Journal: PLoS ONE

Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling

doi: 10.1371/journal.pone.0033889

Figure Lengend Snippet: ( A ) GluA1 current amplitudes in oocytes before and after acute injection of a water-soluble analog of PI(3,5)P 2 . Significant change (p<0.05) is indicated by * (p = 0.0015). ( B ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with Rab11 or the dominant negative form of Rab11 (Rab11DN), before and after acute injection of a water-soluble analog of PI (3,5)P 2 . Numbers of oocytes are n = 7–28. Significant change (p<0.05) is indicated by * (p = 0.025). ( C ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with SGK3, myosin Vb, or the mutated form of myosin Vb (myosin del). Number of oocytes are n = 14–28.

Article Snippet: PI(3,5)P 2 -DIC8 (P4508) was purchased from Echelon Bioscience Inc. (Salt Lake City, USA).

Techniques: Injection, Expressing, Dominant Negative Mutation

Journal: PLoS ONE

Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling

doi: 10.1371/journal.pone.0033889

Figure Lengend Snippet: Upon stimulation of NMDA receptors SGK3 is transcriptionally upregulated. SGK3 in turn phosphorylates/activates PIKfyve, which leads to local production of PI(3,5)P 2 in PIKfyve-containing recycling vesicles. PI(3,5)P 2 in turn stimulates Rab11-dependent plasma membrane-directed trafficking of GluA1-containing vesicles.

Article Snippet: PI(3,5)P 2 -DIC8 (P4508) was purchased from Echelon Bioscience Inc. (Salt Lake City, USA).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: The interdependent transport of yeast vacuole Ca 2+ and H + and the role of phosphatidylinositol 3,5-bisphosphate

doi: 10.1016/j.jbc.2022.102672

Figure Lengend Snippet: Modifying PI(3,5)P 2 levels alters vacuole acidification. Vacuoles were used for vacuole acidification measured by AO fluorescence. Reactions were incubated with or without ATP-regenerating system added at 30 s and incubated for a total of 600 s, at which point FCCP was added to equilibrate the H + gradient. AO fluorescence was normalized to the initial value set to 1. A , WT and fab1 T2250A vacuoles were compared in their efficiency to acidify vacuoles. In parallel, fab1 T2250A vacuoles were incubated in the presence of 125 μM apilimod. B , quantitation of multiple experiments in panel ( A ) showed a significant effect of expressing fab1 T2250A compared to WT [F(2,15) = 36; ∗∗∗∗ p < 0.0001] (One way ANOVA for multiple comparisons). Error bars are mean ± SD. Tukey’s multiple comparison test was used for individual p values. (n ≥ 4). C , WT and fab1 EEE vacuoles were compared in their acidification efficiency. Separately, fab1 EEE vacuoles were incubated in C8-PI(3,5)P 2 . D , quantitation of multiple experiments in panel ( C ) showed a significant effect of expressing fab1 EEE compared to WT [F(2,17) = 62.05; ∗∗∗∗ p < 0.0001] (One way ANOVA for multiple comparisons). Error bars are mean ± SD. Tukey’s multiple comparison test was used for individual p values (n ≥ 4). E , WT, fab1 T2250A , and fab1 EEE cells were stained with quinacrine to label acidified compartments. FM4-64 was used to stain the vacuole membrane and Calcofluor (Calc.) White was used to stain the cell wall. The scale bar represents 5 μm. AO, acridine orange; FCCP, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone.

Article Snippet: C8-PI(3,5)P 2 (1,2-dioctanoyl-phosphatidylinositol 3,5-bisphosphate) was purchased from Echelon Inc. ATP was purchased from RPI.

Techniques: Fluorescence, Incubation, Quantitation Assay, Expressing, Staining

Journal: The Journal of Biological Chemistry

Article Title: The interdependent transport of yeast vacuole Ca 2+ and H + and the role of phosphatidylinositol 3,5-bisphosphate

doi: 10.1016/j.jbc.2022.102672

Figure Lengend Snippet: Sequestering or producing PI(3,5)P 2 inhibits vacuole acidification. A , WT vacuoles were incubated with a dose curve of the PI(3,5)P 2 binding domain GST-ML1N and AO fluorescence was measured. B , quantitation of multiple experiments in panel ( A ) showed a significant effect of treating reactions with ML1N [F(2,9) = 44.48; ∗∗∗∗ p < 0.0001] (One way ANOVA for multiple comparisons with no treatment (0 μM) as a control). Error bars are mean ± SD. Dunnett multiple comparison test was used for individual p values (n = 4). ∗ p < 0.05, ∗∗∗∗ p < 0.001. C , WT type vacuoles were incubated with GST-FYVE and AO quenching was measured. D , average of multiple experiments in panel ( C ). E , vacuoles were treated with 125 μM apilimod, 250 μM verapamil, or DMSO (solvent) in the presence of AO at the beginning of the reaction. AO fluorescence was measured for 250 s and FCCP was added seconds to collapse the H + gradient ( arrow ). F , quantitation of multiple experiments in panel ( E ) showed a significant effect of treating reactions with apilimod and verapamil [F(6,14) = 56.02; ∗∗∗∗ p < 0.0001] (One way ANOVA for multiple comparisons with no treatment (0 μM) as a control). Error bars are mean ± SD. Dunnett multiple comparison test was used for individual p values (n = 3). ∗∗∗∗ p < 0.001. G , effect of late additions of 125 μM apilimod, 250 μM verapamil, 100 nM bafilomycin, and DMSO on H + transport ( arrow , Inhibitor) added at 350 s. FCCP was added at 720 s to collapse the H + gradient ( arrow , FCCP). H , quantitation of multiple experiments in panel ( G ) showed a significant effect of treating reactions with bafilomycin, apilimod, and verapamil [F(4,10) = 65.72; ∗∗∗∗ p < 0.0001] (One way ANOVA for multiple comparisons with no treatment (Late PS) as a control). Error bars are mean ± SD. Dunnett multiple comparison test was used for individual p values (n = 3). ∗∗∗∗ p < 0.001. AO, acridine orange; DMSO, dimethyl sulfoxide; FCCP, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone.

Article Snippet: C8-PI(3,5)P 2 (1,2-dioctanoyl-phosphatidylinositol 3,5-bisphosphate) was purchased from Echelon Inc. ATP was purchased from RPI.

Techniques: Incubation, Binding Assay, Fluorescence, Quantitation Assay

Journal: Nature Communications

Article Title: Segregated cation flux by TPC2 biases Ca 2+ signaling through lysosomes

doi: 10.1038/s41467-022-31959-0

Figure Lengend Snippet: a Effect of TPC2-A1-N (N; 30 µM), TPC2-A1-P (P; 30 µM) or a combination of the two (N+P) on Na + levels of individual SBFI-loaded HEK cells stably expressing TPC2 L11A/L12A . Each trace is the normalised fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. b Pooled data (mean ± sem) quantifying the rate of Na + influx from 4-8 experiments in response to the indicated concentration of agonists. n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). c , d Comparison of TPC2-A1-N and TPC2-A1-P responses on cytosolic Na + and Ca 2+ in TPC2 L11A/L12A -expressing cells loaded with SBFI and Fura-2, respectively. Data are mean ± sem from 4 to 10 experiments. e – h Effect of sequential agonist additions on currents from HEK cells stably expressing TPC2 L11A/L12A under bi-ionic conditions. Macropatches were stimulated with 10 µM TPC2-A1-N and 10 µM TPC2-A1-P ( e ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( g ) in the indicated order. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −100 mV and outward Na + currents at +100 mV from 3 to 6 experiments before (basal) and after agonist addition are shown in f and h . * p < 0.05, *** p = 0.0003, **** p < 0.0001, n.s. not significant (Paired t-test, two-tailed); * p = 0.04, ** p = 0.002, n.s. not significant (Unpaired t-test, two-tailed, in red). i – l Effect of agonist additions on currents from HEK cells stably expressing TPC2 in lysosomes under bi-ionic conditions. Cells were stimulated with 10 µM TPC2-A1-P or 10 µM TPC2-A1-N ( i ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( k ) either alone or in combination. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −60 mV or outward Na + currents at +100 mV from 8 to 13 experiments in response to the agonists are shown in j and l , respectively. * p = 0.02, ** p = 0.005 (One-way ANOVA followed by Dunnett’s post hoc test) ( j ); * p = 0.02, **** p < 0.0001 (One-way ANOVA followed by Dunnett’s post hoc test) ( l ); n.s. not significant (Unpaired t-test, two-tailed, j and l ). m , n Effect of TPC2 agonists on reversal potentials. Pooled data (mean ± sem from 3 to 13 experiments) quantifying the effect of TPC2 agonists on E rev in HEK cells stably expressing TPC2 L11A/L12A at the cell surface ( m ) or TPC2 in lysosomes ( n ). Values were derived from the bi-ionic experiments described in e – l . * p = 0.01, ** p = 0.007, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Tukey’s post hoc test) ( m ); *** p = 0.0005, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test) ( n ). Source data are provided as a Source Data file.

Article Snippet: TPC2-A1-N, TPC2-A1-P, PI(3,5)P 2 (diC8 form; Echelon Biosciences) and NAADP (Bio-Techne) were applied by complete exchange of the cytoplasmic solution.

Techniques: Stable Transfection, Expressing, Fluorescence, Concentration Assay, Two Tailed Test, Derivative Assay

Journal: Nature Communications

Article Title: Segregated cation flux by TPC2 biases Ca 2+ signaling through lysosomes

doi: 10.1038/s41467-022-31959-0

Figure Lengend Snippet: a Effect of subthreshold concentration of TPC2-A1-N (10 µM), the indicated concentration of TPC2-A1-P or a combination on Ca 2+ levels of individual HeLa or SK-MEL-5 cells loaded with Fura-2. Data are presented as mean ± sem from 3 to 9 experiments. b , c Effect of subthreshold concentration of TPC2-A1-N (20 µM), TPC2-A1-P (20 µM) or a combination of the two on Ca 2+ levels of individual primary mouse pancreatic acinar cells loaded with Fura-2 b . Each trace is the normalized fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. Pooled data (mean ± sem) quantifying the peak change in ratio from 6 experiments are shown in c . **** p < 0.0001 (Unpaired t-test, two-tailed). d – f Effect of TPC2-A1-N (30 µM), TPC2-A1-P (30 µM) or a combination of the two on subcellular Ca 2+ levels of individual HEK cells loaded with Cal-520. Typical TIRF images with elementary events highlighted by circles are shown in d . Representative time courses of fluorescence changes from the centre of single tuff sites (1 × 1 μm) in response to the indicated agent. e Pooled data (mean ± sem) quantifying the number of events and sites detected per cell, and the peak response from 8 to 10 experiments are shown in f . * p < 0.05, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). g , h Effect of TPC2-A1-N (30 µM) and TPC2-A1-P (60 µM) on lysosomal pH of individual SK-MEL-5 ( g ) or HeLa ( h ) cells loaded with fluorescein-dextran (FDx). Each trace is the fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. i, j , Effect of the indicated combinations of TPC2-A1-N and TPC2-A1-P on lysosomal pH of SK-MEL-5 cells i . Data are presented as mean ± sem from 3 to 5 experiments. Pooled data quantifying the peak change in ratio from multiple experiments using the indicated cell type and agonist combination are shown in j . * p = 0.02, ** p < 0.01 (One-way ANOVA followed by Dunnett’s post hoc test). k – m Effect of TPC2-A1-N and TPC2-A1-P on lysosomal motility. Images show maximum projections of motility calculated from differences in pixel-wise intensity on a frame-by-frame basis over an early (120–240 s) and late (570−690 s) period following addition of TPC2-A1-N (30 µM) or TPC2-A1-P (60 µM) to HeLa cells k . Intense signals represent large changes over time equating to more lysosome movement. Full time-courses presented as mean ± sem from 3 to 8 experiments in response to the indicated agonist combination are shown in l . Pooled data quantifying motility at 700 s from multiple experiments using the indicated cell type and agonist combination m . * p < 0.05, ** p = 0.008 (One-way ANOVA followed by Dunnett’s post hoc test). n Model showing that NAADP and PI(3,5)P 2 work in a synergistic manner to selectively optimize Ca 2+ signalling from lysosomes to regulate lysosomal pH and motility leaving Na + signals unperturbed. Source data are provided as a Source Data file.

Article Snippet: TPC2-A1-N, TPC2-A1-P, PI(3,5)P 2 (diC8 form; Echelon Biosciences) and NAADP (Bio-Techne) were applied by complete exchange of the cytoplasmic solution.

Techniques: Concentration Assay, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Structural plasticity of Atg18 oligomers: organization of assembled tubes and scaffolds at the isolation membrane

doi: 10.1101/2022.07.26.501514

Figure Lengend Snippet: (A) Atg18-PR72AA refined atomic model with surface colored by electrostatics, FRRG motif in pink and phosphate binding sites from aligned PDB-ID 5LTD. (B) Diamond shaped tetramer unit (green) with FRRG motifs (red) and their adjacent phosphate binding sites, which are buried in Atg18 tubes. (C) Pelletation assay (top) and negative stain of pellet fractions from Atg18-WT (left) and Atg18-FGGG (right) (D) Top. Chemical formulas of soluble PIP derivates diC8-PI3P, diC8-PI(3,5)P 2 and diC8-PI(4,5)P 2 used in binding experiments. Bottom. Negative stain electron micrographs of Atg18 nontreated or treated with 4x molar excess of the indicated PIP derivate preventing tube formation. (E) Pelletation assay with corresponding SDS-PAGE of the supernatant (SN) and the pellet (Pe) fraction and the negative stain EM micrographs of two pellets. (F) Tubes from the negative control were treated with PIP derivates. Only PI(3,5)P 2 resulted in tube disassembly.

Article Snippet: To test the effect of soluble lipids on Atg18 tube formation, diC8-PI3P and diC8-PI(3,5)P 2 (Echelon) were reconstituted in water (1 mM final concentration) and added in 4x molar excess to Atg18 in gel filtration buffer prior to the dialysis into tube buffer.

Techniques: Binding Assay, Staining, SDS Page, Negative Control

Journal: bioRxiv

Article Title: Structural plasticity of Atg18 oligomers: organization of assembled tubes and scaffolds at the isolation membrane

doi: 10.1101/2022.07.26.501514

Figure Lengend Snippet: (A) Gel filtration profile of Atg18-WT (black) and Atg18-FGGG (rose). Uncropped and unedited Coomassie-stained SDS-PAGE gels of pelletation assay used for preparing : (B) FGGG mutant (left) in comparison with Atg18-WT (left) and (C) Atg18 incubated with water (control), soluble DiC8-PI3P, DiC8-PI(3,5)P 2 and DiC8-PI(4,5)P 2 as indicated on top of the lanes.

Article Snippet: To test the effect of soluble lipids on Atg18 tube formation, diC8-PI3P and diC8-PI(3,5)P 2 (Echelon) were reconstituted in water (1 mM final concentration) and added in 4x molar excess to Atg18 in gel filtration buffer prior to the dialysis into tube buffer.

Techniques: Filtration, Staining, SDS Page, Mutagenesis, Incubation

Journal: bioRxiv

Article Title: Structural plasticity of Atg18 oligomers: organization of assembled tubes and scaffolds at the isolation membrane

doi: 10.1101/2022.07.26.501514

Figure Lengend Snippet: (A) Volume density rendering of a reconstructed tomogram containing Atg18-WT and PI(3,5)P 2 -containing large unilamellar vesicles (LUVs). (B) Central slice of the corresponding tomogram acquired with Volta phase plate (averaged five slices to display increased contrast). (C) Examples of rotational 2D classification after subvolume extraction of double-membrane subtomograms. The 60 Å thick membrane bilayers are spaced apart by 80 Å. (D) Initial subvolume average dominated by membrane density contributions. (E) Focused 3D classes after membrane subtraction reveal protein densities. (F) Refined structure of the selected 3D class (C1 symmetry) shows organized stacking of eight disc-shaped molecules. (G) Density fit with four Atg18-WT dimers formed by I-interface previously observed in Atg18 tubular assemblies and soluble dimers. The Atg18 molecule within the dimer is colored orange and magenta, respectively. The remaining six Atg18 molecules are colored grey. FRRG motif is highlighted in red and allows for direct binding of opposing membranes. (H) Slice through reconstructed tomogram reveals Atg18 dimer stacking with the approx. 45° angle relative to the membrane bilayer planes.

Article Snippet: To test the effect of soluble lipids on Atg18 tube formation, diC8-PI3P and diC8-PI(3,5)P 2 (Echelon) were reconstituted in water (1 mM final concentration) and added in 4x molar excess to Atg18 in gel filtration buffer prior to the dialysis into tube buffer.

Techniques: Binding Assay