α-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt) (Brain PIP 2 ) (Avanti Polar Lipids).

Techniques: Filtration, Labeling

Journal: bioRxiv

Article Title: Supported membrane assay probes PLCγ1 activity in LAT condensates

doi: 10.64898/2026.01.23.701188

Figure Lengend Snippet: (A) TIRF images (5 x 5 µm, scale bars = 2 µm) of PLCγ1-mNG in different conditions corresponding to the distributions below. From left to right: PLCγ1-mNG only (with 2 µL control lysate), PLCγ1-mNG + Gads:SLP76 (from lysate, 1 µL each), PLCγ1-mNG + Grb2:SOS-PR (purified, 6 µM:2 µM respectively), and PLCγ1-mNG + all previously described adapters. LAT-Ax555 (10% labeled) was used in this experiment but was determined to have negligible contribution in the PLCγ1-mNG channel. (B) Half-violin plots showing distributions of relative v obs (grey, left axis) and relative PLCγ1-mNG density (green, right axis), normalized to PLCγ1 only conditions. Compact box plots overlay the distributions to convey statistics. Median values for v obs (m v ) and PLCγ1-mNG density (m PLCγ1 ) are expressly denoted for clarity. (C) TIRF images (5 x 5 µm, scale bars = 2 µm) of PLCγ1-mNG in different conditions corresponding to the distributions below. From left to right: PLCγ1-mNG only (no control lysate), PLCγ1-mNG pre-phosphorylated by 1 µM BTK-KD in solution for 1 hr, and similarly pre-phosphorylated PLCγ1-mNG + Grb2:SOS-PR (purified, 6 µM:2 µM respectively). Hck was on the membrane for all experiments. Unlabeled LAT was used in this experiment. (D) Half-violin plots showing distributions of relative v obs (grey, left axis) and relative PLCγ1-mNG density (green, right axis), normalized to PLCγ1 only conditions (no pre-phosphorylation). Compact box plots overlay the distributions to convey statistics. Median values for v obs (m v ) and PLCγ1-mNG density (m ρ ) are expressly denoted for clarity. Lipid compositions for all experiments (mol%): 94:4:2 DOPC:Ni-DGS:PIP 2 . LAT densities: ∼1500 µm -2 .

Article Snippet: Small unilamellar vesicles (SUVs) were made using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-[(N-carboxypentyl)iminidiacetic acid)succinyl] (nickel salt) (Ni2+-NTA-DGS), and L-α-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt) (Brain PIP 2 ) (Avanti Polar Lipids).

Techniques: Control, Purification, Labeling, Membrane, Phospho-proteomics