Journal: The Journal of General Physiology

Article Title: Disruption of membrane cholesterol organization impairs the activity of PIEZO1 channel clusters

doi: 10.1085/jgp.201912515

Figure Lengend Snippet: Effect of cholesterol depletion via MBCD on transiently transfected WT human PIEZO1. (A) Boltzmann curve fits of normalized I/I max values from all control and MBCD-treated cells stably or transiently expressing human PIEZO1 proteins. Data for the stable cell line is quantified in . (B) Quantification of midpoint activation pressure for transiently expressed WT human PIEZO1. PIEZO1 WT ( n = 8 cells), PIEZO1 WT + MBCD ( n = 7 cells). (C) Quantification of channel inactivation expressed as normalized steady-state current for transiently expressed WT human PIEZO1. PIEZO1 WT ( n = 9 cells), PIEZO1 WT + 5 mM MBCD ( n = 7 cells). Statistical analysis was performed using Student’s t test; ****, P < 0.0001. (D) TIRF imaging of transiently expressed hP1-GFP in PIEZO1-KO HEK293T cells (left) and stably expressed hP1-GFP from hP1-GFP-CellLine (right). Scale bar represents 10 µm. (E) Average GFP intensity detected in the basal membrane of transiently or stably expressed PIEZO1-GFP. Transient hP1-GFP ( n = 662 cells), stable hP1-GFP ( n = 756 cells), imaged over three separate sample preparations. Error bars indicate SEM; ****, P < 0.0001. (F) Normalized cell count histogram of the range of observed fluorescence intensities exhibited by cells over the course of the imaging experiments (bin size = 25 A.U.).

Article Snippet: STORM images were acquired on a TIRF microscope (Elyra; Zeiss) using a 100× α Plan APO oil-immersion objective lens (NA 1.46).

Techniques: Transfection, Control, Stable Transfection, Expressing, Activation Assay, Imaging, Membrane, Cell Counting, Fluorescence

Journal: The Journal of General Physiology

Article Title: Disruption of membrane cholesterol organization impairs the activity of PIEZO1 channel clusters

doi: 10.1085/jgp.201912515

Figure Lengend Snippet: Effect of MBCD on cell viability and cholesterol levels. (A) Normalized cell viability of PIEZO1-GFP HEK293T cells in the presence and absence of the hypoxia-mimetic CoCl 2 as a positive control to see a reduction in viability on cell death. Error bars represent mean ± SEM. (B) Effect of 30-min MBCD pretreatment on cell viability. Error bars represent mean ± SEM. (C) HEK293T cells were transfected with D4H-mCherry, a marker of cholesterol in the plasma membrane, and live-imaged in TIRF mode over 45 min during the 5-mM MBCD treatment (scale bar, 5 µm). (D) D4H-mCherry fluorescence intensity monitored in 2.5-s time blocks. Each colored line represents a consecutive fluorescence recording at different time points for the same cell. (E) Time-course of D4H-mCherry fluorescence, normalized to the average fluorescence intensity at time = 0. Each colored plot represents a separate cell undergoing the 5-mM MBCD treatment.

Article Snippet: STORM images were acquired on a TIRF microscope (Elyra; Zeiss) using a 100× α Plan APO oil-immersion objective lens (NA 1.46).

Techniques: Positive Control, Transfection, Marker, Clinical Proteomics, Membrane, Fluorescence

Journal: The Journal of General Physiology

Article Title: Disruption of membrane cholesterol organization impairs the activity of PIEZO1 channel clusters

doi: 10.1085/jgp.201912515

Figure Lengend Snippet: Filipin III staining. (A) TIRF microscopy images of HEK293T cells untreated, cholesterol depleted (5 mM MBCD), or cholesterol enriched (50 µg/ml water-soluble cholesterol). Fields of view including multiple cells are shown on the top row, and images including single, isolated cells on the bottom row. All the images are displayed using the same spectral minimum/maximum values (minimum = 1,600 A.U.; maximum = 3,060 A.U.). Scale bars, 10 μM. (B) Mean fluorescence intensity quantification of Filipin III fluorescence. Intensity calculations were performed using Fiji. A threshold was applied to all images to remove the pixels of intensity <1,600 A.U. before calculating the mean Filipin III intensity (one image represents n = 1). Control ( n = 35), MBCD ( n = 45), cholesterol ( n = 50). Error bars represent mean ± SEM. Statistical comparison performed using one-way ANOVA; **, P < 0.05; ***, P < 0.001.

Article Snippet: STORM images were acquired on a TIRF microscope (Elyra; Zeiss) using a 100× α Plan APO oil-immersion objective lens (NA 1.46).

Techniques: Staining, Microscopy, Isolation, Fluorescence, Control, Comparison

Journal: The Journal of General Physiology

Article Title: Disruption of membrane cholesterol organization impairs the activity of PIEZO1 channel clusters

doi: 10.1085/jgp.201912515

Figure Lengend Snippet: Cholesterol removal alters PIEZO1-GFP dynamics in situ. (A) Left: TIRF images are the initial frames acquired during live-cell imaging. Right: Representative time course of PIEZO1-GFP diffusion dynamics within the highlighted square area, shown next to each of the conditions tested. The four time points (t1–t4) represent the time course of the fluctuations in fluorescence intensity within the 1-µm 2 portion of the image indicated by the white arrow, collected at 150-frame intervals (∼5-s intervals between consecutive time points). Scale bars, 5 μM. (B) Cartoon describing the limited diffusion experienced by PIEZO1-GFP channels in the plasma membrane. (C) Quantification of lateral unbinding rate (s −1 ) of PIEZO1-GFP entities in control ( n = 5 trials, 205 cells), 5 mM MBCD-treated ( n = 5 trials, 193 cells), and 50 µg/ml cholesterol-treated cells ( n = 5 trials, 77 cells). (D) Effective diffusion (µm 2 /s) of PIEZO1-GFP clusters and group of clusters (fluorescent entities) in control, 5 mM MBCD-, and 50 µg/ml cholesterol-treated cells. Both lateral unbinding rates and effective diffusion are increased significantly by cholesterol depletion via MBCD. Data represent mean ± SEM analyzed with one-way ANOVA; ****, P < 0.00005).

Article Snippet: STORM images were acquired on a TIRF microscope (Elyra; Zeiss) using a 100× α Plan APO oil-immersion objective lens (NA 1.46).

Techniques: In Situ, Live Cell Imaging, Diffusion-based Assay, Fluorescence, Clinical Proteomics, Membrane, Control

Journal: The Journal of General Physiology

Article Title: Disruption of membrane cholesterol organization impairs the activity of PIEZO1 channel clusters

doi: 10.1085/jgp.201912515

Figure Lengend Snippet: Modulation of PIEZO1-GFP clusters determined by STORM superresolution microscopy. (A) TIRF microscopy of PIEZO1-GFP cells immunolabeled with an anti-GFP antibody conjugated to Alexa Fluor 647. Images of control cells and treated cells (cell boundary highlighted by a dashed line) show the PIEZO1-GFP signal (green) superimposed on the STORM signal (yellow arrowheads) from Alexa Fluor 647 (red; insets). Scale bar represents 5 µm or 1 μm in inset. The physical properties of the clusters shown in the insets were analyzed by cluster decomposition and are shown in the adjacent cluster maps (black arrowheads). (B) Left: Quantification of PIEZO1-GFP cluster area as violin plots in control and treatment conditions. Right: Relative frequency of cluster areas in square nanometers. (C) Left: Quantification of PIEZO1-GFP cluster perimeters as violin plots in control and treatment conditions. Right: Relative frequency of cluster perimeters in nanometers. For cluster area and perimeter: control PIEZO1-GFP ( n = 34,741), MBCD ( n = 19,159), cholesterol ( n = 12,097), dynasore ( n = 19,982); n = 1 represents a single PIEZO1-GFP cluster. (D) Left: Quantification of PIEZO1-GFP cluster density as violin plots in control and treatment conditions. Right: Relative frequency of cluster density in clusters per square micrometer. Control PIEZO1-GFP ( n = 173), MBCD ( n = 193), cholesterol ( n = 126), dynasore ( n = 82); n = 1 represents a single cell. All violin plots are marked with median (thick dashed line) flanked by 25th and 75th percentile (thin dashed lines). The median value of each plot is reported on the righthand side of each graph. One-way ANOVA; *, P < 0.05; ****, P < 0.00005; ns, not significant.

Article Snippet: STORM images were acquired on a TIRF microscope (Elyra; Zeiss) using a 100× α Plan APO oil-immersion objective lens (NA 1.46).

Techniques: Microscopy, Immunolabeling, Control

Journal: The Journal of General Physiology

Article Title: Disruption of membrane cholesterol organization impairs the activity of PIEZO1 channel clusters

doi: 10.1085/jgp.201912515

Figure Lengend Snippet: Colocalization of PIEZO1-GFP and CtxB raft marker using TIRF. (A) TIRF microscopy of cholesterol-rich domains labeled with CtxB–Alexa Fluor 555 (A555; red) on the basal membrane of PIEZO1-GFP–expressing HEK293T cells (GFP fluorescence in green). (B) Average number of PIEZO1-GFP entities per square micrometer detected after 30-min treatments. (C) Average density of colocalized entities per square micrometer detected after 30-min treatments. Control ( n = 6 trials, 42 cells), MBCD ( n = 6 trials, 81 cells), cholesterol ( n = 6 trials, 62 cells), and dynasore ( n = 6 trials, 55 cells). (D) Effective diffusion rate of colocalized PIEZO1-GFP/CtxB–A555 at the micrometer scale, before and after treatments. Examples of STICCS correlation function time courses are shown in . One-way ANOVA; ***, P < 0.0005; ****, P < 0.0001; error bars represent SEM. Scale bars represent 5 µm.

Article Snippet: STORM images were acquired on a TIRF microscope (Elyra; Zeiss) using a 100× α Plan APO oil-immersion objective lens (NA 1.46).

Techniques: Marker, Microscopy, Labeling, Membrane, Expressing, Fluorescence, Control, Diffusion-based Assay