Journal: bioRxiv

Article Title: Local PI(4,5)P 2 generation controls fusion pore expansion during exocytosis

doi: 10.1101/2022.01.26.477824

Figure Lengend Snippet: (A) Representative example of an INS1 cell expressing the PI(4,5)P 2 sensor PH-PLCδ1 in unstimulated conditions (max pixel intensities for 10 s before stimulation, left), and during stimulation with elevated K + (max pixel intensities during first 20 s of stimulation, right). (B) Three examples (1-3) of single granule exocytosis in K + stimulated cells, as in A. Image frames (upper) show granule (gr) and PI(4,5)P 2 sensor (PIP 2 ) images at the exocytosis site (1s/frame, scalebar 1μm), and traces the quantification of gr (middle) and PIP 2 signal (lower) temporally aligned to the same events. Note temporary increase of gr fluorescence (indicating FR events with arrested fusion pore) and associated PI(4,5)P 2 -signal in examples 1-2, but not 3 (IR events). Black arrows indicate moment of fusion pore opening and content release, red the fusion pore lifetime. (C) Cartoons illustrating the interpretation of events in B. (D-F) Dynamics of phosphatidylinositol species at FR (black) and IR (grey) exocytosis events, quantified as average ΔF/S (see ) for (D) the PI(4)P sensor EGFP-P4M-SidM (107 FR events and 84 IR events in 26 INS1-cells, 3 preps), (E) the PI(4,5)P 2 sensor EGFP-PH-PLCδ1 (164 FR events, 83 IR events in 24 INS1-cells, 3 preps), and (F) the PI(3,4,5)P 3 sensor 4EGFP-GRP1 (83 events in 15 INS1-cells, 3 preps). All signals were aligned to the moment of release (t=0); positive ΔF/S indicates excess sensor fluorescence at the exocytosis site. (G-H) As in D-F but for human islets cells, and aligned to the moment of fusion. (G) PI(4,5)P 2 (228 FR events, 102 IR events in 33 cells), (H) PI(4)P (191 FR events, 234 IR events in 30 cells, 3 preps). (I) PI(4,5)P 2 accumulation (ΔΔF/S) as function of fusion pore lifetime in INS1-cells (I) and human islet cells (J); same experiments as in D and H; circle size indicates events per bin.

Article Snippet: 300 μM of BODIPY ® FL Phosphatidylinositol 4,5-bisphosphate (BODIPY FL-PI(4,5)P 2 ; Echelon Biosciences, Salt Lake City, USA), was freshly prepared and incubated with 100 μM Histone H1 Carrier (Echelon Biosciences, Salt Lake City, USA) for 10 min at room temperature after vigorous pipetting to facilitate complex formation ( ).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Local PI(4,5)P 2 generation controls fusion pore expansion during exocytosis

doi: 10.1101/2022.01.26.477824

Figure Lengend Snippet: (A) Cartoon illustrating phosphatidylinositol synthesis steps. (B) Quantification of PIP5k1c expression in cells expressing scrambled control (scr) or PIP5k1c specific shRNA (K5KD). (C) Exocytosis during 40 s of K + -stimulation quantified in cells expressing NPY. The cells coexpressed combinations of scr, K5KD, EGFP-PH-PLCδ1, and amphiphysin-mCherry, as indicated (scr+PLCδ1, 18 cells; K5KD+PLCδ1, 38 cells; scr+amphiphysin, 16 cells; K5KD+amphiphysin, 38 cells. Values are p-values for scr vs K5KD, non-paired t-test. (D) Fraction of FR events in experiments in C, Values indicate p-values, u-test. (E) Cumulative frequency histograms of NPY release times in C, for scr+PH-PLCδ1 (grey) and K5KD+PH-PLCδ1 (light blue). P=0.006, KS test (F) As in (E) but for scr or K5KD + amphiphysin. P=1.8E-5, KS test. (G) Examples of single granule exocytosis (top), average time course of PH-PLCδ1 signal (ΔF/S ±SEM; lower left), and distribution of the peak amplitudes (ΔΔF/S, non-paired t-test; lower right) for scr+PH-PLCδ1 (grey; 210 FR events in 18 cells, 3 preps) and K5KD+PH-PLCδ1 (light blue; 40 FR events in 38 cells). Same experiments as in C-E. (H) As in (G) but for scr+amphiphysin (grey; 249 FR events, 16 cells) and K5KD+amphiphysin (brown; 32 events, 38 cells).

Article Snippet: 300 μM of BODIPY ® FL Phosphatidylinositol 4,5-bisphosphate (BODIPY FL-PI(4,5)P 2 ; Echelon Biosciences, Salt Lake City, USA), was freshly prepared and incubated with 100 μM Histone H1 Carrier (Echelon Biosciences, Salt Lake City, USA) for 10 min at room temperature after vigorous pipetting to facilitate complex formation ( ).

Techniques: Expressing, shRNA

Journal: bioRxiv

Article Title: Local PI(4,5)P 2 generation controls fusion pore expansion during exocytosis

doi: 10.1101/2022.01.26.477824

Figure Lengend Snippet: (A) Cartoon illustrating phosphatidylinositol synthesis. (B) Quantification of mirRNA based knockout of PI4K2a (K4KD) compared with control INS1 cells. (C-E) Average EGFP-PH-PLCδ1 time course (ΔF/S ±SEM) during K + -stimulation in cells co-expressing NPY-mOrange2; for ctrl (E, 225 events), K4KD (D, 291 events), or ctrl with kinase-4 inhibitor A1 (E, 110 events, 100nM A1 for 10 minutes). All events are spatiotemporally aligned to exocytosis events in the NPY-mOrange2 signal. (F) Distribution of peak amplitudes (ΔΔF/S ±SEM) for transients in C-E, p-values shown are for one-way ANOVA, Fisher posthoc test. (G) Exocytosis during 40 s K + -stimulation in C-E, for control (27 cells), K4KD (33 cells), and A1 (13 cells). p-values are for one-way ANOVA, Fisher posthoc test. (H-L) As in C-G, but for cells expressing EGFP-P4M-SidM in control (H, 264 events, 32 cells), K4KD (I, 245 events, 31 cells), or A1 (J, 103 events, 9 cells). (M-N) Average amphiphysin-mCherry time course for FR exocytosis events (ΔF/S ±SEM, aligned to rising phase of co-expressed NPYmOrange2 signal) in ctrl (M, 291 FR events) and with A1 (N, 142 FR events). (O) Fraction of FR events in cells expressing NPY-EGFP alone (C), or together with amphiphysin-mCherry (Amph), and in presence or absence of A1; ctrl (23 cells), control with A1 (13 cells), Amph (14 cells) or Amph with A1 (13 cells); p-values shown are for u-test. (P) Distribution of peak amplitudes (ΔΔF/S) for events in M-N (p-values for non-paired t-test). (R) Cumulative frequency histograms of NPY release times in (O). P=0.39 for ctrl vs A1, P=0.12 for Amph+A1 vs Amph, and P=1.5E-11 for Amph vs ctrl, KS-test).

Article Snippet: 300 μM of BODIPY ® FL Phosphatidylinositol 4,5-bisphosphate (BODIPY FL-PI(4,5)P 2 ; Echelon Biosciences, Salt Lake City, USA), was freshly prepared and incubated with 100 μM Histone H1 Carrier (Echelon Biosciences, Salt Lake City, USA) for 10 min at room temperature after vigorous pipetting to facilitate complex formation ( ).

Techniques: Knock-Out, Expressing

Journal: Oncotarget

Article Title: Insight into the PTEN – p85α interaction and lipid binding properties of the p85α BH domain

doi: 10.18632/oncotarget.26432

Figure Lengend Snippet: ( A ) PIP strip nitrocellulose membrane with spotted lipids (100 pmol per spot). ( B ) PIP strips were blocked and probed with the indicated purified p85α protein (37 nM). Bound proteins were detected with an anti-p85α BH antibody, followed with an infrared secondary antibody and visualized using a LICOR Odyssey infrared scanner. PI = phosphatidylinositol; PI3P = phosphatidylinositol 3-phosphate; PI4P = phosphatidylinositol 4-phosphate; PI5P = phosphatidylinositol 5-phosphate; PI3,4P 2 = phosphatidylinositol 3,4-bisphosphate; PI3,5P 2 = phosphatidylinositol 3,5-bisphosphate; PI4,5P 2 = phosphatidylinositol 4,5-bisphosphate; PI3,4,5P 3 = phosphatidylinositol 3,4,5-trisphosphate.

Article Snippet: Reaction mixtures (70 µl), contained Bodipy-FL labelled phosphatidylinositol lipids (50 nm; Echelon Biosciences Cat#: C-00F6, C-03F6, C45F6, and C39F6) in 50 mM Tris pH 7.0, 150 mM NaCl, 1 mM EDTA buffer.

Techniques: Stripping Membranes, Purification

Journal: Oncotarget

Article Title: Insight into the PTEN – p85α interaction and lipid binding properties of the p85α BH domain

doi: 10.18632/oncotarget.26432

Figure Lengend Snippet: ( A ) Changes in fluorescence polarization were measured for Bodipy-FL labelled phosphatidylinositol lipids: phosphatidylinositol (PI), phosphatidylinositol 3-phosphate (PI3P), phosphatidylinositol 4,5-bisphosphate (PI4,5P 2 ), and phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P 3 ), upon mixing with increasing concentrations of wild type p85α BH (105–319) protein. ( B–D ) Changes in fluorescence polarization for the indicated bodipy-labeled lipid were measured for the wild type or p85α BH (105–319) mutant proteins as in panel A; PI3P (B), PI4,5P 2 (C), PI3,4,5P 3 (D). Mean ± SEM is shown for each data point from 3 independent experiments.

Article Snippet: Reaction mixtures (70 µl), contained Bodipy-FL labelled phosphatidylinositol lipids (50 nm; Echelon Biosciences Cat#: C-00F6, C-03F6, C45F6, and C39F6) in 50 mM Tris pH 7.0, 150 mM NaCl, 1 mM EDTA buffer.

Techniques: Fluorescence, Labeling, Mutagenesis

Journal: Structure (London, England : 1993)

Article Title: The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain

doi: 10.1016/j.str.2017.11.001

Figure Lengend Snippet:

Article Snippet: BODIPY® FL Phosphatidylinositol , Echelon Biosciences , C-00F6.

Techniques: Recombinant, Protease Inhibitor, Clone Assay, Mutagenesis, Plasmid Preparation, Software