Journal: bioRxiv

Article Title: The phosphoinositide PI5P impairs mitochondrial function through endosome-mitochondria proximity

doi: 10.1101/2025.09.22.677701

Figure Lengend Snippet: (A) Overexpression of IpgD induces mitochondrial fragmentation. Cells expressing myc-IpgD WT, C438S mutant or GFP-Inp54p were fixed and stained for mitochondria (left panel), and the mitochondrial fragmentation index was quantified across 3 independent experiments (right panel). Bar is 10µM in all figures unless otherwise stated. (B-C) Exogenous PI5P induces mitochondrial fragmentation and increases mitochondrial ROS (mtROS) production and decreased mitochondrial membrane potential (mtΔΨ). Cells were treated overnight with 15µM of PI5P or PI4P, fixed and stained for mitochondria (B) or incubated with MitoSOX (for mtROS production) or with TMRE (for mtΔΨ) and live-imaged (C). Quantifications from 3 independent experiments are shown on the right. (D) Bioenergetic profile of cells treated with PI5P. Treated cells were processed for a Mito Stress Test on a Seahorse flux analyser. A representative Seahorse trace including five technical replicates is shown. (E) Quantification of Basal respiration, Maximal respiration and Spare respiratory capacity from 3 independent experiments are shown. (F) Energy map from control or PI5P-treated cells showing a decrease in the oxidative capacity of the PI5P-treated cells, both in basal and stressed (FCCP treated) conditions. (G) Lysates from control or PI5P-treated cells were probed for the indicated antibodies (left panel). Quantifications are shown on the right. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc analysis for multiple comparisons or unpaired t-test. *p<0.05, **p < 0.01, ***p < 0.001,****p < 0.0001, ns non-significant.

Article Snippet: Cells were treated with STA (100nM), DSMO (vehicle only) or cell permeant di-C4 PI5P (P-5004, Echelon Biosciences) or PI4P (P-4004, Echelon Biosciences) as indicated.

Techniques: Over Expression, Expressing, Mutagenesis, Staining, Membrane, Incubation, Control

Journal: bioRxiv

Article Title: The phosphoinositide PI5P impairs mitochondrial function through endosome-mitochondria proximity

doi: 10.1101/2025.09.22.677701

Figure Lengend Snippet: (A) H9C2 cells were fixed and stained for mitochondria (in green) or for PI5P (in green). Zoomed region (i) and (ii) are shown below. (B) Line scan of fluorescence intensity along each arrow from inserts (i) and (ii) from (A). (C) Proportion of mitochondrial vs. non-mitochondrial PI5P. Results are from 19 cells across 3 independent experiments. (D) Cells were treated with STA (100nM for the indicated time) or with DMSO (vehicle only), fixed and stained for mitochondria (in green) or PI5P (in red). Right panel show the quantification of PI5P fluorescence intensity in the mitochondrial (mito.) or non-mitochondrial mask (non-mito.). Results are from 13-27 cells across 3 independent experiments. Statistical significance was determined by unpaired t-test. *p<0.05, ***p < 0.001.

Article Snippet: Cells were treated with STA (100nM), DSMO (vehicle only) or cell permeant di-C4 PI5P (P-5004, Echelon Biosciences) or PI4P (P-4004, Echelon Biosciences) as indicated.

Techniques: Staining, Fluorescence

Journal: bioRxiv

Article Title: The phosphoinositide PI5P impairs mitochondrial function through endosome-mitochondria proximity

doi: 10.1101/2025.09.22.677701

Figure Lengend Snippet: (A) Cells were treated with 2DG (50mM) in complete medium for 4h, fixed and stained for mitochondria (in green) or PI5P (in red). Quantifications of total PI5P fluorescence, PI5P fluorescence intensity in the mitochondrial (mito.) or non-mitochondrial mask (non-mito.) from 3 independent experiments are shown on the bottom panel. (B) Cells were treated as in (A) except for a pre-treatment with STA (30min, 100nM) as indicated. Results are from 3 independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc analysis for multiple comparisons or unpaired t-test. *p<0.05, **p < 0.01, ***p < 0.001,****p < 0.0001.

Article Snippet: Cells were treated with STA (100nM), DSMO (vehicle only) or cell permeant di-C4 PI5P (P-5004, Echelon Biosciences) or PI4P (P-4004, Echelon Biosciences) as indicated.

Techniques: Staining, Fluorescence

Journal: Plant Signaling & Behavior

Article Title: Phosphoinositide 5-Phosphate and Phosphoinositide 4-Phosphate Trigger Distinct Specific Responses of Arabidopsis Genes

doi:

Figure Lengend Snippet: Table 1

Article Snippet: C-05R16 BODIPY-PI5P-tagged product from (Echelon Biosciences Inc., Salt Lake) was used to illustrate internalization and colocalization of exogenous PtdIns(5)P.

Techniques:

Journal: PLoS ONE

Article Title: A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation

doi: 10.1371/journal.pone.0054127

Figure Lengend Snippet: Schematic representation of the 2∶1 DPPS:PI5P lipid preparation protocol.

Article Snippet: DPPS (1,2-dipalmitoyl- sn -glycero-3-phosphoserine) and PI5P (D-myo-phosphatidylinositol 5-phosphate diC16) were purchased from Echelon Biosciences (Salt Lake City, UT, USA).

Techniques:

Journal: PLoS ONE

Article Title: A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation

doi: 10.1371/journal.pone.0054127

Figure Lengend Snippet: (A) The overnight (16 hour) stability of the assay reagents at 4°C when the enzyme and lipid were premixed, stored separately or made up fresh as compared to a no enzyme and 5 µM ADP (representing 0% and 100% conversion, respectively). The error bars represent the standard deviation (N = 2). (B) The PI5P lipid dependence of the PI5P4Kα enzyme reaction. The error bars represent the standard deviation (N = 2) and are not discernable on the plot. (C) and (D) Tyrphostin AG-82 (AG82) was identified as a weak inhibitor of PI5P4Kα (decreases the enzyme activity by 75%) by a radiometric assay that uses γ- 32 P-ATP and PI5P and measures the radiolabeled enzymatic product, PI(4,5)P 2 after the separation by thin layer chromatography. Five additional compounds were tested and found not to significantly inhibit PI5P4Kα (AG17 = tyrphostin AG-17, AG18 = tyrphostin AG-18, MP = mycophenolate, PVB = purvalanol B and SU6668). All compounds were tested at 100 µM, except for PVB, which was tested at 10 µM due to solubility limitations at higher concentrations. The raw image and the extracted data are shown in (C) and (D), respectively. The commercial PI5P substrate predominantly contains two palmitate groups with a very small amount of deacylated lipid lyso-PI5P that contains only one palmitate group. The intense top spots in (C) represent the PI(4,5)P 2 product with two palmitate groups, and the faint spots below represent the product with just one palmitate group.

Article Snippet: DPPS (1,2-dipalmitoyl- sn -glycero-3-phosphoserine) and PI5P (D-myo-phosphatidylinositol 5-phosphate diC16) were purchased from Echelon Biosciences (Salt Lake City, UT, USA).

Techniques: Standard Deviation, Activity Assay, Thin Layer Chromatography, Solubility

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Recruitment of Irgb6 to the membrane is a direct trigger for membrane deformation

doi: 10.3389/fcimb.2022.992198

Figure Lengend Snippet: Irgb6 deforms PS or PI5P-containing liposomes. Negative stain EM of 10% PI5P-, PS- or PC-containing liposomes (0.1 mg/ml) incubated with 1 μM Irgb6-WT or K275A/R371A (A–D) . Liposome alone were shown (E–G) . Note that remarkable membrane deformation of PI5P- or PS-containing liposomes (A, B) . Scale bar: 1000 nm (A–G) .

Article Snippet: Ten % (mol/mol) PI5P:phosphatidylinositol-5-phosphate (Cat#P-5016, Echelon Biosciences), 80% phosphatidylethanolamine (PE; Cat#840022C, Avanti Polar Lipids), 10% cholesterol (Chol; Cat#700000, Avanti Polar Lipids) were mixed in chloroform-methanol mixture (1:3 v/v).

Techniques: Staining, Incubation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Recruitment of Irgb6 to the membrane is a direct trigger for membrane deformation

doi: 10.3389/fcimb.2022.992198

Figure Lengend Snippet: Effect of nucleotide on membrane deformation of Irgb6-WT. Negative stain EM showing deformation of PI5P-containing liposomes by Irgb6-WT under nucleotide conditions as indicated. PI5P-containing liposomes (0.1 mg/ml) incubated 1 μM Irgb6-WT as in Figure 1 were absorbed on the grids. Then, buffer alone (A, E, I) , GMP-PNP at 0.5 mM (B, F, J) , GTP at 0.1 mM (C, G) or GDP at 0.1 mM (D, H) was added and incubated for 5 min as is in Supplemental Figure 1 . Scale bar: 1000 nm for upper panels (A–D) , 200 nm for middle panels (E–H) , 75 nm in bottom panels (I , J) .

Article Snippet: Ten % (mol/mol) PI5P:phosphatidylinositol-5-phosphate (Cat#P-5016, Echelon Biosciences), 80% phosphatidylethanolamine (PE; Cat#840022C, Avanti Polar Lipids), 10% cholesterol (Chol; Cat#700000, Avanti Polar Lipids) were mixed in chloroform-methanol mixture (1:3 v/v).

Techniques: Staining, Incubation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Recruitment of Irgb6 to the membrane is a direct trigger for membrane deformation

doi: 10.3389/fcimb.2022.992198

Figure Lengend Snippet: PI5P-containing liposomes stimulate Irgb6 GTPase activity. (A) GTPase activity of Irgb6-WT under low (15 mM NaCl) or high (100 mM NaCl) ionic strength conditions. Irgb6-WT at indicated concentrations was incubated in the buffer containing 15 mM or 100 mM NaCl at 37°C for 30 min. Data are means ± S.E.M. of three independent experiments. (*P < 0.05). (B) PI5P-containing liposomes stimulate GTPase activity of Irgb6-WT. Irgb6-WT at indicated concentrations were incubated with 0.1 mg/ml PI5P-containing liposomes in high ionic strength condition (100 mM NaCl) at 37°C for 30 min. Data are means ± S.E.M. of three independent experiments. (**P < 0.01).

Article Snippet: Ten % (mol/mol) PI5P:phosphatidylinositol-5-phosphate (Cat#P-5016, Echelon Biosciences), 80% phosphatidylethanolamine (PE; Cat#840022C, Avanti Polar Lipids), 10% cholesterol (Chol; Cat#700000, Avanti Polar Lipids) were mixed in chloroform-methanol mixture (1:3 v/v).

Techniques: Activity Assay, Incubation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Recruitment of Irgb6 to the membrane is a direct trigger for membrane deformation

doi: 10.3389/fcimb.2022.992198

Figure Lengend Snippet: Nucleotide-dependent binding of Irgb6 to PI5P-containing lipid monolayers. (A–J) Increased binding of Irgb6 to the lipid monolayers containing PI5P in the presence of GMP-PNP. Three μM Irgb6-WT was absorbed to the lipid monolayers without nucleotide (B, G) , or with 1 mM GMP-PNP (C, H) , GTP (D, I) or GDP (E, J) at room temperature for 3 h. TEM image of monolayer without Irgb6 is shown as a negative control (A, F) . TEM images taken at 300× (upper panels) or at 30000× (bottom panels) were shown. Note that binding of Irgb6-WT to monolayers was remarkedly increased in the presence of GMP-PNP (H) , and some appeared bulged (arrowheads in H ). Scale bar: 8.6 μm in upper panels (A–E) , 100 nm in bottom panels (F–J) . (K) Quantification of binding of Irgb6-WT to monolayers. Irgb6-WT on the lipid monolayers could be visible in negatively stained TEM images (512 x 512 pixel) taken as in (A–E) at low magnification. The area corresponding Irgb6-WT in TEM image was quantified using Image J from three to four independent grids. (***P < 0.0001). (L) Model for the molecular machinery of Irgb6 in PVM disruption. Recruitment of Irgb6 to the membrane leads to membrane deformation. The membrane binding is increased at GTP-binding state because of GTP-dependent polymerization of Irgb6. Dense packing of Irgb6 on lipid membrane results in the stimulation of GTPase activity, and upon GTP hydrolysis Irgb6 detaches from the membrane accompanying membrane damage.

Article Snippet: Ten % (mol/mol) PI5P:phosphatidylinositol-5-phosphate (Cat#P-5016, Echelon Biosciences), 80% phosphatidylethanolamine (PE; Cat#840022C, Avanti Polar Lipids), 10% cholesterol (Chol; Cat#700000, Avanti Polar Lipids) were mixed in chloroform-methanol mixture (1:3 v/v).

Techniques: Binding Assay, Negative Control, Staining, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

doi: 10.1016/j.jbc.2022.102264

Figure Lengend Snippet: TMEM16A Ca 2+ -evoked Cl − currents rundown in excised patches and are recovered by a diC8-PI(4,5)P 2 application. A , example currents recorded at the indicated times during 150 ms steps to −60 and +60 mV, recorded using excised inside–out macropatches from Xenopus laevis oocytes. B , normalized plot of current measured at −60 mV versus time, fit with a single exponential ( red line ). C , box plot distribution of the rate of current decay (τ), measured by fitting plots of relative current versus time with single exponentials (N = 11). The central line denotes the median, the box denotes 25 to 75% of the data, and the whiskers represent 10 to 90% of the data. D , a soluble synthetic analog of PI(4,5)P 2 , diC8-PI(4,5)P 2 , was applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. E , box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P 2 with Ca 2+ (N = 8). diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A.

Article Snippet: The following dioctanoyl phospholipids were obtained from Echelon Biosciences: diC8-PI3P, diC8-PI4P, diC8-PI5P, diC8-PI(3,4)P 2 , diC8-PI(4,5)P 2 , diC8-PI(3,5)P 2 , and diC8-PIP 3 .

Techniques: Stable Transfection

Journal: The Journal of Biological Chemistry

Article Title: Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

doi: 10.1016/j.jbc.2022.102264

Figure Lengend Snippet: Phospholipid analogs differentially recovered TMEM16A. Soluble synthetic analogs of PIP 3 (diC8-PIP 3 ), PI(3,4)P 2 (diC8-PI(3,4)P 2 ), and PI(3,5)P 2 were applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. A , box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P 2 (N = 9), diC8-PIP 3 (N = 7), diC8-PI(3,4)P 2 (N = 6), or diC8-PI(3,5)P 2 (N = 5). Representative plots of normalized currents versus time, before and during application of 100 μM diC8-PIP 3 ( B ), diC8-PI(3,4)P 2 ( C ), or diC8-PI(3,5)P 2 ( D ). ∗ represents p < 0.025 as determined by ANOVA and Tukey's HSD post hoc tests. diC8-PI(3,4)P 2 , dioctanoyl phosphatidylinositol 3,4-bisphosphate; diC8-PI(3,5)P 2 , dioctanoyl phosphatidylinositol 3,5-bisphosphate; diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; diC8-HSD, honestly significant difference; PIP 3, dioctanoyl phosphatidylinositol 3,4,5-trisphosphate; PI(3,4)P 2 , phosphatidylinositol 3,4-bisphosphate; PI(3,5)P 2 , dioctanoyl phosphatidylinositol 3,5-bisphosphate; PIP 3, phosphatidyl 3,4,5-trisphosphate; TMEM16A, TransMEMbrane 16A.

Article Snippet: The following dioctanoyl phospholipids were obtained from Echelon Biosciences: diC8-PI3P, diC8-PI4P, diC8-PI5P, diC8-PI(3,4)P 2 , diC8-PI(4,5)P 2 , diC8-PI(3,5)P 2 , and diC8-PIP 3 .

Techniques: Stable Transfection

Journal: The Journal of Biological Chemistry

Article Title: Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

doi: 10.1016/j.jbc.2022.102264

Figure Lengend Snippet: Phospholipids with phosphates at position 4′ of the inositol ring recover current. Soluble synthetic analogs of PI3P, PI4P, and PI5P were applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. Representative plots of normalized currents versus time, before and during application of 100 μM diC8-PI3P ( A ), 100 μM diC8-PI5P ( B ), or 100 μM diC8-PI4P ( D ). C , box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P 2 (N = 9), diC8-PI3P (N = 7), diC8-PI4P (N = 7), or diC8-PI5P (N = 5). ∗ denotes p < 0.05 between indicated treatment and diC8-PI(4,5)P 2 , as determined by ANOVA and Tukey’s HSD post hoc tests. diC8-PI3P, dioctanoyl 3-monophosphate; diC8-PI4P, dioctanoyl 4-monophosphate; diC8-PI5P, dioctanoyl 5-monophosphate; HSD, honestly significant difference.

Article Snippet: The following dioctanoyl phospholipids were obtained from Echelon Biosciences: diC8-PI3P, diC8-PI4P, diC8-PI5P, diC8-PI(3,4)P 2 , diC8-PI(4,5)P 2 , diC8-PI(3,5)P 2 , and diC8-PIP 3 .

Techniques: Stable Transfection

Journal: The Journal of Biological Chemistry

Article Title: Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

doi: 10.1016/j.jbc.2022.102264

Figure Lengend Snippet: Docking suggests key PI(4,5)P 2 phosphate interactions with TMEM16A. Docking was performed with either diC8-PI(4,5)P 2 or IP 3 into a homology model of Xenopus laevis TMEM16A (xTMEM16A). A , position of diC8-PI(4,5)P 2 shown against the homology model of xTMEM16A. Lines indicating the position of the intracellular and extracellular boundaries of the plasma membrane were created using the OPM entry for mouse TMEM16A (PDB: 5OYB ). B , detailed view of the hypothesized PI(4,5)P 2 –xTMEM16A interaction. Interacting residues (E442, K446, R450, K592, and K912 from the other chain) and phosphates (positions 2′–5′) are highlighted. C , superposition of docked IP 3 (foreground) on the PI(4,5)P 2 –xTMEM16A (transparency) interaction. diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; PDB, Protein Data Bank; PI(4,5)P 2, dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A; xTMEM16A, Xenopus laevis TransMEMbrane 16A.

Article Snippet: The following dioctanoyl phospholipids were obtained from Echelon Biosciences: diC8-PI3P, diC8-PI4P, diC8-PI5P, diC8-PI(3,4)P 2 , diC8-PI(4,5)P 2 , diC8-PI(3,5)P 2 , and diC8-PIP 3 .

Techniques: