Structured Review

Croda International Plc pi 4 5 p 2
(A-C) Cells transiently expressing the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81) along with low levels of mEGFP-tagged 2xBtk(2-166) (A), 2xBtk(2-166)-R28C (B), or Btk(2-166) (C) were imaged at the bottom surfaces by TIRF microscopy. Left: Representative images of the mEGFP-tagged Btk sensor and the PI(4,5)P 2 sensor (inserts). Right: The intensity profile heatmap of the mEGFP-tagged Btk sensor around the cell periphery from multiple cells (n = 60 cells each). (D) Box plots showing the standard deviation of mEGFP-tagged sensor distribution at the plasma membrane (box: median with the 25th and 75th percentiles; whiskers: 1.5-fold the interquartile range; n = 62, 62, 63, and 60 cells). (E) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81) were imaged at 10-s intervals for 30 min. Representative montage shows PI(3,4,5)P 3 and PI(4,5)P 2 sensors at the indicated times. The fluorescence intensity profile of the PI(3,4,5)P 3 sensor around the cell periphery over time is shown in the right panel. (F) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and mCherry-FAK were imaged at the bottom surfaces at 10-s intervals by TIRF microscopy. Left: Images of PI(3,4,5)P 3 sensor and mCherry-FAK from a single frame of a representative time series. Middle: Images showing FA, Non-FA, and Random regions of the cells. Right: Box plots showing the mean fluorescence intensity of the PI(3,4,5)P 3 sensor on each FA, Non-FA, or Random regions; scatter plots showing the relationship between the fluorescence intensity of the PI(3,4,5)P 3 sensor and the size/intensity of each FA (red lines represent the average intensity within the indicated area ranges). (G) The montage shows selected frames of the boxed regions in (F) (FAs detected are plotted at the bottom). Kymographs were generated along the white line (on the 10-min frame) for the time series. The plots below the kymographs show the relative intensity profile along the line on the single frame. Top right: FAs detected in the time series are overlaid, with each frame represented by a different color. Bottom right: Plots showing the relative intensity of the PI(3,4,5)P 3 sensor and mCherry-FAK on FAs over time. (H) Top: Images of a cell transiently expressing the PI(3,4,5)P 3 sensor Halo-2xBtk(2-166) (labeled with JFX 650 -HaloTag ligand), mEGFP-2xBtk(2-166), and mCherry-FAK. Bottom: Images of a cell transiently expressing the PI(4,5)P 2 sensor Halo-PH(PLC81) (labeled with JFX 650 -HaloTag ligand), the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166), and mCherry-FAK. (I) Top: Images of the maximum-intensity projection of a representative time series. Bottom: Scatter plots showing the relationship between the intensity of the PI(3,4,5)P 3 sensor and the integrated number of p110α-mNeonGreen molecules (left) or the area/intensity of mCherry-FAK (right) on each FA in the time series. (J) Dual gene-edited mEGFP-p85α +/+ and EGFR-Halo (pool, labeled with JFX 650 -HaloTag ligand) cells transiently expressing mCherry-FAK were imaged at 10-s intervals, with EGF added (set as 0 s) during continuous imaging. Representative images show the recruitment of mEGFP-p85α to the plasma membrane and colocalization with EGFR (arrows) after EGF treatment. (K) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and Halo-FAK (labeled with JFX 650 -HaloTag ligand) were imaged at 10-s intervals with EGF added (set as 0 s) during continuous imaging. Images from a representative time series at 0 s and 120 s after EGF treatment are shown. Kymographs were generated along the white line for the time series. Plots show the relative intensity of the PI(3,4,5)P 3 sensor at FA and Non-FA regions, as well as Halo-FAK at FAs over time. Cells were imaged at the bottom surface by TIRF microscopy in (A-K). Statistical analysis in (D) and (F) was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons test; ** P < 0.001; **** P < 0.0001. Scale bars, 10 μm.
Pi 4 5 P 2, supplied by Croda International Plc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Spatial organization of PI3K-PI(3,4,5)P3-AKT signaling by focal adhesions"

Article Title: Spatial organization of PI3K-PI(3,4,5)P3-AKT signaling by focal adhesions

Journal: bioRxiv

doi: 10.1101/2024.07.05.602013

(A-C) Cells transiently expressing the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81) along with low levels of mEGFP-tagged 2xBtk(2-166) (A), 2xBtk(2-166)-R28C (B), or Btk(2-166) (C) were imaged at the bottom surfaces by TIRF microscopy. Left: Representative images of the mEGFP-tagged Btk sensor and the PI(4,5)P 2 sensor (inserts). Right: The intensity profile heatmap of the mEGFP-tagged Btk sensor around the cell periphery from multiple cells (n = 60 cells each). (D) Box plots showing the standard deviation of mEGFP-tagged sensor distribution at the plasma membrane (box: median with the 25th and 75th percentiles; whiskers: 1.5-fold the interquartile range; n = 62, 62, 63, and 60 cells). (E) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81) were imaged at 10-s intervals for 30 min. Representative montage shows PI(3,4,5)P 3 and PI(4,5)P 2 sensors at the indicated times. The fluorescence intensity profile of the PI(3,4,5)P 3 sensor around the cell periphery over time is shown in the right panel. (F) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and mCherry-FAK were imaged at the bottom surfaces at 10-s intervals by TIRF microscopy. Left: Images of PI(3,4,5)P 3 sensor and mCherry-FAK from a single frame of a representative time series. Middle: Images showing FA, Non-FA, and Random regions of the cells. Right: Box plots showing the mean fluorescence intensity of the PI(3,4,5)P 3 sensor on each FA, Non-FA, or Random regions; scatter plots showing the relationship between the fluorescence intensity of the PI(3,4,5)P 3 sensor and the size/intensity of each FA (red lines represent the average intensity within the indicated area ranges). (G) The montage shows selected frames of the boxed regions in (F) (FAs detected are plotted at the bottom). Kymographs were generated along the white line (on the 10-min frame) for the time series. The plots below the kymographs show the relative intensity profile along the line on the single frame. Top right: FAs detected in the time series are overlaid, with each frame represented by a different color. Bottom right: Plots showing the relative intensity of the PI(3,4,5)P 3 sensor and mCherry-FAK on FAs over time. (H) Top: Images of a cell transiently expressing the PI(3,4,5)P 3 sensor Halo-2xBtk(2-166) (labeled with JFX 650 -HaloTag ligand), mEGFP-2xBtk(2-166), and mCherry-FAK. Bottom: Images of a cell transiently expressing the PI(4,5)P 2 sensor Halo-PH(PLC81) (labeled with JFX 650 -HaloTag ligand), the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166), and mCherry-FAK. (I) Top: Images of the maximum-intensity projection of a representative time series. Bottom: Scatter plots showing the relationship between the intensity of the PI(3,4,5)P 3 sensor and the integrated number of p110α-mNeonGreen molecules (left) or the area/intensity of mCherry-FAK (right) on each FA in the time series. (J) Dual gene-edited mEGFP-p85α +/+ and EGFR-Halo (pool, labeled with JFX 650 -HaloTag ligand) cells transiently expressing mCherry-FAK were imaged at 10-s intervals, with EGF added (set as 0 s) during continuous imaging. Representative images show the recruitment of mEGFP-p85α to the plasma membrane and colocalization with EGFR (arrows) after EGF treatment. (K) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and Halo-FAK (labeled with JFX 650 -HaloTag ligand) were imaged at 10-s intervals with EGF added (set as 0 s) during continuous imaging. Images from a representative time series at 0 s and 120 s after EGF treatment are shown. Kymographs were generated along the white line for the time series. Plots show the relative intensity of the PI(3,4,5)P 3 sensor at FA and Non-FA regions, as well as Halo-FAK at FAs over time. Cells were imaged at the bottom surface by TIRF microscopy in (A-K). Statistical analysis in (D) and (F) was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons test; ** P < 0.001; **** P < 0.0001. Scale bars, 10 μm.
Figure Legend Snippet: (A-C) Cells transiently expressing the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81) along with low levels of mEGFP-tagged 2xBtk(2-166) (A), 2xBtk(2-166)-R28C (B), or Btk(2-166) (C) were imaged at the bottom surfaces by TIRF microscopy. Left: Representative images of the mEGFP-tagged Btk sensor and the PI(4,5)P 2 sensor (inserts). Right: The intensity profile heatmap of the mEGFP-tagged Btk sensor around the cell periphery from multiple cells (n = 60 cells each). (D) Box plots showing the standard deviation of mEGFP-tagged sensor distribution at the plasma membrane (box: median with the 25th and 75th percentiles; whiskers: 1.5-fold the interquartile range; n = 62, 62, 63, and 60 cells). (E) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81) were imaged at 10-s intervals for 30 min. Representative montage shows PI(3,4,5)P 3 and PI(4,5)P 2 sensors at the indicated times. The fluorescence intensity profile of the PI(3,4,5)P 3 sensor around the cell periphery over time is shown in the right panel. (F) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and mCherry-FAK were imaged at the bottom surfaces at 10-s intervals by TIRF microscopy. Left: Images of PI(3,4,5)P 3 sensor and mCherry-FAK from a single frame of a representative time series. Middle: Images showing FA, Non-FA, and Random regions of the cells. Right: Box plots showing the mean fluorescence intensity of the PI(3,4,5)P 3 sensor on each FA, Non-FA, or Random regions; scatter plots showing the relationship between the fluorescence intensity of the PI(3,4,5)P 3 sensor and the size/intensity of each FA (red lines represent the average intensity within the indicated area ranges). (G) The montage shows selected frames of the boxed regions in (F) (FAs detected are plotted at the bottom). Kymographs were generated along the white line (on the 10-min frame) for the time series. The plots below the kymographs show the relative intensity profile along the line on the single frame. Top right: FAs detected in the time series are overlaid, with each frame represented by a different color. Bottom right: Plots showing the relative intensity of the PI(3,4,5)P 3 sensor and mCherry-FAK on FAs over time. (H) Top: Images of a cell transiently expressing the PI(3,4,5)P 3 sensor Halo-2xBtk(2-166) (labeled with JFX 650 -HaloTag ligand), mEGFP-2xBtk(2-166), and mCherry-FAK. Bottom: Images of a cell transiently expressing the PI(4,5)P 2 sensor Halo-PH(PLC81) (labeled with JFX 650 -HaloTag ligand), the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166), and mCherry-FAK. (I) Top: Images of the maximum-intensity projection of a representative time series. Bottom: Scatter plots showing the relationship between the intensity of the PI(3,4,5)P 3 sensor and the integrated number of p110α-mNeonGreen molecules (left) or the area/intensity of mCherry-FAK (right) on each FA in the time series. (J) Dual gene-edited mEGFP-p85α +/+ and EGFR-Halo (pool, labeled with JFX 650 -HaloTag ligand) cells transiently expressing mCherry-FAK were imaged at 10-s intervals, with EGF added (set as 0 s) during continuous imaging. Representative images show the recruitment of mEGFP-p85α to the plasma membrane and colocalization with EGFR (arrows) after EGF treatment. (K) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and Halo-FAK (labeled with JFX 650 -HaloTag ligand) were imaged at 10-s intervals with EGF added (set as 0 s) during continuous imaging. Images from a representative time series at 0 s and 120 s after EGF treatment are shown. Kymographs were generated along the white line for the time series. Plots show the relative intensity of the PI(3,4,5)P 3 sensor at FA and Non-FA regions, as well as Halo-FAK at FAs over time. Cells were imaged at the bottom surface by TIRF microscopy in (A-K). Statistical analysis in (D) and (F) was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons test; ** P < 0.001; **** P < 0.0001. Scale bars, 10 μm.

Techniques Used: Expressing, Microscopy, Standard Deviation, Membrane, Fluorescence, Generated, Labeling, Imaging

(A) Sequencing results of genomic DNA from the parental SUM159 cells ( PIK3CA WT/H1047L ) and gene-edited SUM159 cells homozygous for H1047 ( PIK3CA WT/WT ) or H1047L ( PIK3CA H1047L/H1047L ) of PIK3CA . (B) PIK3CA WT/H1047L , PIK3CA WT/WT , PIK3CA H1047L/H1047L , or PTEN-KO SUM159 cells were serum-starved overnight and then subjected to western blot analysis using the indicated antibodies. The ratios of phosphorylated AKT to total AKT are shown for four independent experiments. (C) Quantification of PI(3,4,5)P 3 and PI(4,5)P 2 levels in the four cell lines by mass spectrometry. (D) PIK3CA WT/H1047L , PIK3CA WT/WT , or PIK3CA H1047L/H1047L cells were transiently transfected with the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81). Shown are representative images and intensity profile heatmaps of the PI(3,4,5)P 3 sensor in the three cell lines (n = 60 cells each). Box plots show the standard deviation of PI(3,4,5)P 3 sensor distribution at the plasma membrane (box: median with the 25th and 75th percentiles; whiskers: 1.5-fold the interquartile range; n = 69, 61, and 67 cells). (E) SUM159 cells transiently co-expressing mCherry-FAK with p110α(H1047L)-mNeonGreen were imaged at 0.2-s intervals for 301 frames. Left: Images of a single frame and the maximum-intensity projection of a representative time series are shown. Right: Kymographs generated along the line showing recruitment of p110α(H1047L) to FAs. (F) PTEN-KO SUM159 cells were transiently expressed with the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166), or mEGFP-2xBtk(2-166) together with mScarlet-I-PTEN or mScarlet-I-PTEN-G129E (inserts). Shown are representative images and intensity profile heatmaps of the PI(3,4,5)P 3 sensor (n = 60 cells each). (G) PTEN-KO SUM159 cells transiently co-expressing mCherry-FAK and p110α-mNeonGreen were imaged at 0.2-s intervals for 301 frames. Left: Images of a single frame and the maximum-intensity projection of a representative time series are shown. Right: Kymographs were generated along the line. (H) PIK3CA WT/H1047L , PIK3CA WT/WT , PIK3CA H1047L/H1047L , or PTEN-KO cells transiently expressing mNeonGreen-AKT1, mCherry-FAK and the PI(3,4,5)P 3 sensor Halo-2xBtk(2-166) were imaged at 0.3-s intervals. Images of a single frame of a representative time series are shown. (I) PIK3CA WT/H1047L , PIK3CA WT/WT , PIK3CA H1047L/H1047L , or PTEN-KO cells were treated with or without defactinib for 10 min. The total and phosphorylated AKT and FAK levels were analyzed by western blot. Statistical analysis was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons test; * P < 0.05; **** P < 0.0001. Scale bars, 10 μm.
Figure Legend Snippet: (A) Sequencing results of genomic DNA from the parental SUM159 cells ( PIK3CA WT/H1047L ) and gene-edited SUM159 cells homozygous for H1047 ( PIK3CA WT/WT ) or H1047L ( PIK3CA H1047L/H1047L ) of PIK3CA . (B) PIK3CA WT/H1047L , PIK3CA WT/WT , PIK3CA H1047L/H1047L , or PTEN-KO SUM159 cells were serum-starved overnight and then subjected to western blot analysis using the indicated antibodies. The ratios of phosphorylated AKT to total AKT are shown for four independent experiments. (C) Quantification of PI(3,4,5)P 3 and PI(4,5)P 2 levels in the four cell lines by mass spectrometry. (D) PIK3CA WT/H1047L , PIK3CA WT/WT , or PIK3CA H1047L/H1047L cells were transiently transfected with the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81). Shown are representative images and intensity profile heatmaps of the PI(3,4,5)P 3 sensor in the three cell lines (n = 60 cells each). Box plots show the standard deviation of PI(3,4,5)P 3 sensor distribution at the plasma membrane (box: median with the 25th and 75th percentiles; whiskers: 1.5-fold the interquartile range; n = 69, 61, and 67 cells). (E) SUM159 cells transiently co-expressing mCherry-FAK with p110α(H1047L)-mNeonGreen were imaged at 0.2-s intervals for 301 frames. Left: Images of a single frame and the maximum-intensity projection of a representative time series are shown. Right: Kymographs generated along the line showing recruitment of p110α(H1047L) to FAs. (F) PTEN-KO SUM159 cells were transiently expressed with the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166), or mEGFP-2xBtk(2-166) together with mScarlet-I-PTEN or mScarlet-I-PTEN-G129E (inserts). Shown are representative images and intensity profile heatmaps of the PI(3,4,5)P 3 sensor (n = 60 cells each). (G) PTEN-KO SUM159 cells transiently co-expressing mCherry-FAK and p110α-mNeonGreen were imaged at 0.2-s intervals for 301 frames. Left: Images of a single frame and the maximum-intensity projection of a representative time series are shown. Right: Kymographs were generated along the line. (H) PIK3CA WT/H1047L , PIK3CA WT/WT , PIK3CA H1047L/H1047L , or PTEN-KO cells transiently expressing mNeonGreen-AKT1, mCherry-FAK and the PI(3,4,5)P 3 sensor Halo-2xBtk(2-166) were imaged at 0.3-s intervals. Images of a single frame of a representative time series are shown. (I) PIK3CA WT/H1047L , PIK3CA WT/WT , PIK3CA H1047L/H1047L , or PTEN-KO cells were treated with or without defactinib for 10 min. The total and phosphorylated AKT and FAK levels were analyzed by western blot. Statistical analysis was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons test; * P < 0.05; **** P < 0.0001. Scale bars, 10 μm.

Techniques Used: Sequencing, Western Blot, Mass Spectrometry, Transfection, Standard Deviation, Membrane, Expressing, Generated

pi 4 5 p 2 dic8 echelon  (Echelon Biosciences)


Bioz Verified Symbol Echelon Biosciences is a verified supplier
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    Structured Review

    Echelon Biosciences pi 4 5 p 2 dic8 echelon
    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.
    Pi 4 5 P 2 Dic8 Echelon, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pi 4 5 p 2 dic8 echelon/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pi 4 5 p 2 dic8 echelon - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "TRPML1 gating modulation by allosteric mutations and lipids (Design of allosteric mutations that recapitulate the gating of TRPML1)"

    Article Title: TRPML1 gating modulation by allosteric mutations and lipids (Design of allosteric mutations that recapitulate the gating of TRPML1)

    Journal: bioRxiv

    doi: 10.1101/2024.07.04.602033

    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.
    Figure Legend Snippet: (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.

    Techniques Used: Binding Assay, Comparison

    (a) Overall structure of PI(4,5)P 2 -bound TRPML1 and the zoomed-in view of the lipid-binding site. The lipid density is shown as blue surface and modeled as sphingomyelin (SM). The side chains of lipid-interacting residues are shown as yellow sticks. (b) SM inhibition effect on SF-51-activated wild-type TRPML1. (c) SM activation effect on ML-SI1-inhibited Y404W mutant. Currents shown in (b) and (c) were recorded using patch clamp in whole-cell configuration with pH 4.6 in the bath solution as the adverse effect of SM on agonist or antagonist is subtle and is measurable only at low luminal pH.
    Figure Legend Snippet: (a) Overall structure of PI(4,5)P 2 -bound TRPML1 and the zoomed-in view of the lipid-binding site. The lipid density is shown as blue surface and modeled as sphingomyelin (SM). The side chains of lipid-interacting residues are shown as yellow sticks. (b) SM inhibition effect on SF-51-activated wild-type TRPML1. (c) SM activation effect on ML-SI1-inhibited Y404W mutant. Currents shown in (b) and (c) were recorded using patch clamp in whole-cell configuration with pH 4.6 in the bath solution as the adverse effect of SM on agonist or antagonist is subtle and is measurable only at low luminal pH.

    Techniques Used: Binding Assay, Inhibition, Activation Assay, Mutagenesis, Patch Clamp

    pi 4 5 p 2  (Thermo Fisher)


    Bioz Verified Symbol Thermo Fisher is a verified supplier
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    Thermo Fisher pi 4 5 p 2
    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.
    Pi 4 5 P 2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pi 4 5 p 2/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pi 4 5 p 2 - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "TRPML1 gating modulation by allosteric mutations and lipids (Design of allosteric mutations that recapitulate the gating of TRPML1)"

    Article Title: TRPML1 gating modulation by allosteric mutations and lipids (Design of allosteric mutations that recapitulate the gating of TRPML1)

    Journal: bioRxiv

    doi: 10.1101/2024.07.04.602033

    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.
    Figure Legend Snippet: (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.

    Techniques Used: Binding Assay, Comparison

    (a) Overall structure of PI(4,5)P 2 -bound TRPML1 and the zoomed-in view of the lipid-binding site. The lipid density is shown as blue surface and modeled as sphingomyelin (SM). The side chains of lipid-interacting residues are shown as yellow sticks. (b) SM inhibition effect on SF-51-activated wild-type TRPML1. (c) SM activation effect on ML-SI1-inhibited Y404W mutant. Currents shown in (b) and (c) were recorded using patch clamp in whole-cell configuration with pH 4.6 in the bath solution as the adverse effect of SM on agonist or antagonist is subtle and is measurable only at low luminal pH.
    Figure Legend Snippet: (a) Overall structure of PI(4,5)P 2 -bound TRPML1 and the zoomed-in view of the lipid-binding site. The lipid density is shown as blue surface and modeled as sphingomyelin (SM). The side chains of lipid-interacting residues are shown as yellow sticks. (b) SM inhibition effect on SF-51-activated wild-type TRPML1. (c) SM activation effect on ML-SI1-inhibited Y404W mutant. Currents shown in (b) and (c) were recorded using patch clamp in whole-cell configuration with pH 4.6 in the bath solution as the adverse effect of SM on agonist or antagonist is subtle and is measurable only at low luminal pH.

    Techniques Used: Binding Assay, Inhibition, Activation Assay, Mutagenesis, Patch Clamp

    pi 4 5 p 2  (Echelon Biosciences)


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    Echelon Biosciences pi 4 5 p 2
    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.
    Pi 4 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    1) Product Images from "TRPML1 gating modulation by allosteric mutations and lipids (Design of allosteric mutations that recapitulate the gating of TRPML1)"

    Article Title: TRPML1 gating modulation by allosteric mutations and lipids (Design of allosteric mutations that recapitulate the gating of TRPML1)

    Journal: bioRxiv

    doi: 10.1101/2024.07.04.602033

    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.
    Figure Legend Snippet: (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.

    Techniques Used: Binding Assay, Comparison

    (a) Overall structure of PI(4,5)P 2 -bound TRPML1 and the zoomed-in view of the lipid-binding site. The lipid density is shown as blue surface and modeled as sphingomyelin (SM). The side chains of lipid-interacting residues are shown as yellow sticks. (b) SM inhibition effect on SF-51-activated wild-type TRPML1. (c) SM activation effect on ML-SI1-inhibited Y404W mutant. Currents shown in (b) and (c) were recorded using patch clamp in whole-cell configuration with pH 4.6 in the bath solution as the adverse effect of SM on agonist or antagonist is subtle and is measurable only at low luminal pH.
    Figure Legend Snippet: (a) Overall structure of PI(4,5)P 2 -bound TRPML1 and the zoomed-in view of the lipid-binding site. The lipid density is shown as blue surface and modeled as sphingomyelin (SM). The side chains of lipid-interacting residues are shown as yellow sticks. (b) SM inhibition effect on SF-51-activated wild-type TRPML1. (c) SM activation effect on ML-SI1-inhibited Y404W mutant. Currents shown in (b) and (c) were recorded using patch clamp in whole-cell configuration with pH 4.6 in the bath solution as the adverse effect of SM on agonist or antagonist is subtle and is measurable only at low luminal pH.

    Techniques Used: Binding Assay, Inhibition, Activation Assay, Mutagenesis, Patch Clamp

    pi 4 5 p 2  (Echelon Biosciences)


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    Structured Review

    Echelon Biosciences pi 4 5 p 2
    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.
    Pi 4 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "TRPML1 gating modulation by allosteric mutations and lipids (Design of allosteric mutations that recapitulate the gating of TRPML1)"

    Article Title: TRPML1 gating modulation by allosteric mutations and lipids (Design of allosteric mutations that recapitulate the gating of TRPML1)

    Journal: bioRxiv

    doi: 10.1101/2024.07.04.602033

    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.
    Figure Legend Snippet: (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.

    Techniques Used: Binding Assay, Comparison

    (a) Overall structure of PI(4,5)P 2 -bound TRPML1 and the zoomed-in view of the lipid-binding site. The lipid density is shown as blue surface and modeled as sphingomyelin (SM). The side chains of lipid-interacting residues are shown as yellow sticks. (b) SM inhibition effect on SF-51-activated wild-type TRPML1. (c) SM activation effect on ML-SI1-inhibited Y404W mutant. Currents shown in (b) and (c) were recorded using patch clamp in whole-cell configuration with pH 4.6 in the bath solution as the adverse effect of SM on agonist or antagonist is subtle and is measurable only at low luminal pH.
    Figure Legend Snippet: (a) Overall structure of PI(4,5)P 2 -bound TRPML1 and the zoomed-in view of the lipid-binding site. The lipid density is shown as blue surface and modeled as sphingomyelin (SM). The side chains of lipid-interacting residues are shown as yellow sticks. (b) SM inhibition effect on SF-51-activated wild-type TRPML1. (c) SM activation effect on ML-SI1-inhibited Y404W mutant. Currents shown in (b) and (c) were recorded using patch clamp in whole-cell configuration with pH 4.6 in the bath solution as the adverse effect of SM on agonist or antagonist is subtle and is measurable only at low luminal pH.

    Techniques Used: Binding Assay, Inhibition, Activation Assay, Mutagenesis, Patch Clamp


    Structured Review

    Croda International Plc l α pi 4 5 p 2
    L α Pi 4 5 P 2, supplied by Croda International Plc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Structured Review

    Millipore l α pi 4 5 p 2
    L α Pi 4 5 P 2, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    brain pi 4 5 p 2 l α phosphatidylinositol 4 5 bisphosphate  (Croda International Plc)

     
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    Structured Review

    Croda International Plc brain pi 4 5 p 2 l α phosphatidylinositol 4 5 bisphosphate
    (A) Predicted structural organization of Tg REMIND. (B ) Ribbon representation of the AlphaFold-predicted model of the F-BAR domain of Tg REMIND in a dimeric form. The position of each monomer’s N- and C-terminal ends is indicated. (C) Same view as (B) or view of the concave face of the F-BAR domain with the surface colored by electrostatic potential (red = -16.9 kTe -1 , blue = +16.9 kTe -1 ). (D) Far-UV CD spectrum of purified F-BAR REMIND (3 µM) in 20 mM Tris, pH 7.4, 120 mM NaF buffer recorded at room temperature. The percentage of α-helix, β-sheet, and other structures, derived from the CD spectra analysis and the AlphaFold-predicted model obtained using the DSSP algorithm, is given. MRE: mean residue ellipticity, H: α-helix, E: β-sheet, O: other structures. ( E ) Flotation assay. F-BAR REMIND (0.75 µM) was incubated with liposomes (750 µM total lipids) only made of DOPC and additionally containing 30% DOPS or 10% PIPs (PI(3)P, PI(4)P or <t>PI(4,5)P</t> 2 at the expense of DOPC), extruded through pores of defined size, indicated at the top of the gel. This incubation was performed for 1 h at 25 °C in 50 mM Tris, pH 7.4, 150 mM NaCl (TN) buffer under constant agitation. After centrifugation, the liposomes were recovered at the top of sucrose cushions and analyzed by SDS-PAGE. The amount of membrane-bound F-BAR was determined using the content of the first lane (100% total) as a reference based on the SYPRO Orange signal. The percentage of membrane-bound F-BAR REMIND is represented as the function of the average hydrodynamic radius (R H ) of liposomes. Data are expressed as mean ± s.e.m. (n = 3-5). (F) Flotation assay. N-BAR domain of human amphiphysin (N-BAR Amph , 0.75 µM) was incubated with liposomes (750 µM lipids), only made of DOPC and additionally containing 30% DOPS or 10% PI(4,5)P 2 , extruded through pores of defined size, for 1 h at 25 °C in 50 mM Tris, pH 7.4, 150 mM NaCl (TN) buffer. The percentage of membrane-bound N-BAR Amph is shown as the function of the radius of liposomes (n = 3). ( G ) Negative-staining EM. Liposomes made of Folch fraction I lipids (30 µM lipids) and extruded through 0.4 µm pores were incubated with F-BAR REMIND or N-BAR Amph (1.9 µM) for 2 h at room temperature. A control picture of liposomes alone is shown. Scale bar = 100 nm. ( H ) Length distributions of membrane tubules induced by F-BAR REMIND and N-BAR Amph (n =105 and 26, respectively). (I) Diameter distribution of tubules and N-BAR Amph (n =105 and 26, respectively). The structure of the F-BAR domain of Tg REMIND seems adapted to the diameter of tubules that have been experimentally measured. (J) Cryo-EM. Folch fraction liposomes (90 µM lipids), extruded through 0.4 µm pores, were mixed with F-BAR REMIND (6 µM) at P/L= 1/15 and dialyzed three times under constant stirring in TN buffer for 30 min at cold temperature. Enlargements: (1) Outer leaflet destabilization (white arrowhead) by F-BAR REMIND , (2) Intact bilayer, (3) Outer leaflet destabilization (white arrowhead) by F-BAR REMIND . Green arrows point to some individual F-BAR REMIND molecules. White arrows indicate local membrane disruption (both leaflets)
    Brain Pi 4 5 P 2 L α Phosphatidylinositol 4 5 Bisphosphate, supplied by Croda International Plc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Characterization of atypical BAR domain-containing proteins coded by Toxoplasma gondii"

    Article Title: Characterization of atypical BAR domain-containing proteins coded by Toxoplasma gondii

    Journal: bioRxiv

    doi: 10.1101/2024.06.13.598837

    (A) Predicted structural organization of Tg REMIND. (B ) Ribbon representation of the AlphaFold-predicted model of the F-BAR domain of Tg REMIND in a dimeric form. The position of each monomer’s N- and C-terminal ends is indicated. (C) Same view as (B) or view of the concave face of the F-BAR domain with the surface colored by electrostatic potential (red = -16.9 kTe -1 , blue = +16.9 kTe -1 ). (D) Far-UV CD spectrum of purified F-BAR REMIND (3 µM) in 20 mM Tris, pH 7.4, 120 mM NaF buffer recorded at room temperature. The percentage of α-helix, β-sheet, and other structures, derived from the CD spectra analysis and the AlphaFold-predicted model obtained using the DSSP algorithm, is given. MRE: mean residue ellipticity, H: α-helix, E: β-sheet, O: other structures. ( E ) Flotation assay. F-BAR REMIND (0.75 µM) was incubated with liposomes (750 µM total lipids) only made of DOPC and additionally containing 30% DOPS or 10% PIPs (PI(3)P, PI(4)P or PI(4,5)P 2 at the expense of DOPC), extruded through pores of defined size, indicated at the top of the gel. This incubation was performed for 1 h at 25 °C in 50 mM Tris, pH 7.4, 150 mM NaCl (TN) buffer under constant agitation. After centrifugation, the liposomes were recovered at the top of sucrose cushions and analyzed by SDS-PAGE. The amount of membrane-bound F-BAR was determined using the content of the first lane (100% total) as a reference based on the SYPRO Orange signal. The percentage of membrane-bound F-BAR REMIND is represented as the function of the average hydrodynamic radius (R H ) of liposomes. Data are expressed as mean ± s.e.m. (n = 3-5). (F) Flotation assay. N-BAR domain of human amphiphysin (N-BAR Amph , 0.75 µM) was incubated with liposomes (750 µM lipids), only made of DOPC and additionally containing 30% DOPS or 10% PI(4,5)P 2 , extruded through pores of defined size, for 1 h at 25 °C in 50 mM Tris, pH 7.4, 150 mM NaCl (TN) buffer. The percentage of membrane-bound N-BAR Amph is shown as the function of the radius of liposomes (n = 3). ( G ) Negative-staining EM. Liposomes made of Folch fraction I lipids (30 µM lipids) and extruded through 0.4 µm pores were incubated with F-BAR REMIND or N-BAR Amph (1.9 µM) for 2 h at room temperature. A control picture of liposomes alone is shown. Scale bar = 100 nm. ( H ) Length distributions of membrane tubules induced by F-BAR REMIND and N-BAR Amph (n =105 and 26, respectively). (I) Diameter distribution of tubules and N-BAR Amph (n =105 and 26, respectively). The structure of the F-BAR domain of Tg REMIND seems adapted to the diameter of tubules that have been experimentally measured. (J) Cryo-EM. Folch fraction liposomes (90 µM lipids), extruded through 0.4 µm pores, were mixed with F-BAR REMIND (6 µM) at P/L= 1/15 and dialyzed three times under constant stirring in TN buffer for 30 min at cold temperature. Enlargements: (1) Outer leaflet destabilization (white arrowhead) by F-BAR REMIND , (2) Intact bilayer, (3) Outer leaflet destabilization (white arrowhead) by F-BAR REMIND . Green arrows point to some individual F-BAR REMIND molecules. White arrows indicate local membrane disruption (both leaflets)
    Figure Legend Snippet: (A) Predicted structural organization of Tg REMIND. (B ) Ribbon representation of the AlphaFold-predicted model of the F-BAR domain of Tg REMIND in a dimeric form. The position of each monomer’s N- and C-terminal ends is indicated. (C) Same view as (B) or view of the concave face of the F-BAR domain with the surface colored by electrostatic potential (red = -16.9 kTe -1 , blue = +16.9 kTe -1 ). (D) Far-UV CD spectrum of purified F-BAR REMIND (3 µM) in 20 mM Tris, pH 7.4, 120 mM NaF buffer recorded at room temperature. The percentage of α-helix, β-sheet, and other structures, derived from the CD spectra analysis and the AlphaFold-predicted model obtained using the DSSP algorithm, is given. MRE: mean residue ellipticity, H: α-helix, E: β-sheet, O: other structures. ( E ) Flotation assay. F-BAR REMIND (0.75 µM) was incubated with liposomes (750 µM total lipids) only made of DOPC and additionally containing 30% DOPS or 10% PIPs (PI(3)P, PI(4)P or PI(4,5)P 2 at the expense of DOPC), extruded through pores of defined size, indicated at the top of the gel. This incubation was performed for 1 h at 25 °C in 50 mM Tris, pH 7.4, 150 mM NaCl (TN) buffer under constant agitation. After centrifugation, the liposomes were recovered at the top of sucrose cushions and analyzed by SDS-PAGE. The amount of membrane-bound F-BAR was determined using the content of the first lane (100% total) as a reference based on the SYPRO Orange signal. The percentage of membrane-bound F-BAR REMIND is represented as the function of the average hydrodynamic radius (R H ) of liposomes. Data are expressed as mean ± s.e.m. (n = 3-5). (F) Flotation assay. N-BAR domain of human amphiphysin (N-BAR Amph , 0.75 µM) was incubated with liposomes (750 µM lipids), only made of DOPC and additionally containing 30% DOPS or 10% PI(4,5)P 2 , extruded through pores of defined size, for 1 h at 25 °C in 50 mM Tris, pH 7.4, 150 mM NaCl (TN) buffer. The percentage of membrane-bound N-BAR Amph is shown as the function of the radius of liposomes (n = 3). ( G ) Negative-staining EM. Liposomes made of Folch fraction I lipids (30 µM lipids) and extruded through 0.4 µm pores were incubated with F-BAR REMIND or N-BAR Amph (1.9 µM) for 2 h at room temperature. A control picture of liposomes alone is shown. Scale bar = 100 nm. ( H ) Length distributions of membrane tubules induced by F-BAR REMIND and N-BAR Amph (n =105 and 26, respectively). (I) Diameter distribution of tubules and N-BAR Amph (n =105 and 26, respectively). The structure of the F-BAR domain of Tg REMIND seems adapted to the diameter of tubules that have been experimentally measured. (J) Cryo-EM. Folch fraction liposomes (90 µM lipids), extruded through 0.4 µm pores, were mixed with F-BAR REMIND (6 µM) at P/L= 1/15 and dialyzed three times under constant stirring in TN buffer for 30 min at cold temperature. Enlargements: (1) Outer leaflet destabilization (white arrowhead) by F-BAR REMIND , (2) Intact bilayer, (3) Outer leaflet destabilization (white arrowhead) by F-BAR REMIND . Green arrows point to some individual F-BAR REMIND molecules. White arrows indicate local membrane disruption (both leaflets)

    Techniques Used: Purification, Derivative Assay, Circular Dichroism, Residue, Incubation, Liposomes, Centrifugation, SDS Page, Membrane, Negative Staining, Cryo-EM Sample Prep, Disruption

    (A) Localisation of arginine and lysine residues that constitute a basic cluster in the concave face of F-BAR REMIND (in blue) and localization of solvent-exposed hydrophobic residues in the lateral side of this domain (in orange). These residues were substituted by anionic residues (aspartate or glutamate) to test their contribution to the F-BAR REMIND /membrane interaction. (B) Electrostatic features of the molecular surface of F-BAR REMIND and its mutated forms (red = -16.9 kTe -1 , blue = +16.9 kTe -1 ). (C) Flotation assay. F-BAR REMIND or K210D/R214E, R214E/R217E or R217E/R250E mutant (0.75 µM) was incubated with liposomes composed of DOPC/PI(4,5)P 2 (90:10, 750 µM lipids), with a defined radius, for 1 h at 25 °C. Data are represented as mean ± s.e.m. (n = 3-5). (D) Flotation assay. F-BAR REMIND or L201D/M212E mutant (0.75 µM) was incubated with liposomes (750 µM) composed of DOPC or DOPC/PI(4,5)P 2 (90:10), with a defined radius, for 1 h at 25 °C. Mean ± s.e.m. (n = 3-4). (E) Negative-staining EM. Liposomes made of Folch fraction I lipids (30 µM) were incubated with F-BAR REMIND or its mutated form (1.9 µM) for 2 h at room temperature (scale bar = 100 nm). A control picture of liposomes only is shown
    Figure Legend Snippet: (A) Localisation of arginine and lysine residues that constitute a basic cluster in the concave face of F-BAR REMIND (in blue) and localization of solvent-exposed hydrophobic residues in the lateral side of this domain (in orange). These residues were substituted by anionic residues (aspartate or glutamate) to test their contribution to the F-BAR REMIND /membrane interaction. (B) Electrostatic features of the molecular surface of F-BAR REMIND and its mutated forms (red = -16.9 kTe -1 , blue = +16.9 kTe -1 ). (C) Flotation assay. F-BAR REMIND or K210D/R214E, R214E/R217E or R217E/R250E mutant (0.75 µM) was incubated with liposomes composed of DOPC/PI(4,5)P 2 (90:10, 750 µM lipids), with a defined radius, for 1 h at 25 °C. Data are represented as mean ± s.e.m. (n = 3-5). (D) Flotation assay. F-BAR REMIND or L201D/M212E mutant (0.75 µM) was incubated with liposomes (750 µM) composed of DOPC or DOPC/PI(4,5)P 2 (90:10), with a defined radius, for 1 h at 25 °C. Mean ± s.e.m. (n = 3-4). (E) Negative-staining EM. Liposomes made of Folch fraction I lipids (30 µM) were incubated with F-BAR REMIND or its mutated form (1.9 µM) for 2 h at room temperature (scale bar = 100 nm). A control picture of liposomes only is shown

    Techniques Used: Solvent, Membrane, Mutagenesis, Incubation, Liposomes, Negative Staining

    ( A ) Ribbon representation of the three-dimensional model of the REMIND domain (region 500-836 of Tg REMIND) established by AlphaFold (in grey) and of the final conformation of this domain after 1 µs-MD simulation in water (in blue). The N- and C-terminal ends of the domain are indicated. ( B ) RMSD of the Cα atoms with respect to the starting and equilibrated structure of the REMIND domain as a function of time. (C) RMSF values of atomic positions of Cα atoms, indicative of the internal protein motions, are shown as a function of residue number. The localization of α-helix and β-sheet along the sequence is indicated. (D) Predicted and experimental CD spectra of the REMIND domain. Spectra were predicted from configurations of the REMIND domain collected every 10 ns during the MD simulation (grey spectra) using the PDBMD2CD algorithm. An average spectrum is represented in black. For comparison, the far-UV CD spectrum of purified Tg REMIND[495-840] construct (REMIND, 2 µM) in 20 mM Tris, pH 7.4, 120 mM NaF buffer at room temperature is shown (in red). (E) The intrinsic fluorescence of REMIND (1µM) was measured in TN buffer at 30 °C. A spectrum was measured with free L-tryptophane (4 µM) as a comparison (F) Flotation assay. REMIND (0.75 µM) was incubated with liposomes (750 µM lipids) of different mean radii, composed of DOPC or DOPC/PI(4,5)P 2 (90:10) for 1 h at 25 °C under agitation. Data are represented as mean ± s.e.m (n = 3-4). (G) Negative staining-EM images of liposomes (30 µM lipids), composed of Folch fraction I lipids, mixed or not with REMIND (1.9 µM). Scale bar = 500 nm.
    Figure Legend Snippet: ( A ) Ribbon representation of the three-dimensional model of the REMIND domain (region 500-836 of Tg REMIND) established by AlphaFold (in grey) and of the final conformation of this domain after 1 µs-MD simulation in water (in blue). The N- and C-terminal ends of the domain are indicated. ( B ) RMSD of the Cα atoms with respect to the starting and equilibrated structure of the REMIND domain as a function of time. (C) RMSF values of atomic positions of Cα atoms, indicative of the internal protein motions, are shown as a function of residue number. The localization of α-helix and β-sheet along the sequence is indicated. (D) Predicted and experimental CD spectra of the REMIND domain. Spectra were predicted from configurations of the REMIND domain collected every 10 ns during the MD simulation (grey spectra) using the PDBMD2CD algorithm. An average spectrum is represented in black. For comparison, the far-UV CD spectrum of purified Tg REMIND[495-840] construct (REMIND, 2 µM) in 20 mM Tris, pH 7.4, 120 mM NaF buffer at room temperature is shown (in red). (E) The intrinsic fluorescence of REMIND (1µM) was measured in TN buffer at 30 °C. A spectrum was measured with free L-tryptophane (4 µM) as a comparison (F) Flotation assay. REMIND (0.75 µM) was incubated with liposomes (750 µM lipids) of different mean radii, composed of DOPC or DOPC/PI(4,5)P 2 (90:10) for 1 h at 25 °C under agitation. Data are represented as mean ± s.e.m (n = 3-4). (G) Negative staining-EM images of liposomes (30 µM lipids), composed of Folch fraction I lipids, mixed or not with REMIND (1.9 µM). Scale bar = 500 nm.

    Techniques Used: Residue, Sequencing, Circular Dichroism, Comparison, Purification, Construct, Fluorescence, Incubation, Liposomes, Negative Staining

    (A) Flotation assays. Tg REMIND (0.75 µM) was incubated with liposomes (750 µM lipids) of different sizes, composed of DOPC/PI(4,5)P 2 (90:10) for 1 h at 25 °C under agitation. Data are represented as mean ± s.e.m. (n = 3). (B) F-BAR REMIND was mixed alone or together with a stoichiometric amount of REMIND with liposomes of different sizes, composed of DOPC only or DOPC/PI(4,5)P 2 (90:10) for 1 h at 25 °C under agitation. Mean ± s.e.m. (n = 3-5). (C) Negative staining EM. Representative images of Folch fraction liposomes incubated with F-BAR REMIND alone or mixed with REMIND. A large view shows that no tubule emanates from liposomes when the REMIND domain is present. (D) A three- dimensional model of the full-length Tg REMIND established by AlphaFold shows the association between the REMIND domain and the tip of the F-BAR domain. Only one monomer is shown. (E) Close-up view of F-BAR binding site predicted at the surface of the REMIND domain showing the degree of amino acid conservation based on 37 distinct sequences from diverse Apicomplexan species. Residues that are highly conserved and/or able to form one or more hydrogen bonds with residues of the BAR domain are indicated (a star indicates whether a residue forms hydrogen bond(s)). (F) A heat map based on a proximity matrix shows that the 214-258 region of Tg REMIND (the extremity of the F-BAR domain) is closely associated with different residues of the REMIND domain. The average distance between two residues was calculated based on conformations observed in one 250-ns MD trajectory. Values higher than 2 nanometers are not shown. (G) The average number of H-bonds per configuration between two given residues belonging to the BAR domain and the REMIND domain was calculated from three independent 250-ns MD trajectories (green bars). The values obtained independently from each trajectory are also shown (black dots).
    Figure Legend Snippet: (A) Flotation assays. Tg REMIND (0.75 µM) was incubated with liposomes (750 µM lipids) of different sizes, composed of DOPC/PI(4,5)P 2 (90:10) for 1 h at 25 °C under agitation. Data are represented as mean ± s.e.m. (n = 3). (B) F-BAR REMIND was mixed alone or together with a stoichiometric amount of REMIND with liposomes of different sizes, composed of DOPC only or DOPC/PI(4,5)P 2 (90:10) for 1 h at 25 °C under agitation. Mean ± s.e.m. (n = 3-5). (C) Negative staining EM. Representative images of Folch fraction liposomes incubated with F-BAR REMIND alone or mixed with REMIND. A large view shows that no tubule emanates from liposomes when the REMIND domain is present. (D) A three- dimensional model of the full-length Tg REMIND established by AlphaFold shows the association between the REMIND domain and the tip of the F-BAR domain. Only one monomer is shown. (E) Close-up view of F-BAR binding site predicted at the surface of the REMIND domain showing the degree of amino acid conservation based on 37 distinct sequences from diverse Apicomplexan species. Residues that are highly conserved and/or able to form one or more hydrogen bonds with residues of the BAR domain are indicated (a star indicates whether a residue forms hydrogen bond(s)). (F) A heat map based on a proximity matrix shows that the 214-258 region of Tg REMIND (the extremity of the F-BAR domain) is closely associated with different residues of the REMIND domain. The average distance between two residues was calculated based on conformations observed in one 250-ns MD trajectory. Values higher than 2 nanometers are not shown. (G) The average number of H-bonds per configuration between two given residues belonging to the BAR domain and the REMIND domain was calculated from three independent 250-ns MD trajectories (green bars). The values obtained independently from each trajectory are also shown (black dots).

    Techniques Used: Incubation, Liposomes, Negative Staining, Binding Assay, Residue

    (A) Structural organization of Tg BAR2 and AlphaFold-predicted model of its BAR domain in a dimeric form. Its intrinsic curvature seems adapted to the recognition of 22 nm diameter tubules. The position of the N- and C-terminal ends of each monomer is shown. The structure is represented in ribbon mode. (B) Electrostatic potential of the dimeric BAR domain red (red = -16.9 kTe -1 , blue = +16.9 kTe -1 ). (C) Flotation assays. Tg BAR2 (0.75 µM) was incubated with liposomes of different radii (750 µM lipids), only made of DOPC or additionally containing 30% DOPS or 10% PI(4,5)P 2 , in TN buffer for 1 h at 25 °C under agitation. The percentage of membrane-bound Tg BAR2 is represented as the function of the average hydrodynamic radius (R H ) of liposomes. Data corresponds to mean ± s.e.m. (n = 3). (D) Flotation assays. Membrane-bound fraction of Tg BAR2 or F-BAR REMIND (0.75 µM) incubated with liposomes extruded through 0.1 µm pores (750 µM lipids), only made of DOPC or additionally containing 30% DOPS and 5% PI(4,5)P 2 . Data corresponds to mean ± s.e.m. (n = 3). (E) Negative- staining EM. Liposomes made of Folch fraction I lipids (30 µM) and extruded through 0.4 µm pores were incubated with Tg BAR2 (1.9 µM). Control experiments were conducted with liposomes only. Representative pictures are shown. Scale bar = 200 nm. (F) Diameter and length distribution of narrow and broader membrane tubules induced by Tg BAR2 (narrow tubules, n = 50; wide tubules, n = 112). The indicated values correspond to mean ± s.e.m. (G) Cryo-EM. Liposomes made of Folch fraction I lipids (150 µM lipids) and extruded through 0.4 µm pores were mixed with Tg BAR2 (5 µM) at P/L= 1/30 and dialyzed three times under agitation in TN buffer for 30 min at cold temperature. A control picture showing liposomes without Tg BAR2 is shown (1). Tg BAR2 can transform liposomes into tubular micelles ( G, picture 2 and 2’ black arrow) and bilayered tubules ( G , picture 3 and 3’, white arrow). Tg BAR2 coats the membrane surfaces and can form transmembrane densities (yellow arrows). (G) Lower panels are general interpretations of membrane destabilization phenomena by Tg BAR2; left: tubular micelles; right: bilayered tubules (H) Diameter distribution of membrane tubules with average values observed by cryo-EM. The indicated value corresponds to mean ± s.e.m. (tubular micelle, n = 16; bilayered tubules, n = 17).
    Figure Legend Snippet: (A) Structural organization of Tg BAR2 and AlphaFold-predicted model of its BAR domain in a dimeric form. Its intrinsic curvature seems adapted to the recognition of 22 nm diameter tubules. The position of the N- and C-terminal ends of each monomer is shown. The structure is represented in ribbon mode. (B) Electrostatic potential of the dimeric BAR domain red (red = -16.9 kTe -1 , blue = +16.9 kTe -1 ). (C) Flotation assays. Tg BAR2 (0.75 µM) was incubated with liposomes of different radii (750 µM lipids), only made of DOPC or additionally containing 30% DOPS or 10% PI(4,5)P 2 , in TN buffer for 1 h at 25 °C under agitation. The percentage of membrane-bound Tg BAR2 is represented as the function of the average hydrodynamic radius (R H ) of liposomes. Data corresponds to mean ± s.e.m. (n = 3). (D) Flotation assays. Membrane-bound fraction of Tg BAR2 or F-BAR REMIND (0.75 µM) incubated with liposomes extruded through 0.1 µm pores (750 µM lipids), only made of DOPC or additionally containing 30% DOPS and 5% PI(4,5)P 2 . Data corresponds to mean ± s.e.m. (n = 3). (E) Negative- staining EM. Liposomes made of Folch fraction I lipids (30 µM) and extruded through 0.4 µm pores were incubated with Tg BAR2 (1.9 µM). Control experiments were conducted with liposomes only. Representative pictures are shown. Scale bar = 200 nm. (F) Diameter and length distribution of narrow and broader membrane tubules induced by Tg BAR2 (narrow tubules, n = 50; wide tubules, n = 112). The indicated values correspond to mean ± s.e.m. (G) Cryo-EM. Liposomes made of Folch fraction I lipids (150 µM lipids) and extruded through 0.4 µm pores were mixed with Tg BAR2 (5 µM) at P/L= 1/30 and dialyzed three times under agitation in TN buffer for 30 min at cold temperature. A control picture showing liposomes without Tg BAR2 is shown (1). Tg BAR2 can transform liposomes into tubular micelles ( G, picture 2 and 2’ black arrow) and bilayered tubules ( G , picture 3 and 3’, white arrow). Tg BAR2 coats the membrane surfaces and can form transmembrane densities (yellow arrows). (G) Lower panels are general interpretations of membrane destabilization phenomena by Tg BAR2; left: tubular micelles; right: bilayered tubules (H) Diameter distribution of membrane tubules with average values observed by cryo-EM. The indicated value corresponds to mean ± s.e.m. (tubular micelle, n = 16; bilayered tubules, n = 17).

    Techniques Used: Incubation, Liposomes, Membrane, Negative Staining, Cryo-EM Sample Prep

    anti pi 4 5 p 2  (Echelon Biosciences)


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    Structured Review

    Echelon Biosciences anti pi 4 5 p 2
    A IC 50 values of phenothiazine compounds in the indicated cell lines after 48 h of treatment. B Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. C IC 50 of phenothiazines 48 h after drug treatment. D Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. E Western blot analysis of empty vector (EV) and PTEN overexpression U87-MG cells treated with either vehicle or 10 µM perphenazine for 24 h. F Western blot analysis of T98G and LN18 cells treated with either vehicle or 10 µM perphenazine for 48 h. G Western blot analysis of Akt and RPS6 activation at 4 and 24 h after treatment with 10 µM perphenazine. H , I Images showing PI(3,4,5)P 3 and PI(4,5)P 2 levels in U87-MG-PTEN and U87-MG cells treated with either vehicle or 10 µM perphenazine for 48 h. J Kaplan-Meier survival curves of mice intracranially implanted with 100,000 U87-MG-PTEN cells and treated with either vehicle or 10 mg/kg perphenazine. A total of ten treatments were administered starting on day 8 post-implantation. Treatments were given for five consecutive days followed by a 2-day break for 2 weeks.
    Anti Pi 4 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Antipsychotics possess anti-glioblastoma activity by disrupting lysosomal function and inhibiting oncogenic signaling by stabilizing PTEN"

    Article Title: Antipsychotics possess anti-glioblastoma activity by disrupting lysosomal function and inhibiting oncogenic signaling by stabilizing PTEN

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-024-06779-3

    A IC 50 values of phenothiazine compounds in the indicated cell lines after 48 h of treatment. B Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. C IC 50 of phenothiazines 48 h after drug treatment. D Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. E Western blot analysis of empty vector (EV) and PTEN overexpression U87-MG cells treated with either vehicle or 10 µM perphenazine for 24 h. F Western blot analysis of T98G and LN18 cells treated with either vehicle or 10 µM perphenazine for 48 h. G Western blot analysis of Akt and RPS6 activation at 4 and 24 h after treatment with 10 µM perphenazine. H , I Images showing PI(3,4,5)P 3 and PI(4,5)P 2 levels in U87-MG-PTEN and U87-MG cells treated with either vehicle or 10 µM perphenazine for 48 h. J Kaplan-Meier survival curves of mice intracranially implanted with 100,000 U87-MG-PTEN cells and treated with either vehicle or 10 mg/kg perphenazine. A total of ten treatments were administered starting on day 8 post-implantation. Treatments were given for five consecutive days followed by a 2-day break for 2 weeks.
    Figure Legend Snippet: A IC 50 values of phenothiazine compounds in the indicated cell lines after 48 h of treatment. B Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. C IC 50 of phenothiazines 48 h after drug treatment. D Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. E Western blot analysis of empty vector (EV) and PTEN overexpression U87-MG cells treated with either vehicle or 10 µM perphenazine for 24 h. F Western blot analysis of T98G and LN18 cells treated with either vehicle or 10 µM perphenazine for 48 h. G Western blot analysis of Akt and RPS6 activation at 4 and 24 h after treatment with 10 µM perphenazine. H , I Images showing PI(3,4,5)P 3 and PI(4,5)P 2 levels in U87-MG-PTEN and U87-MG cells treated with either vehicle or 10 µM perphenazine for 48 h. J Kaplan-Meier survival curves of mice intracranially implanted with 100,000 U87-MG-PTEN cells and treated with either vehicle or 10 mg/kg perphenazine. A total of ten treatments were administered starting on day 8 post-implantation. Treatments were given for five consecutive days followed by a 2-day break for 2 weeks.

    Techniques Used: Western Blot, Plasmid Preparation, Over Expression, Activation Assay

    pi 4 5 p 2 biosensor  (Thermo Fisher)


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    Thermo Fisher pi 4 5 p 2 biosensor
    Phosphoinositide staining following inhibition of lipid kinases. (a) HeLa cells were treated with a kinase inhibitor cocktail of Wortmannin, PI-273, GSK-A1, PI4KIIIβ-IN 10, and Apilimod, each at 500 nM for 30 min before being fixed and stained using recombinant biosensors against PI, PI(4)P and PI(3)P conjugated to Alexa488, 546, or 647, respectively. Linescan shows the signal of PI on the membrane of large vacuoles that can be found throughout the cell. (b) HeLa cells were treated with either GSK-A1, PI-273, or PI4KIIIβ-IN 10 at 100 nM each for 30 min before being fixed and stained against PI(4)P using recombinant biosensors against PI(4)P conjugated to Alexa488. (c) HeLa cells were treated with LY 294022 at 1 µM for 30 min, stimulated with 5 ng/ml human EGF for 10 min, and fixed and stained using recombinant biosensors against PI(4,5)P 2 , PI(3,4)P 2 , and PI(3,4,5)P 3 conjugated to Alexa488, 546, or 647, respectively. Linescans show signals of PI(4,5)P 2 , PI(3,4)P 2 , and PI(3,4,5)P 3 in DMSO and LY294002 treated cells at membrane ruffles as well as the cytosolic background.
    Pi 4 5 P 2 Biosensor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides"

    Article Title: Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.202310095

    Phosphoinositide staining following inhibition of lipid kinases. (a) HeLa cells were treated with a kinase inhibitor cocktail of Wortmannin, PI-273, GSK-A1, PI4KIIIβ-IN 10, and Apilimod, each at 500 nM for 30 min before being fixed and stained using recombinant biosensors against PI, PI(4)P and PI(3)P conjugated to Alexa488, 546, or 647, respectively. Linescan shows the signal of PI on the membrane of large vacuoles that can be found throughout the cell. (b) HeLa cells were treated with either GSK-A1, PI-273, or PI4KIIIβ-IN 10 at 100 nM each for 30 min before being fixed and stained against PI(4)P using recombinant biosensors against PI(4)P conjugated to Alexa488. (c) HeLa cells were treated with LY 294022 at 1 µM for 30 min, stimulated with 5 ng/ml human EGF for 10 min, and fixed and stained using recombinant biosensors against PI(4,5)P 2 , PI(3,4)P 2 , and PI(3,4,5)P 3 conjugated to Alexa488, 546, or 647, respectively. Linescans show signals of PI(4,5)P 2 , PI(3,4)P 2 , and PI(3,4,5)P 3 in DMSO and LY294002 treated cells at membrane ruffles as well as the cytosolic background.
    Figure Legend Snippet: Phosphoinositide staining following inhibition of lipid kinases. (a) HeLa cells were treated with a kinase inhibitor cocktail of Wortmannin, PI-273, GSK-A1, PI4KIIIβ-IN 10, and Apilimod, each at 500 nM for 30 min before being fixed and stained using recombinant biosensors against PI, PI(4)P and PI(3)P conjugated to Alexa488, 546, or 647, respectively. Linescan shows the signal of PI on the membrane of large vacuoles that can be found throughout the cell. (b) HeLa cells were treated with either GSK-A1, PI-273, or PI4KIIIβ-IN 10 at 100 nM each for 30 min before being fixed and stained against PI(4)P using recombinant biosensors against PI(4)P conjugated to Alexa488. (c) HeLa cells were treated with LY 294022 at 1 µM for 30 min, stimulated with 5 ng/ml human EGF for 10 min, and fixed and stained using recombinant biosensors against PI(4,5)P 2 , PI(3,4)P 2 , and PI(3,4,5)P 3 conjugated to Alexa488, 546, or 647, respectively. Linescans show signals of PI(4,5)P 2 , PI(3,4)P 2 , and PI(3,4,5)P 3 in DMSO and LY294002 treated cells at membrane ruffles as well as the cytosolic background.

    Techniques Used: Staining, Inhibition, Recombinant, Membrane

    Staining using one step His purification. The recombinant biosensor against PI(4,5)P 2 (6xHis-SNAP-PLCδ1) was purified from BL21 E. coli using a HisTrap column and eluted against an increasing gradient of 250 mM Imidazole. Peak fractions were pooled and either directly labeled with SNAP-Surface Alexa488 or first incubated with recombinant PreScission Protease to cleave the 6xHis tag and used to stain HeLa cells as previously described. Source data are available for this figure:  .
    Figure Legend Snippet: Staining using one step His purification. The recombinant biosensor against PI(4,5)P 2 (6xHis-SNAP-PLCδ1) was purified from BL21 E. coli using a HisTrap column and eluted against an increasing gradient of 250 mM Imidazole. Peak fractions were pooled and either directly labeled with SNAP-Surface Alexa488 or first incubated with recombinant PreScission Protease to cleave the 6xHis tag and used to stain HeLa cells as previously described. Source data are available for this figure: .

    Techniques Used: Staining, Purification, Recombinant, Labeling, Incubation

    Multiplex staining of phosphoinositides across scales. (a) HeLa cells were fixed, permeabilized, and stained using recombinant biosensors against PI(3)P, PI(4)P, and PI(4,5)P 2 , conjugated respectively to Alexa488, 546, and 647. (b) NMuMG spheroids were grown in Matrigel and stained with the same combination. (c) Drosophila pupal wings were dissected and stained with the PI(4,5)P 2 and PI(4)P biosensors conjugated to Alexa647 and 546, together with Phalloidin conjugated to Alexa488, to visualize the actin cytoskeleton and cellular junctions.
    Figure Legend Snippet: Multiplex staining of phosphoinositides across scales. (a) HeLa cells were fixed, permeabilized, and stained using recombinant biosensors against PI(3)P, PI(4)P, and PI(4,5)P 2 , conjugated respectively to Alexa488, 546, and 647. (b) NMuMG spheroids were grown in Matrigel and stained with the same combination. (c) Drosophila pupal wings were dissected and stained with the PI(4,5)P 2 and PI(4)P biosensors conjugated to Alexa647 and 546, together with Phalloidin conjugated to Alexa488, to visualize the actin cytoskeleton and cellular junctions.

    Techniques Used: Multiplex Assay, Staining, Recombinant

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    Croda International Plc pi 4 5 p 2
    (A-C) Cells transiently expressing the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81) along with low levels of mEGFP-tagged 2xBtk(2-166) (A), 2xBtk(2-166)-R28C (B), or Btk(2-166) (C) were imaged at the bottom surfaces by TIRF microscopy. Left: Representative images of the mEGFP-tagged Btk sensor and the PI(4,5)P 2 sensor (inserts). Right: The intensity profile heatmap of the mEGFP-tagged Btk sensor around the cell periphery from multiple cells (n = 60 cells each). (D) Box plots showing the standard deviation of mEGFP-tagged sensor distribution at the plasma membrane (box: median with the 25th and 75th percentiles; whiskers: 1.5-fold the interquartile range; n = 62, 62, 63, and 60 cells). (E) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81) were imaged at 10-s intervals for 30 min. Representative montage shows PI(3,4,5)P 3 and PI(4,5)P 2 sensors at the indicated times. The fluorescence intensity profile of the PI(3,4,5)P 3 sensor around the cell periphery over time is shown in the right panel. (F) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and mCherry-FAK were imaged at the bottom surfaces at 10-s intervals by TIRF microscopy. Left: Images of PI(3,4,5)P 3 sensor and mCherry-FAK from a single frame of a representative time series. Middle: Images showing FA, Non-FA, and Random regions of the cells. Right: Box plots showing the mean fluorescence intensity of the PI(3,4,5)P 3 sensor on each FA, Non-FA, or Random regions; scatter plots showing the relationship between the fluorescence intensity of the PI(3,4,5)P 3 sensor and the size/intensity of each FA (red lines represent the average intensity within the indicated area ranges). (G) The montage shows selected frames of the boxed regions in (F) (FAs detected are plotted at the bottom). Kymographs were generated along the white line (on the 10-min frame) for the time series. The plots below the kymographs show the relative intensity profile along the line on the single frame. Top right: FAs detected in the time series are overlaid, with each frame represented by a different color. Bottom right: Plots showing the relative intensity of the PI(3,4,5)P 3 sensor and mCherry-FAK on FAs over time. (H) Top: Images of a cell transiently expressing the PI(3,4,5)P 3 sensor Halo-2xBtk(2-166) (labeled with JFX 650 -HaloTag ligand), mEGFP-2xBtk(2-166), and mCherry-FAK. Bottom: Images of a cell transiently expressing the PI(4,5)P 2 sensor Halo-PH(PLC81) (labeled with JFX 650 -HaloTag ligand), the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166), and mCherry-FAK. (I) Top: Images of the maximum-intensity projection of a representative time series. Bottom: Scatter plots showing the relationship between the intensity of the PI(3,4,5)P 3 sensor and the integrated number of p110α-mNeonGreen molecules (left) or the area/intensity of mCherry-FAK (right) on each FA in the time series. (J) Dual gene-edited mEGFP-p85α +/+ and EGFR-Halo (pool, labeled with JFX 650 -HaloTag ligand) cells transiently expressing mCherry-FAK were imaged at 10-s intervals, with EGF added (set as 0 s) during continuous imaging. Representative images show the recruitment of mEGFP-p85α to the plasma membrane and colocalization with EGFR (arrows) after EGF treatment. (K) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and Halo-FAK (labeled with JFX 650 -HaloTag ligand) were imaged at 10-s intervals with EGF added (set as 0 s) during continuous imaging. Images from a representative time series at 0 s and 120 s after EGF treatment are shown. Kymographs were generated along the white line for the time series. Plots show the relative intensity of the PI(3,4,5)P 3 sensor at FA and Non-FA regions, as well as Halo-FAK at FAs over time. Cells were imaged at the bottom surface by TIRF microscopy in (A-K). Statistical analysis in (D) and (F) was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons test; ** P < 0.001; **** P < 0.0001. Scale bars, 10 μm.
    Pi 4 5 P 2, supplied by Croda International Plc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences pi 4 5 p 2 dic8 echelon
    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.
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    Thermo Fisher pi 4 5 p 2
    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.
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    Echelon Biosciences pi 4 5 p 2
    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.
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    Croda International Plc l α pi 4 5 p 2
    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.
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    Millipore l α pi 4 5 p 2
    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.
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    Croda International Plc brain pi 4 5 p 2 l α phosphatidylinositol 4 5 bisphosphate
    (A) Predicted structural organization of Tg REMIND. (B ) Ribbon representation of the AlphaFold-predicted model of the F-BAR domain of Tg REMIND in a dimeric form. The position of each monomer’s N- and C-terminal ends is indicated. (C) Same view as (B) or view of the concave face of the F-BAR domain with the surface colored by electrostatic potential (red = -16.9 kTe -1 , blue = +16.9 kTe -1 ). (D) Far-UV CD spectrum of purified F-BAR REMIND (3 µM) in 20 mM Tris, pH 7.4, 120 mM NaF buffer recorded at room temperature. The percentage of α-helix, β-sheet, and other structures, derived from the CD spectra analysis and the AlphaFold-predicted model obtained using the DSSP algorithm, is given. MRE: mean residue ellipticity, H: α-helix, E: β-sheet, O: other structures. ( E ) Flotation assay. F-BAR REMIND (0.75 µM) was incubated with liposomes (750 µM total lipids) only made of DOPC and additionally containing 30% DOPS or 10% PIPs (PI(3)P, PI(4)P or <t>PI(4,5)P</t> 2 at the expense of DOPC), extruded through pores of defined size, indicated at the top of the gel. This incubation was performed for 1 h at 25 °C in 50 mM Tris, pH 7.4, 150 mM NaCl (TN) buffer under constant agitation. After centrifugation, the liposomes were recovered at the top of sucrose cushions and analyzed by SDS-PAGE. The amount of membrane-bound F-BAR was determined using the content of the first lane (100% total) as a reference based on the SYPRO Orange signal. The percentage of membrane-bound F-BAR REMIND is represented as the function of the average hydrodynamic radius (R H ) of liposomes. Data are expressed as mean ± s.e.m. (n = 3-5). (F) Flotation assay. N-BAR domain of human amphiphysin (N-BAR Amph , 0.75 µM) was incubated with liposomes (750 µM lipids), only made of DOPC and additionally containing 30% DOPS or 10% PI(4,5)P 2 , extruded through pores of defined size, for 1 h at 25 °C in 50 mM Tris, pH 7.4, 150 mM NaCl (TN) buffer. The percentage of membrane-bound N-BAR Amph is shown as the function of the radius of liposomes (n = 3). ( G ) Negative-staining EM. Liposomes made of Folch fraction I lipids (30 µM lipids) and extruded through 0.4 µm pores were incubated with F-BAR REMIND or N-BAR Amph (1.9 µM) for 2 h at room temperature. A control picture of liposomes alone is shown. Scale bar = 100 nm. ( H ) Length distributions of membrane tubules induced by F-BAR REMIND and N-BAR Amph (n =105 and 26, respectively). (I) Diameter distribution of tubules and N-BAR Amph (n =105 and 26, respectively). The structure of the F-BAR domain of Tg REMIND seems adapted to the diameter of tubules that have been experimentally measured. (J) Cryo-EM. Folch fraction liposomes (90 µM lipids), extruded through 0.4 µm pores, were mixed with F-BAR REMIND (6 µM) at P/L= 1/15 and dialyzed three times under constant stirring in TN buffer for 30 min at cold temperature. Enlargements: (1) Outer leaflet destabilization (white arrowhead) by F-BAR REMIND , (2) Intact bilayer, (3) Outer leaflet destabilization (white arrowhead) by F-BAR REMIND . Green arrows point to some individual F-BAR REMIND molecules. White arrows indicate local membrane disruption (both leaflets)
    Brain Pi 4 5 P 2 L α Phosphatidylinositol 4 5 Bisphosphate, supplied by Croda International Plc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences anti pi 4 5 p 2
    A IC 50 values of phenothiazine compounds in the indicated cell lines after 48 h of treatment. B Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. C IC 50 of phenothiazines 48 h after drug treatment. D Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. E Western blot analysis of empty vector (EV) and PTEN overexpression U87-MG cells treated with either vehicle or 10 µM perphenazine for 24 h. F Western blot analysis of T98G and LN18 cells treated with either vehicle or 10 µM perphenazine for 48 h. G Western blot analysis of Akt and RPS6 activation at 4 and 24 h after treatment with 10 µM perphenazine. H , I Images showing PI(3,4,5)P 3 and PI(4,5)P 2 levels in U87-MG-PTEN and U87-MG cells treated with either vehicle or 10 µM perphenazine for 48 h. J Kaplan-Meier survival curves of mice intracranially implanted with 100,000 U87-MG-PTEN cells and treated with either vehicle or 10 mg/kg perphenazine. A total of ten treatments were administered starting on day 8 post-implantation. Treatments were given for five consecutive days followed by a 2-day break for 2 weeks.
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    Thermo Fisher pi 4 5 p 2 biosensor
    Phosphoinositide staining following inhibition of lipid kinases. (a) HeLa cells were treated with a kinase inhibitor cocktail of Wortmannin, PI-273, GSK-A1, PI4KIIIβ-IN 10, and Apilimod, each at 500 nM for 30 min before being fixed and stained using recombinant biosensors against PI, PI(4)P and PI(3)P conjugated to Alexa488, 546, or 647, respectively. Linescan shows the signal of PI on the membrane of large vacuoles that can be found throughout the cell. (b) HeLa cells were treated with either GSK-A1, PI-273, or PI4KIIIβ-IN 10 at 100 nM each for 30 min before being fixed and stained against PI(4)P using recombinant biosensors against PI(4)P conjugated to Alexa488. (c) HeLa cells were treated with LY 294022 at 1 µM for 30 min, stimulated with 5 ng/ml human EGF for 10 min, and fixed and stained using recombinant biosensors against PI(4,5)P 2 , PI(3,4)P 2 , and PI(3,4,5)P 3 conjugated to Alexa488, 546, or 647, respectively. Linescans show signals of PI(4,5)P 2 , PI(3,4)P 2 , and PI(3,4,5)P 3 in DMSO and LY294002 treated cells at membrane ruffles as well as the cytosolic background.
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    Image Search Results


    (A-C) Cells transiently expressing the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81) along with low levels of mEGFP-tagged 2xBtk(2-166) (A), 2xBtk(2-166)-R28C (B), or Btk(2-166) (C) were imaged at the bottom surfaces by TIRF microscopy. Left: Representative images of the mEGFP-tagged Btk sensor and the PI(4,5)P 2 sensor (inserts). Right: The intensity profile heatmap of the mEGFP-tagged Btk sensor around the cell periphery from multiple cells (n = 60 cells each). (D) Box plots showing the standard deviation of mEGFP-tagged sensor distribution at the plasma membrane (box: median with the 25th and 75th percentiles; whiskers: 1.5-fold the interquartile range; n = 62, 62, 63, and 60 cells). (E) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81) were imaged at 10-s intervals for 30 min. Representative montage shows PI(3,4,5)P 3 and PI(4,5)P 2 sensors at the indicated times. The fluorescence intensity profile of the PI(3,4,5)P 3 sensor around the cell periphery over time is shown in the right panel. (F) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and mCherry-FAK were imaged at the bottom surfaces at 10-s intervals by TIRF microscopy. Left: Images of PI(3,4,5)P 3 sensor and mCherry-FAK from a single frame of a representative time series. Middle: Images showing FA, Non-FA, and Random regions of the cells. Right: Box plots showing the mean fluorescence intensity of the PI(3,4,5)P 3 sensor on each FA, Non-FA, or Random regions; scatter plots showing the relationship between the fluorescence intensity of the PI(3,4,5)P 3 sensor and the size/intensity of each FA (red lines represent the average intensity within the indicated area ranges). (G) The montage shows selected frames of the boxed regions in (F) (FAs detected are plotted at the bottom). Kymographs were generated along the white line (on the 10-min frame) for the time series. The plots below the kymographs show the relative intensity profile along the line on the single frame. Top right: FAs detected in the time series are overlaid, with each frame represented by a different color. Bottom right: Plots showing the relative intensity of the PI(3,4,5)P 3 sensor and mCherry-FAK on FAs over time. (H) Top: Images of a cell transiently expressing the PI(3,4,5)P 3 sensor Halo-2xBtk(2-166) (labeled with JFX 650 -HaloTag ligand), mEGFP-2xBtk(2-166), and mCherry-FAK. Bottom: Images of a cell transiently expressing the PI(4,5)P 2 sensor Halo-PH(PLC81) (labeled with JFX 650 -HaloTag ligand), the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166), and mCherry-FAK. (I) Top: Images of the maximum-intensity projection of a representative time series. Bottom: Scatter plots showing the relationship between the intensity of the PI(3,4,5)P 3 sensor and the integrated number of p110α-mNeonGreen molecules (left) or the area/intensity of mCherry-FAK (right) on each FA in the time series. (J) Dual gene-edited mEGFP-p85α +/+ and EGFR-Halo (pool, labeled with JFX 650 -HaloTag ligand) cells transiently expressing mCherry-FAK were imaged at 10-s intervals, with EGF added (set as 0 s) during continuous imaging. Representative images show the recruitment of mEGFP-p85α to the plasma membrane and colocalization with EGFR (arrows) after EGF treatment. (K) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and Halo-FAK (labeled with JFX 650 -HaloTag ligand) were imaged at 10-s intervals with EGF added (set as 0 s) during continuous imaging. Images from a representative time series at 0 s and 120 s after EGF treatment are shown. Kymographs were generated along the white line for the time series. Plots show the relative intensity of the PI(3,4,5)P 3 sensor at FA and Non-FA regions, as well as Halo-FAK at FAs over time. Cells were imaged at the bottom surface by TIRF microscopy in (A-K). Statistical analysis in (D) and (F) was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons test; ** P < 0.001; **** P < 0.0001. Scale bars, 10 μm.

    Journal: bioRxiv

    Article Title: Spatial organization of PI3K-PI(3,4,5)P3-AKT signaling by focal adhesions

    doi: 10.1101/2024.07.05.602013

    Figure Lengend Snippet: (A-C) Cells transiently expressing the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81) along with low levels of mEGFP-tagged 2xBtk(2-166) (A), 2xBtk(2-166)-R28C (B), or Btk(2-166) (C) were imaged at the bottom surfaces by TIRF microscopy. Left: Representative images of the mEGFP-tagged Btk sensor and the PI(4,5)P 2 sensor (inserts). Right: The intensity profile heatmap of the mEGFP-tagged Btk sensor around the cell periphery from multiple cells (n = 60 cells each). (D) Box plots showing the standard deviation of mEGFP-tagged sensor distribution at the plasma membrane (box: median with the 25th and 75th percentiles; whiskers: 1.5-fold the interquartile range; n = 62, 62, 63, and 60 cells). (E) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81) were imaged at 10-s intervals for 30 min. Representative montage shows PI(3,4,5)P 3 and PI(4,5)P 2 sensors at the indicated times. The fluorescence intensity profile of the PI(3,4,5)P 3 sensor around the cell periphery over time is shown in the right panel. (F) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and mCherry-FAK were imaged at the bottom surfaces at 10-s intervals by TIRF microscopy. Left: Images of PI(3,4,5)P 3 sensor and mCherry-FAK from a single frame of a representative time series. Middle: Images showing FA, Non-FA, and Random regions of the cells. Right: Box plots showing the mean fluorescence intensity of the PI(3,4,5)P 3 sensor on each FA, Non-FA, or Random regions; scatter plots showing the relationship between the fluorescence intensity of the PI(3,4,5)P 3 sensor and the size/intensity of each FA (red lines represent the average intensity within the indicated area ranges). (G) The montage shows selected frames of the boxed regions in (F) (FAs detected are plotted at the bottom). Kymographs were generated along the white line (on the 10-min frame) for the time series. The plots below the kymographs show the relative intensity profile along the line on the single frame. Top right: FAs detected in the time series are overlaid, with each frame represented by a different color. Bottom right: Plots showing the relative intensity of the PI(3,4,5)P 3 sensor and mCherry-FAK on FAs over time. (H) Top: Images of a cell transiently expressing the PI(3,4,5)P 3 sensor Halo-2xBtk(2-166) (labeled with JFX 650 -HaloTag ligand), mEGFP-2xBtk(2-166), and mCherry-FAK. Bottom: Images of a cell transiently expressing the PI(4,5)P 2 sensor Halo-PH(PLC81) (labeled with JFX 650 -HaloTag ligand), the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166), and mCherry-FAK. (I) Top: Images of the maximum-intensity projection of a representative time series. Bottom: Scatter plots showing the relationship between the intensity of the PI(3,4,5)P 3 sensor and the integrated number of p110α-mNeonGreen molecules (left) or the area/intensity of mCherry-FAK (right) on each FA in the time series. (J) Dual gene-edited mEGFP-p85α +/+ and EGFR-Halo (pool, labeled with JFX 650 -HaloTag ligand) cells transiently expressing mCherry-FAK were imaged at 10-s intervals, with EGF added (set as 0 s) during continuous imaging. Representative images show the recruitment of mEGFP-p85α to the plasma membrane and colocalization with EGFR (arrows) after EGF treatment. (K) Cells transiently expressing the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and Halo-FAK (labeled with JFX 650 -HaloTag ligand) were imaged at 10-s intervals with EGF added (set as 0 s) during continuous imaging. Images from a representative time series at 0 s and 120 s after EGF treatment are shown. Kymographs were generated along the white line for the time series. Plots show the relative intensity of the PI(3,4,5)P 3 sensor at FA and Non-FA regions, as well as Halo-FAK at FAs over time. Cells were imaged at the bottom surface by TIRF microscopy in (A-K). Statistical analysis in (D) and (F) was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons test; ** P < 0.001; **** P < 0.0001. Scale bars, 10 μm.

    Article Snippet: Internal standard cocktail containing 17:0/20:4 PI3P, 17:0/20:4 PI4P, 17:0/20:4 PI5P, 17:0/20:4 PI(3,4)P 2 , 17:0/20:4 PI(3,5)P 2 , 17:0/20:4 PI(4,5)P 2 and 17:0/20:4 PI(3,4,5)P 3 (Avanti Polar Lipids) was added into individual samples during extraction.

    Techniques: Expressing, Microscopy, Standard Deviation, Membrane, Fluorescence, Generated, Labeling, Imaging

    (A) Sequencing results of genomic DNA from the parental SUM159 cells ( PIK3CA WT/H1047L ) and gene-edited SUM159 cells homozygous for H1047 ( PIK3CA WT/WT ) or H1047L ( PIK3CA H1047L/H1047L ) of PIK3CA . (B) PIK3CA WT/H1047L , PIK3CA WT/WT , PIK3CA H1047L/H1047L , or PTEN-KO SUM159 cells were serum-starved overnight and then subjected to western blot analysis using the indicated antibodies. The ratios of phosphorylated AKT to total AKT are shown for four independent experiments. (C) Quantification of PI(3,4,5)P 3 and PI(4,5)P 2 levels in the four cell lines by mass spectrometry. (D) PIK3CA WT/H1047L , PIK3CA WT/WT , or PIK3CA H1047L/H1047L cells were transiently transfected with the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81). Shown are representative images and intensity profile heatmaps of the PI(3,4,5)P 3 sensor in the three cell lines (n = 60 cells each). Box plots show the standard deviation of PI(3,4,5)P 3 sensor distribution at the plasma membrane (box: median with the 25th and 75th percentiles; whiskers: 1.5-fold the interquartile range; n = 69, 61, and 67 cells). (E) SUM159 cells transiently co-expressing mCherry-FAK with p110α(H1047L)-mNeonGreen were imaged at 0.2-s intervals for 301 frames. Left: Images of a single frame and the maximum-intensity projection of a representative time series are shown. Right: Kymographs generated along the line showing recruitment of p110α(H1047L) to FAs. (F) PTEN-KO SUM159 cells were transiently expressed with the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166), or mEGFP-2xBtk(2-166) together with mScarlet-I-PTEN or mScarlet-I-PTEN-G129E (inserts). Shown are representative images and intensity profile heatmaps of the PI(3,4,5)P 3 sensor (n = 60 cells each). (G) PTEN-KO SUM159 cells transiently co-expressing mCherry-FAK and p110α-mNeonGreen were imaged at 0.2-s intervals for 301 frames. Left: Images of a single frame and the maximum-intensity projection of a representative time series are shown. Right: Kymographs were generated along the line. (H) PIK3CA WT/H1047L , PIK3CA WT/WT , PIK3CA H1047L/H1047L , or PTEN-KO cells transiently expressing mNeonGreen-AKT1, mCherry-FAK and the PI(3,4,5)P 3 sensor Halo-2xBtk(2-166) were imaged at 0.3-s intervals. Images of a single frame of a representative time series are shown. (I) PIK3CA WT/H1047L , PIK3CA WT/WT , PIK3CA H1047L/H1047L , or PTEN-KO cells were treated with or without defactinib for 10 min. The total and phosphorylated AKT and FAK levels were analyzed by western blot. Statistical analysis was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons test; * P < 0.05; **** P < 0.0001. Scale bars, 10 μm.

    Journal: bioRxiv

    Article Title: Spatial organization of PI3K-PI(3,4,5)P3-AKT signaling by focal adhesions

    doi: 10.1101/2024.07.05.602013

    Figure Lengend Snippet: (A) Sequencing results of genomic DNA from the parental SUM159 cells ( PIK3CA WT/H1047L ) and gene-edited SUM159 cells homozygous for H1047 ( PIK3CA WT/WT ) or H1047L ( PIK3CA H1047L/H1047L ) of PIK3CA . (B) PIK3CA WT/H1047L , PIK3CA WT/WT , PIK3CA H1047L/H1047L , or PTEN-KO SUM159 cells were serum-starved overnight and then subjected to western blot analysis using the indicated antibodies. The ratios of phosphorylated AKT to total AKT are shown for four independent experiments. (C) Quantification of PI(3,4,5)P 3 and PI(4,5)P 2 levels in the four cell lines by mass spectrometry. (D) PIK3CA WT/H1047L , PIK3CA WT/WT , or PIK3CA H1047L/H1047L cells were transiently transfected with the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166) and the PI(4,5)P 2 sensor mScarlet-I-PH(PLC81). Shown are representative images and intensity profile heatmaps of the PI(3,4,5)P 3 sensor in the three cell lines (n = 60 cells each). Box plots show the standard deviation of PI(3,4,5)P 3 sensor distribution at the plasma membrane (box: median with the 25th and 75th percentiles; whiskers: 1.5-fold the interquartile range; n = 69, 61, and 67 cells). (E) SUM159 cells transiently co-expressing mCherry-FAK with p110α(H1047L)-mNeonGreen were imaged at 0.2-s intervals for 301 frames. Left: Images of a single frame and the maximum-intensity projection of a representative time series are shown. Right: Kymographs generated along the line showing recruitment of p110α(H1047L) to FAs. (F) PTEN-KO SUM159 cells were transiently expressed with the PI(3,4,5)P 3 sensor mEGFP-2xBtk(2-166), or mEGFP-2xBtk(2-166) together with mScarlet-I-PTEN or mScarlet-I-PTEN-G129E (inserts). Shown are representative images and intensity profile heatmaps of the PI(3,4,5)P 3 sensor (n = 60 cells each). (G) PTEN-KO SUM159 cells transiently co-expressing mCherry-FAK and p110α-mNeonGreen were imaged at 0.2-s intervals for 301 frames. Left: Images of a single frame and the maximum-intensity projection of a representative time series are shown. Right: Kymographs were generated along the line. (H) PIK3CA WT/H1047L , PIK3CA WT/WT , PIK3CA H1047L/H1047L , or PTEN-KO cells transiently expressing mNeonGreen-AKT1, mCherry-FAK and the PI(3,4,5)P 3 sensor Halo-2xBtk(2-166) were imaged at 0.3-s intervals. Images of a single frame of a representative time series are shown. (I) PIK3CA WT/H1047L , PIK3CA WT/WT , PIK3CA H1047L/H1047L , or PTEN-KO cells were treated with or without defactinib for 10 min. The total and phosphorylated AKT and FAK levels were analyzed by western blot. Statistical analysis was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons test; * P < 0.05; **** P < 0.0001. Scale bars, 10 μm.

    Article Snippet: Internal standard cocktail containing 17:0/20:4 PI3P, 17:0/20:4 PI4P, 17:0/20:4 PI5P, 17:0/20:4 PI(3,4)P 2 , 17:0/20:4 PI(3,5)P 2 , 17:0/20:4 PI(4,5)P 2 and 17:0/20:4 PI(3,4,5)P 3 (Avanti Polar Lipids) was added into individual samples during extraction.

    Techniques: Sequencing, Western Blot, Mass Spectrometry, Transfection, Standard Deviation, Membrane, Expressing, Generated

    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.

    Journal: bioRxiv

    Article Title: TRPML1 gating modulation by allosteric mutations and lipids (Design of allosteric mutations that recapitulate the gating of TRPML1)

    doi: 10.1101/2024.07.04.602033

    Figure Lengend Snippet: (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.

    Article Snippet: For PI(4,5)P 2 -bound structure, purified protein was incubated with 0.5mM PI(4,5)P 2 on ice for 4 h. The lipid ligand used in this study is PI(4,5)P 2 diC8 (Echelon)

    Techniques: Binding Assay, Comparison

    (a) Overall structure of PI(4,5)P 2 -bound TRPML1 and the zoomed-in view of the lipid-binding site. The lipid density is shown as blue surface and modeled as sphingomyelin (SM). The side chains of lipid-interacting residues are shown as yellow sticks. (b) SM inhibition effect on SF-51-activated wild-type TRPML1. (c) SM activation effect on ML-SI1-inhibited Y404W mutant. Currents shown in (b) and (c) were recorded using patch clamp in whole-cell configuration with pH 4.6 in the bath solution as the adverse effect of SM on agonist or antagonist is subtle and is measurable only at low luminal pH.

    Journal: bioRxiv

    Article Title: TRPML1 gating modulation by allosteric mutations and lipids (Design of allosteric mutations that recapitulate the gating of TRPML1)

    doi: 10.1101/2024.07.04.602033

    Figure Lengend Snippet: (a) Overall structure of PI(4,5)P 2 -bound TRPML1 and the zoomed-in view of the lipid-binding site. The lipid density is shown as blue surface and modeled as sphingomyelin (SM). The side chains of lipid-interacting residues are shown as yellow sticks. (b) SM inhibition effect on SF-51-activated wild-type TRPML1. (c) SM activation effect on ML-SI1-inhibited Y404W mutant. Currents shown in (b) and (c) were recorded using patch clamp in whole-cell configuration with pH 4.6 in the bath solution as the adverse effect of SM on agonist or antagonist is subtle and is measurable only at low luminal pH.

    Article Snippet: For PI(4,5)P 2 -bound structure, purified protein was incubated with 0.5mM PI(4,5)P 2 on ice for 4 h. The lipid ligand used in this study is PI(4,5)P 2 diC8 (Echelon)

    Techniques: Binding Assay, Inhibition, Activation Assay, Mutagenesis, Patch Clamp

    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.

    Journal: bioRxiv

    Article Title: TRPML1 gating modulation by allosteric mutations and lipids (Design of allosteric mutations that recapitulate the gating of TRPML1)

    doi: 10.1101/2024.07.04.602033

    Figure Lengend Snippet: (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.

    Article Snippet: The total exposure time was between 5 to 6 s. For the PI(4,5)P 2- bound dataset, micrographs were acquired on a Titan Krios microscope (FEI) operated at 300 kV with a Falcon4 electron detector (Thermo Fisher), using a slit width of 20 eV on a post-column Selectris X energy filter (Thermo Fisher Scientific).

    Techniques: Binding Assay, Comparison

    (a) Overall structure of PI(4,5)P 2 -bound TRPML1 and the zoomed-in view of the lipid-binding site. The lipid density is shown as blue surface and modeled as sphingomyelin (SM). The side chains of lipid-interacting residues are shown as yellow sticks. (b) SM inhibition effect on SF-51-activated wild-type TRPML1. (c) SM activation effect on ML-SI1-inhibited Y404W mutant. Currents shown in (b) and (c) were recorded using patch clamp in whole-cell configuration with pH 4.6 in the bath solution as the adverse effect of SM on agonist or antagonist is subtle and is measurable only at low luminal pH.

    Journal: bioRxiv

    Article Title: TRPML1 gating modulation by allosteric mutations and lipids (Design of allosteric mutations that recapitulate the gating of TRPML1)

    doi: 10.1101/2024.07.04.602033

    Figure Lengend Snippet: (a) Overall structure of PI(4,5)P 2 -bound TRPML1 and the zoomed-in view of the lipid-binding site. The lipid density is shown as blue surface and modeled as sphingomyelin (SM). The side chains of lipid-interacting residues are shown as yellow sticks. (b) SM inhibition effect on SF-51-activated wild-type TRPML1. (c) SM activation effect on ML-SI1-inhibited Y404W mutant. Currents shown in (b) and (c) were recorded using patch clamp in whole-cell configuration with pH 4.6 in the bath solution as the adverse effect of SM on agonist or antagonist is subtle and is measurable only at low luminal pH.

    Article Snippet: The total exposure time was between 5 to 6 s. For the PI(4,5)P 2- bound dataset, micrographs were acquired on a Titan Krios microscope (FEI) operated at 300 kV with a Falcon4 electron detector (Thermo Fisher), using a slit width of 20 eV on a post-column Selectris X energy filter (Thermo Fisher Scientific).

    Techniques: Binding Assay, Inhibition, Activation Assay, Mutagenesis, Patch Clamp

    (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.

    Journal: bioRxiv

    Article Title: TRPML1 gating modulation by allosteric mutations and lipids (Design of allosteric mutations that recapitulate the gating of TRPML1)

    doi: 10.1101/2024.07.04.602033

    Figure Lengend Snippet: (a) Overall structure of PI(4,5)P 2 - bound TRPML1 with the front subunit shown in orange cartoon and the rest shown as grey surface representation. Density for PI(4,5)P 2 head group is shown in blue surface. (b) Zoomed-in view of the PI(4,5)P 2 -binding pocket with the density of its IP3 head group shown in blue surface. (c) Zoomed-in view of the PI(4,5)P 2 -binding pocket with side chains of IP3-interacting residues shown as yellow sticks. (d) Zoomed-in view of the IP3 position in the PI(3,5)P 2 -bound open TRPML1 structure. The C3 phosphate group directly interacts with Y355 and R403. (e) Comparison of the head group positions in PI(3,5)P 2 -bound open (green) and PI(4,5)P 2 -bound closed (orange) structures. The inositol rings PI(3,5)P 2 and PI(4,5)P 2 are colored yellow and cyan, respectively. The red arrow marks the upward movement of S1 from closed to open conformation.

    Article Snippet: For PI(4,5)P 2 -bound structure, purified protein was incubated with 0.5mM PI(4,5)P 2 on ice for 4 h. The lipid ligand used in this study is PI(4,5)P 2 diC8 (Echelon)

    Techniques: Binding Assay, Comparison

    (a) Overall structure of PI(4,5)P 2 -bound TRPML1 and the zoomed-in view of the lipid-binding site. The lipid density is shown as blue surface and modeled as sphingomyelin (SM). The side chains of lipid-interacting residues are shown as yellow sticks. (b) SM inhibition effect on SF-51-activated wild-type TRPML1. (c) SM activation effect on ML-SI1-inhibited Y404W mutant. Currents shown in (b) and (c) were recorded using patch clamp in whole-cell configuration with pH 4.6 in the bath solution as the adverse effect of SM on agonist or antagonist is subtle and is measurable only at low luminal pH.

    Journal: bioRxiv

    Article Title: TRPML1 gating modulation by allosteric mutations and lipids (Design of allosteric mutations that recapitulate the gating of TRPML1)

    doi: 10.1101/2024.07.04.602033

    Figure Lengend Snippet: (a) Overall structure of PI(4,5)P 2 -bound TRPML1 and the zoomed-in view of the lipid-binding site. The lipid density is shown as blue surface and modeled as sphingomyelin (SM). The side chains of lipid-interacting residues are shown as yellow sticks. (b) SM inhibition effect on SF-51-activated wild-type TRPML1. (c) SM activation effect on ML-SI1-inhibited Y404W mutant. Currents shown in (b) and (c) were recorded using patch clamp in whole-cell configuration with pH 4.6 in the bath solution as the adverse effect of SM on agonist or antagonist is subtle and is measurable only at low luminal pH.

    Article Snippet: For PI(4,5)P 2 -bound structure, purified protein was incubated with 0.5mM PI(4,5)P 2 on ice for 4 h. The lipid ligand used in this study is PI(4,5)P 2 diC8 (Echelon)

    Techniques: Binding Assay, Inhibition, Activation Assay, Mutagenesis, Patch Clamp

    (A) Predicted structural organization of Tg REMIND. (B ) Ribbon representation of the AlphaFold-predicted model of the F-BAR domain of Tg REMIND in a dimeric form. The position of each monomer’s N- and C-terminal ends is indicated. (C) Same view as (B) or view of the concave face of the F-BAR domain with the surface colored by electrostatic potential (red = -16.9 kTe -1 , blue = +16.9 kTe -1 ). (D) Far-UV CD spectrum of purified F-BAR REMIND (3 µM) in 20 mM Tris, pH 7.4, 120 mM NaF buffer recorded at room temperature. The percentage of α-helix, β-sheet, and other structures, derived from the CD spectra analysis and the AlphaFold-predicted model obtained using the DSSP algorithm, is given. MRE: mean residue ellipticity, H: α-helix, E: β-sheet, O: other structures. ( E ) Flotation assay. F-BAR REMIND (0.75 µM) was incubated with liposomes (750 µM total lipids) only made of DOPC and additionally containing 30% DOPS or 10% PIPs (PI(3)P, PI(4)P or PI(4,5)P 2 at the expense of DOPC), extruded through pores of defined size, indicated at the top of the gel. This incubation was performed for 1 h at 25 °C in 50 mM Tris, pH 7.4, 150 mM NaCl (TN) buffer under constant agitation. After centrifugation, the liposomes were recovered at the top of sucrose cushions and analyzed by SDS-PAGE. The amount of membrane-bound F-BAR was determined using the content of the first lane (100% total) as a reference based on the SYPRO Orange signal. The percentage of membrane-bound F-BAR REMIND is represented as the function of the average hydrodynamic radius (R H ) of liposomes. Data are expressed as mean ± s.e.m. (n = 3-5). (F) Flotation assay. N-BAR domain of human amphiphysin (N-BAR Amph , 0.75 µM) was incubated with liposomes (750 µM lipids), only made of DOPC and additionally containing 30% DOPS or 10% PI(4,5)P 2 , extruded through pores of defined size, for 1 h at 25 °C in 50 mM Tris, pH 7.4, 150 mM NaCl (TN) buffer. The percentage of membrane-bound N-BAR Amph is shown as the function of the radius of liposomes (n = 3). ( G ) Negative-staining EM. Liposomes made of Folch fraction I lipids (30 µM lipids) and extruded through 0.4 µm pores were incubated with F-BAR REMIND or N-BAR Amph (1.9 µM) for 2 h at room temperature. A control picture of liposomes alone is shown. Scale bar = 100 nm. ( H ) Length distributions of membrane tubules induced by F-BAR REMIND and N-BAR Amph (n =105 and 26, respectively). (I) Diameter distribution of tubules and N-BAR Amph (n =105 and 26, respectively). The structure of the F-BAR domain of Tg REMIND seems adapted to the diameter of tubules that have been experimentally measured. (J) Cryo-EM. Folch fraction liposomes (90 µM lipids), extruded through 0.4 µm pores, were mixed with F-BAR REMIND (6 µM) at P/L= 1/15 and dialyzed three times under constant stirring in TN buffer for 30 min at cold temperature. Enlargements: (1) Outer leaflet destabilization (white arrowhead) by F-BAR REMIND , (2) Intact bilayer, (3) Outer leaflet destabilization (white arrowhead) by F-BAR REMIND . Green arrows point to some individual F-BAR REMIND molecules. White arrows indicate local membrane disruption (both leaflets)

    Journal: bioRxiv

    Article Title: Characterization of atypical BAR domain-containing proteins coded by Toxoplasma gondii

    doi: 10.1101/2024.06.13.598837

    Figure Lengend Snippet: (A) Predicted structural organization of Tg REMIND. (B ) Ribbon representation of the AlphaFold-predicted model of the F-BAR domain of Tg REMIND in a dimeric form. The position of each monomer’s N- and C-terminal ends is indicated. (C) Same view as (B) or view of the concave face of the F-BAR domain with the surface colored by electrostatic potential (red = -16.9 kTe -1 , blue = +16.9 kTe -1 ). (D) Far-UV CD spectrum of purified F-BAR REMIND (3 µM) in 20 mM Tris, pH 7.4, 120 mM NaF buffer recorded at room temperature. The percentage of α-helix, β-sheet, and other structures, derived from the CD spectra analysis and the AlphaFold-predicted model obtained using the DSSP algorithm, is given. MRE: mean residue ellipticity, H: α-helix, E: β-sheet, O: other structures. ( E ) Flotation assay. F-BAR REMIND (0.75 µM) was incubated with liposomes (750 µM total lipids) only made of DOPC and additionally containing 30% DOPS or 10% PIPs (PI(3)P, PI(4)P or PI(4,5)P 2 at the expense of DOPC), extruded through pores of defined size, indicated at the top of the gel. This incubation was performed for 1 h at 25 °C in 50 mM Tris, pH 7.4, 150 mM NaCl (TN) buffer under constant agitation. After centrifugation, the liposomes were recovered at the top of sucrose cushions and analyzed by SDS-PAGE. The amount of membrane-bound F-BAR was determined using the content of the first lane (100% total) as a reference based on the SYPRO Orange signal. The percentage of membrane-bound F-BAR REMIND is represented as the function of the average hydrodynamic radius (R H ) of liposomes. Data are expressed as mean ± s.e.m. (n = 3-5). (F) Flotation assay. N-BAR domain of human amphiphysin (N-BAR Amph , 0.75 µM) was incubated with liposomes (750 µM lipids), only made of DOPC and additionally containing 30% DOPS or 10% PI(4,5)P 2 , extruded through pores of defined size, for 1 h at 25 °C in 50 mM Tris, pH 7.4, 150 mM NaCl (TN) buffer. The percentage of membrane-bound N-BAR Amph is shown as the function of the radius of liposomes (n = 3). ( G ) Negative-staining EM. Liposomes made of Folch fraction I lipids (30 µM lipids) and extruded through 0.4 µm pores were incubated with F-BAR REMIND or N-BAR Amph (1.9 µM) for 2 h at room temperature. A control picture of liposomes alone is shown. Scale bar = 100 nm. ( H ) Length distributions of membrane tubules induced by F-BAR REMIND and N-BAR Amph (n =105 and 26, respectively). (I) Diameter distribution of tubules and N-BAR Amph (n =105 and 26, respectively). The structure of the F-BAR domain of Tg REMIND seems adapted to the diameter of tubules that have been experimentally measured. (J) Cryo-EM. Folch fraction liposomes (90 µM lipids), extruded through 0.4 µm pores, were mixed with F-BAR REMIND (6 µM) at P/L= 1/15 and dialyzed three times under constant stirring in TN buffer for 30 min at cold temperature. Enlargements: (1) Outer leaflet destabilization (white arrowhead) by F-BAR REMIND , (2) Intact bilayer, (3) Outer leaflet destabilization (white arrowhead) by F-BAR REMIND . Green arrows point to some individual F-BAR REMIND molecules. White arrows indicate local membrane disruption (both leaflets)

    Article Snippet: 18:1/18:1-PC (1,2-dioleoyl- sn -glycero-3-phosphocholine or DOPC), 18:1/18:1-PS (1,2-dioleoyl- sn - glycero-3-phospho-L-serine or DOPS), brain PI(4)P (L-α-phosphatidylinositol 4-phosphate), brain PI(4,5)P 2 (L-α-phosphatidylinositol 4,5-bisphosphate), NBD-PC (1-palmitoyl-2-(12-[(7-nitro-2-1,3- benzoxadiazol-4-yl)amino]dodecanoyl)-sn-glycero-3-phosphocholine) NBD-PE (1,2-dioleoyl- sn -glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)) were purchased from Avanti Polar Lipids.

    Techniques: Purification, Derivative Assay, Circular Dichroism, Residue, Incubation, Liposomes, Centrifugation, SDS Page, Membrane, Negative Staining, Cryo-EM Sample Prep, Disruption

    (A) Localisation of arginine and lysine residues that constitute a basic cluster in the concave face of F-BAR REMIND (in blue) and localization of solvent-exposed hydrophobic residues in the lateral side of this domain (in orange). These residues were substituted by anionic residues (aspartate or glutamate) to test their contribution to the F-BAR REMIND /membrane interaction. (B) Electrostatic features of the molecular surface of F-BAR REMIND and its mutated forms (red = -16.9 kTe -1 , blue = +16.9 kTe -1 ). (C) Flotation assay. F-BAR REMIND or K210D/R214E, R214E/R217E or R217E/R250E mutant (0.75 µM) was incubated with liposomes composed of DOPC/PI(4,5)P 2 (90:10, 750 µM lipids), with a defined radius, for 1 h at 25 °C. Data are represented as mean ± s.e.m. (n = 3-5). (D) Flotation assay. F-BAR REMIND or L201D/M212E mutant (0.75 µM) was incubated with liposomes (750 µM) composed of DOPC or DOPC/PI(4,5)P 2 (90:10), with a defined radius, for 1 h at 25 °C. Mean ± s.e.m. (n = 3-4). (E) Negative-staining EM. Liposomes made of Folch fraction I lipids (30 µM) were incubated with F-BAR REMIND or its mutated form (1.9 µM) for 2 h at room temperature (scale bar = 100 nm). A control picture of liposomes only is shown

    Journal: bioRxiv

    Article Title: Characterization of atypical BAR domain-containing proteins coded by Toxoplasma gondii

    doi: 10.1101/2024.06.13.598837

    Figure Lengend Snippet: (A) Localisation of arginine and lysine residues that constitute a basic cluster in the concave face of F-BAR REMIND (in blue) and localization of solvent-exposed hydrophobic residues in the lateral side of this domain (in orange). These residues were substituted by anionic residues (aspartate or glutamate) to test their contribution to the F-BAR REMIND /membrane interaction. (B) Electrostatic features of the molecular surface of F-BAR REMIND and its mutated forms (red = -16.9 kTe -1 , blue = +16.9 kTe -1 ). (C) Flotation assay. F-BAR REMIND or K210D/R214E, R214E/R217E or R217E/R250E mutant (0.75 µM) was incubated with liposomes composed of DOPC/PI(4,5)P 2 (90:10, 750 µM lipids), with a defined radius, for 1 h at 25 °C. Data are represented as mean ± s.e.m. (n = 3-5). (D) Flotation assay. F-BAR REMIND or L201D/M212E mutant (0.75 µM) was incubated with liposomes (750 µM) composed of DOPC or DOPC/PI(4,5)P 2 (90:10), with a defined radius, for 1 h at 25 °C. Mean ± s.e.m. (n = 3-4). (E) Negative-staining EM. Liposomes made of Folch fraction I lipids (30 µM) were incubated with F-BAR REMIND or its mutated form (1.9 µM) for 2 h at room temperature (scale bar = 100 nm). A control picture of liposomes only is shown

    Article Snippet: 18:1/18:1-PC (1,2-dioleoyl- sn -glycero-3-phosphocholine or DOPC), 18:1/18:1-PS (1,2-dioleoyl- sn - glycero-3-phospho-L-serine or DOPS), brain PI(4)P (L-α-phosphatidylinositol 4-phosphate), brain PI(4,5)P 2 (L-α-phosphatidylinositol 4,5-bisphosphate), NBD-PC (1-palmitoyl-2-(12-[(7-nitro-2-1,3- benzoxadiazol-4-yl)amino]dodecanoyl)-sn-glycero-3-phosphocholine) NBD-PE (1,2-dioleoyl- sn -glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)) were purchased from Avanti Polar Lipids.

    Techniques: Solvent, Membrane, Mutagenesis, Incubation, Liposomes, Negative Staining

    ( A ) Ribbon representation of the three-dimensional model of the REMIND domain (region 500-836 of Tg REMIND) established by AlphaFold (in grey) and of the final conformation of this domain after 1 µs-MD simulation in water (in blue). The N- and C-terminal ends of the domain are indicated. ( B ) RMSD of the Cα atoms with respect to the starting and equilibrated structure of the REMIND domain as a function of time. (C) RMSF values of atomic positions of Cα atoms, indicative of the internal protein motions, are shown as a function of residue number. The localization of α-helix and β-sheet along the sequence is indicated. (D) Predicted and experimental CD spectra of the REMIND domain. Spectra were predicted from configurations of the REMIND domain collected every 10 ns during the MD simulation (grey spectra) using the PDBMD2CD algorithm. An average spectrum is represented in black. For comparison, the far-UV CD spectrum of purified Tg REMIND[495-840] construct (REMIND, 2 µM) in 20 mM Tris, pH 7.4, 120 mM NaF buffer at room temperature is shown (in red). (E) The intrinsic fluorescence of REMIND (1µM) was measured in TN buffer at 30 °C. A spectrum was measured with free L-tryptophane (4 µM) as a comparison (F) Flotation assay. REMIND (0.75 µM) was incubated with liposomes (750 µM lipids) of different mean radii, composed of DOPC or DOPC/PI(4,5)P 2 (90:10) for 1 h at 25 °C under agitation. Data are represented as mean ± s.e.m (n = 3-4). (G) Negative staining-EM images of liposomes (30 µM lipids), composed of Folch fraction I lipids, mixed or not with REMIND (1.9 µM). Scale bar = 500 nm.

    Journal: bioRxiv

    Article Title: Characterization of atypical BAR domain-containing proteins coded by Toxoplasma gondii

    doi: 10.1101/2024.06.13.598837

    Figure Lengend Snippet: ( A ) Ribbon representation of the three-dimensional model of the REMIND domain (region 500-836 of Tg REMIND) established by AlphaFold (in grey) and of the final conformation of this domain after 1 µs-MD simulation in water (in blue). The N- and C-terminal ends of the domain are indicated. ( B ) RMSD of the Cα atoms with respect to the starting and equilibrated structure of the REMIND domain as a function of time. (C) RMSF values of atomic positions of Cα atoms, indicative of the internal protein motions, are shown as a function of residue number. The localization of α-helix and β-sheet along the sequence is indicated. (D) Predicted and experimental CD spectra of the REMIND domain. Spectra were predicted from configurations of the REMIND domain collected every 10 ns during the MD simulation (grey spectra) using the PDBMD2CD algorithm. An average spectrum is represented in black. For comparison, the far-UV CD spectrum of purified Tg REMIND[495-840] construct (REMIND, 2 µM) in 20 mM Tris, pH 7.4, 120 mM NaF buffer at room temperature is shown (in red). (E) The intrinsic fluorescence of REMIND (1µM) was measured in TN buffer at 30 °C. A spectrum was measured with free L-tryptophane (4 µM) as a comparison (F) Flotation assay. REMIND (0.75 µM) was incubated with liposomes (750 µM lipids) of different mean radii, composed of DOPC or DOPC/PI(4,5)P 2 (90:10) for 1 h at 25 °C under agitation. Data are represented as mean ± s.e.m (n = 3-4). (G) Negative staining-EM images of liposomes (30 µM lipids), composed of Folch fraction I lipids, mixed or not with REMIND (1.9 µM). Scale bar = 500 nm.

    Article Snippet: 18:1/18:1-PC (1,2-dioleoyl- sn -glycero-3-phosphocholine or DOPC), 18:1/18:1-PS (1,2-dioleoyl- sn - glycero-3-phospho-L-serine or DOPS), brain PI(4)P (L-α-phosphatidylinositol 4-phosphate), brain PI(4,5)P 2 (L-α-phosphatidylinositol 4,5-bisphosphate), NBD-PC (1-palmitoyl-2-(12-[(7-nitro-2-1,3- benzoxadiazol-4-yl)amino]dodecanoyl)-sn-glycero-3-phosphocholine) NBD-PE (1,2-dioleoyl- sn -glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)) were purchased from Avanti Polar Lipids.

    Techniques: Residue, Sequencing, Circular Dichroism, Comparison, Purification, Construct, Fluorescence, Incubation, Liposomes, Negative Staining

    (A) Flotation assays. Tg REMIND (0.75 µM) was incubated with liposomes (750 µM lipids) of different sizes, composed of DOPC/PI(4,5)P 2 (90:10) for 1 h at 25 °C under agitation. Data are represented as mean ± s.e.m. (n = 3). (B) F-BAR REMIND was mixed alone or together with a stoichiometric amount of REMIND with liposomes of different sizes, composed of DOPC only or DOPC/PI(4,5)P 2 (90:10) for 1 h at 25 °C under agitation. Mean ± s.e.m. (n = 3-5). (C) Negative staining EM. Representative images of Folch fraction liposomes incubated with F-BAR REMIND alone or mixed with REMIND. A large view shows that no tubule emanates from liposomes when the REMIND domain is present. (D) A three- dimensional model of the full-length Tg REMIND established by AlphaFold shows the association between the REMIND domain and the tip of the F-BAR domain. Only one monomer is shown. (E) Close-up view of F-BAR binding site predicted at the surface of the REMIND domain showing the degree of amino acid conservation based on 37 distinct sequences from diverse Apicomplexan species. Residues that are highly conserved and/or able to form one or more hydrogen bonds with residues of the BAR domain are indicated (a star indicates whether a residue forms hydrogen bond(s)). (F) A heat map based on a proximity matrix shows that the 214-258 region of Tg REMIND (the extremity of the F-BAR domain) is closely associated with different residues of the REMIND domain. The average distance between two residues was calculated based on conformations observed in one 250-ns MD trajectory. Values higher than 2 nanometers are not shown. (G) The average number of H-bonds per configuration between two given residues belonging to the BAR domain and the REMIND domain was calculated from three independent 250-ns MD trajectories (green bars). The values obtained independently from each trajectory are also shown (black dots).

    Journal: bioRxiv

    Article Title: Characterization of atypical BAR domain-containing proteins coded by Toxoplasma gondii

    doi: 10.1101/2024.06.13.598837

    Figure Lengend Snippet: (A) Flotation assays. Tg REMIND (0.75 µM) was incubated with liposomes (750 µM lipids) of different sizes, composed of DOPC/PI(4,5)P 2 (90:10) for 1 h at 25 °C under agitation. Data are represented as mean ± s.e.m. (n = 3). (B) F-BAR REMIND was mixed alone or together with a stoichiometric amount of REMIND with liposomes of different sizes, composed of DOPC only or DOPC/PI(4,5)P 2 (90:10) for 1 h at 25 °C under agitation. Mean ± s.e.m. (n = 3-5). (C) Negative staining EM. Representative images of Folch fraction liposomes incubated with F-BAR REMIND alone or mixed with REMIND. A large view shows that no tubule emanates from liposomes when the REMIND domain is present. (D) A three- dimensional model of the full-length Tg REMIND established by AlphaFold shows the association between the REMIND domain and the tip of the F-BAR domain. Only one monomer is shown. (E) Close-up view of F-BAR binding site predicted at the surface of the REMIND domain showing the degree of amino acid conservation based on 37 distinct sequences from diverse Apicomplexan species. Residues that are highly conserved and/or able to form one or more hydrogen bonds with residues of the BAR domain are indicated (a star indicates whether a residue forms hydrogen bond(s)). (F) A heat map based on a proximity matrix shows that the 214-258 region of Tg REMIND (the extremity of the F-BAR domain) is closely associated with different residues of the REMIND domain. The average distance between two residues was calculated based on conformations observed in one 250-ns MD trajectory. Values higher than 2 nanometers are not shown. (G) The average number of H-bonds per configuration between two given residues belonging to the BAR domain and the REMIND domain was calculated from three independent 250-ns MD trajectories (green bars). The values obtained independently from each trajectory are also shown (black dots).

    Article Snippet: 18:1/18:1-PC (1,2-dioleoyl- sn -glycero-3-phosphocholine or DOPC), 18:1/18:1-PS (1,2-dioleoyl- sn - glycero-3-phospho-L-serine or DOPS), brain PI(4)P (L-α-phosphatidylinositol 4-phosphate), brain PI(4,5)P 2 (L-α-phosphatidylinositol 4,5-bisphosphate), NBD-PC (1-palmitoyl-2-(12-[(7-nitro-2-1,3- benzoxadiazol-4-yl)amino]dodecanoyl)-sn-glycero-3-phosphocholine) NBD-PE (1,2-dioleoyl- sn -glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)) were purchased from Avanti Polar Lipids.

    Techniques: Incubation, Liposomes, Negative Staining, Binding Assay, Residue

    (A) Structural organization of Tg BAR2 and AlphaFold-predicted model of its BAR domain in a dimeric form. Its intrinsic curvature seems adapted to the recognition of 22 nm diameter tubules. The position of the N- and C-terminal ends of each monomer is shown. The structure is represented in ribbon mode. (B) Electrostatic potential of the dimeric BAR domain red (red = -16.9 kTe -1 , blue = +16.9 kTe -1 ). (C) Flotation assays. Tg BAR2 (0.75 µM) was incubated with liposomes of different radii (750 µM lipids), only made of DOPC or additionally containing 30% DOPS or 10% PI(4,5)P 2 , in TN buffer for 1 h at 25 °C under agitation. The percentage of membrane-bound Tg BAR2 is represented as the function of the average hydrodynamic radius (R H ) of liposomes. Data corresponds to mean ± s.e.m. (n = 3). (D) Flotation assays. Membrane-bound fraction of Tg BAR2 or F-BAR REMIND (0.75 µM) incubated with liposomes extruded through 0.1 µm pores (750 µM lipids), only made of DOPC or additionally containing 30% DOPS and 5% PI(4,5)P 2 . Data corresponds to mean ± s.e.m. (n = 3). (E) Negative- staining EM. Liposomes made of Folch fraction I lipids (30 µM) and extruded through 0.4 µm pores were incubated with Tg BAR2 (1.9 µM). Control experiments were conducted with liposomes only. Representative pictures are shown. Scale bar = 200 nm. (F) Diameter and length distribution of narrow and broader membrane tubules induced by Tg BAR2 (narrow tubules, n = 50; wide tubules, n = 112). The indicated values correspond to mean ± s.e.m. (G) Cryo-EM. Liposomes made of Folch fraction I lipids (150 µM lipids) and extruded through 0.4 µm pores were mixed with Tg BAR2 (5 µM) at P/L= 1/30 and dialyzed three times under agitation in TN buffer for 30 min at cold temperature. A control picture showing liposomes without Tg BAR2 is shown (1). Tg BAR2 can transform liposomes into tubular micelles ( G, picture 2 and 2’ black arrow) and bilayered tubules ( G , picture 3 and 3’, white arrow). Tg BAR2 coats the membrane surfaces and can form transmembrane densities (yellow arrows). (G) Lower panels are general interpretations of membrane destabilization phenomena by Tg BAR2; left: tubular micelles; right: bilayered tubules (H) Diameter distribution of membrane tubules with average values observed by cryo-EM. The indicated value corresponds to mean ± s.e.m. (tubular micelle, n = 16; bilayered tubules, n = 17).

    Journal: bioRxiv

    Article Title: Characterization of atypical BAR domain-containing proteins coded by Toxoplasma gondii

    doi: 10.1101/2024.06.13.598837

    Figure Lengend Snippet: (A) Structural organization of Tg BAR2 and AlphaFold-predicted model of its BAR domain in a dimeric form. Its intrinsic curvature seems adapted to the recognition of 22 nm diameter tubules. The position of the N- and C-terminal ends of each monomer is shown. The structure is represented in ribbon mode. (B) Electrostatic potential of the dimeric BAR domain red (red = -16.9 kTe -1 , blue = +16.9 kTe -1 ). (C) Flotation assays. Tg BAR2 (0.75 µM) was incubated with liposomes of different radii (750 µM lipids), only made of DOPC or additionally containing 30% DOPS or 10% PI(4,5)P 2 , in TN buffer for 1 h at 25 °C under agitation. The percentage of membrane-bound Tg BAR2 is represented as the function of the average hydrodynamic radius (R H ) of liposomes. Data corresponds to mean ± s.e.m. (n = 3). (D) Flotation assays. Membrane-bound fraction of Tg BAR2 or F-BAR REMIND (0.75 µM) incubated with liposomes extruded through 0.1 µm pores (750 µM lipids), only made of DOPC or additionally containing 30% DOPS and 5% PI(4,5)P 2 . Data corresponds to mean ± s.e.m. (n = 3). (E) Negative- staining EM. Liposomes made of Folch fraction I lipids (30 µM) and extruded through 0.4 µm pores were incubated with Tg BAR2 (1.9 µM). Control experiments were conducted with liposomes only. Representative pictures are shown. Scale bar = 200 nm. (F) Diameter and length distribution of narrow and broader membrane tubules induced by Tg BAR2 (narrow tubules, n = 50; wide tubules, n = 112). The indicated values correspond to mean ± s.e.m. (G) Cryo-EM. Liposomes made of Folch fraction I lipids (150 µM lipids) and extruded through 0.4 µm pores were mixed with Tg BAR2 (5 µM) at P/L= 1/30 and dialyzed three times under agitation in TN buffer for 30 min at cold temperature. A control picture showing liposomes without Tg BAR2 is shown (1). Tg BAR2 can transform liposomes into tubular micelles ( G, picture 2 and 2’ black arrow) and bilayered tubules ( G , picture 3 and 3’, white arrow). Tg BAR2 coats the membrane surfaces and can form transmembrane densities (yellow arrows). (G) Lower panels are general interpretations of membrane destabilization phenomena by Tg BAR2; left: tubular micelles; right: bilayered tubules (H) Diameter distribution of membrane tubules with average values observed by cryo-EM. The indicated value corresponds to mean ± s.e.m. (tubular micelle, n = 16; bilayered tubules, n = 17).

    Article Snippet: 18:1/18:1-PC (1,2-dioleoyl- sn -glycero-3-phosphocholine or DOPC), 18:1/18:1-PS (1,2-dioleoyl- sn - glycero-3-phospho-L-serine or DOPS), brain PI(4)P (L-α-phosphatidylinositol 4-phosphate), brain PI(4,5)P 2 (L-α-phosphatidylinositol 4,5-bisphosphate), NBD-PC (1-palmitoyl-2-(12-[(7-nitro-2-1,3- benzoxadiazol-4-yl)amino]dodecanoyl)-sn-glycero-3-phosphocholine) NBD-PE (1,2-dioleoyl- sn -glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)) were purchased from Avanti Polar Lipids.

    Techniques: Incubation, Liposomes, Membrane, Negative Staining, Cryo-EM Sample Prep

    A IC 50 values of phenothiazine compounds in the indicated cell lines after 48 h of treatment. B Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. C IC 50 of phenothiazines 48 h after drug treatment. D Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. E Western blot analysis of empty vector (EV) and PTEN overexpression U87-MG cells treated with either vehicle or 10 µM perphenazine for 24 h. F Western blot analysis of T98G and LN18 cells treated with either vehicle or 10 µM perphenazine for 48 h. G Western blot analysis of Akt and RPS6 activation at 4 and 24 h after treatment with 10 µM perphenazine. H , I Images showing PI(3,4,5)P 3 and PI(4,5)P 2 levels in U87-MG-PTEN and U87-MG cells treated with either vehicle or 10 µM perphenazine for 48 h. J Kaplan-Meier survival curves of mice intracranially implanted with 100,000 U87-MG-PTEN cells and treated with either vehicle or 10 mg/kg perphenazine. A total of ten treatments were administered starting on day 8 post-implantation. Treatments were given for five consecutive days followed by a 2-day break for 2 weeks.

    Journal: Cell Death & Disease

    Article Title: Antipsychotics possess anti-glioblastoma activity by disrupting lysosomal function and inhibiting oncogenic signaling by stabilizing PTEN

    doi: 10.1038/s41419-024-06779-3

    Figure Lengend Snippet: A IC 50 values of phenothiazine compounds in the indicated cell lines after 48 h of treatment. B Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. C IC 50 of phenothiazines 48 h after drug treatment. D Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. E Western blot analysis of empty vector (EV) and PTEN overexpression U87-MG cells treated with either vehicle or 10 µM perphenazine for 24 h. F Western blot analysis of T98G and LN18 cells treated with either vehicle or 10 µM perphenazine for 48 h. G Western blot analysis of Akt and RPS6 activation at 4 and 24 h after treatment with 10 µM perphenazine. H , I Images showing PI(3,4,5)P 3 and PI(4,5)P 2 levels in U87-MG-PTEN and U87-MG cells treated with either vehicle or 10 µM perphenazine for 48 h. J Kaplan-Meier survival curves of mice intracranially implanted with 100,000 U87-MG-PTEN cells and treated with either vehicle or 10 mg/kg perphenazine. A total of ten treatments were administered starting on day 8 post-implantation. Treatments were given for five consecutive days followed by a 2-day break for 2 weeks.

    Article Snippet: Anti-PI(4,5)P 2 (Z-P045) was purchased from Echelon Biosciences.

    Techniques: Western Blot, Plasmid Preparation, Over Expression, Activation Assay

    Phosphoinositide staining following inhibition of lipid kinases. (a) HeLa cells were treated with a kinase inhibitor cocktail of Wortmannin, PI-273, GSK-A1, PI4KIIIβ-IN 10, and Apilimod, each at 500 nM for 30 min before being fixed and stained using recombinant biosensors against PI, PI(4)P and PI(3)P conjugated to Alexa488, 546, or 647, respectively. Linescan shows the signal of PI on the membrane of large vacuoles that can be found throughout the cell. (b) HeLa cells were treated with either GSK-A1, PI-273, or PI4KIIIβ-IN 10 at 100 nM each for 30 min before being fixed and stained against PI(4)P using recombinant biosensors against PI(4)P conjugated to Alexa488. (c) HeLa cells were treated with LY 294022 at 1 µM for 30 min, stimulated with 5 ng/ml human EGF for 10 min, and fixed and stained using recombinant biosensors against PI(4,5)P 2 , PI(3,4)P 2 , and PI(3,4,5)P 3 conjugated to Alexa488, 546, or 647, respectively. Linescans show signals of PI(4,5)P 2 , PI(3,4)P 2 , and PI(3,4,5)P 3 in DMSO and LY294002 treated cells at membrane ruffles as well as the cytosolic background.

    Journal: The Journal of Cell Biology

    Article Title: Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides

    doi: 10.1083/jcb.202310095

    Figure Lengend Snippet: Phosphoinositide staining following inhibition of lipid kinases. (a) HeLa cells were treated with a kinase inhibitor cocktail of Wortmannin, PI-273, GSK-A1, PI4KIIIβ-IN 10, and Apilimod, each at 500 nM for 30 min before being fixed and stained using recombinant biosensors against PI, PI(4)P and PI(3)P conjugated to Alexa488, 546, or 647, respectively. Linescan shows the signal of PI on the membrane of large vacuoles that can be found throughout the cell. (b) HeLa cells were treated with either GSK-A1, PI-273, or PI4KIIIβ-IN 10 at 100 nM each for 30 min before being fixed and stained against PI(4)P using recombinant biosensors against PI(4)P conjugated to Alexa488. (c) HeLa cells were treated with LY 294022 at 1 µM for 30 min, stimulated with 5 ng/ml human EGF for 10 min, and fixed and stained using recombinant biosensors against PI(4,5)P 2 , PI(3,4)P 2 , and PI(3,4,5)P 3 conjugated to Alexa488, 546, or 647, respectively. Linescans show signals of PI(4,5)P 2 , PI(3,4)P 2 , and PI(3,4,5)P 3 in DMSO and LY294002 treated cells at membrane ruffles as well as the cytosolic background.

    Article Snippet: Recombinant biosensors were diluted in 0.1% Saponin in PIPES buffer (PI(4)P biosensor at a final concentration of 150 nM and PI(4,5)P 2 biosensor at 500 nM) with 1:200 Alexa488-conjugated Phalloidin (Invitrogen) and incubated with the wings for 1 h. Wings were then washed 12 times in 0.1% Saponin in PIPES buffer, post-fixed for 10 min in 2% PFA, washed a further four times with 0.1% Saponin in PIPES buffer, and mounted in 2.5% DABCO with 10% glycerol in PBS.

    Techniques: Staining, Inhibition, Recombinant, Membrane

    Staining using one step His purification. The recombinant biosensor against PI(4,5)P 2 (6xHis-SNAP-PLCδ1) was purified from BL21 E. coli using a HisTrap column and eluted against an increasing gradient of 250 mM Imidazole. Peak fractions were pooled and either directly labeled with SNAP-Surface Alexa488 or first incubated with recombinant PreScission Protease to cleave the 6xHis tag and used to stain HeLa cells as previously described. Source data are available for this figure:  .

    Journal: The Journal of Cell Biology

    Article Title: Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides

    doi: 10.1083/jcb.202310095

    Figure Lengend Snippet: Staining using one step His purification. The recombinant biosensor against PI(4,5)P 2 (6xHis-SNAP-PLCδ1) was purified from BL21 E. coli using a HisTrap column and eluted against an increasing gradient of 250 mM Imidazole. Peak fractions were pooled and either directly labeled with SNAP-Surface Alexa488 or first incubated with recombinant PreScission Protease to cleave the 6xHis tag and used to stain HeLa cells as previously described. Source data are available for this figure: .

    Article Snippet: Recombinant biosensors were diluted in 0.1% Saponin in PIPES buffer (PI(4)P biosensor at a final concentration of 150 nM and PI(4,5)P 2 biosensor at 500 nM) with 1:200 Alexa488-conjugated Phalloidin (Invitrogen) and incubated with the wings for 1 h. Wings were then washed 12 times in 0.1% Saponin in PIPES buffer, post-fixed for 10 min in 2% PFA, washed a further four times with 0.1% Saponin in PIPES buffer, and mounted in 2.5% DABCO with 10% glycerol in PBS.

    Techniques: Staining, Purification, Recombinant, Labeling, Incubation

    Multiplex staining of phosphoinositides across scales. (a) HeLa cells were fixed, permeabilized, and stained using recombinant biosensors against PI(3)P, PI(4)P, and PI(4,5)P 2 , conjugated respectively to Alexa488, 546, and 647. (b) NMuMG spheroids were grown in Matrigel and stained with the same combination. (c) Drosophila pupal wings were dissected and stained with the PI(4,5)P 2 and PI(4)P biosensors conjugated to Alexa647 and 546, together with Phalloidin conjugated to Alexa488, to visualize the actin cytoskeleton and cellular junctions.

    Journal: The Journal of Cell Biology

    Article Title: Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides

    doi: 10.1083/jcb.202310095

    Figure Lengend Snippet: Multiplex staining of phosphoinositides across scales. (a) HeLa cells were fixed, permeabilized, and stained using recombinant biosensors against PI(3)P, PI(4)P, and PI(4,5)P 2 , conjugated respectively to Alexa488, 546, and 647. (b) NMuMG spheroids were grown in Matrigel and stained with the same combination. (c) Drosophila pupal wings were dissected and stained with the PI(4,5)P 2 and PI(4)P biosensors conjugated to Alexa647 and 546, together with Phalloidin conjugated to Alexa488, to visualize the actin cytoskeleton and cellular junctions.

    Article Snippet: Recombinant biosensors were diluted in 0.1% Saponin in PIPES buffer (PI(4)P biosensor at a final concentration of 150 nM and PI(4,5)P 2 biosensor at 500 nM) with 1:200 Alexa488-conjugated Phalloidin (Invitrogen) and incubated with the wings for 1 h. Wings were then washed 12 times in 0.1% Saponin in PIPES buffer, post-fixed for 10 min in 2% PFA, washed a further four times with 0.1% Saponin in PIPES buffer, and mounted in 2.5% DABCO with 10% glycerol in PBS.

    Techniques: Multiplex Assay, Staining, Recombinant