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

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

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

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