Journal: bioRxiv

Article Title: 3D-MINFLUX nanoscopy reveals distinct allosteric mechanisms for activation and modulation of PIEZO1 by Yoda1

doi: 10.1101/2025.07.10.664100

Figure Lengend Snippet: (A) Cartoon depicting the proposed mechanism of action of Yoda1 on PIEZO1 (left) and close-up view of the THU8–9 interface with previously proposed residues lining putative Yoda1 binding sites highlighted in sphere representation. (B) , time course of Ca 2+ -influx (F/F0) evoked by 10 and 100 µM Yoda1 in cells expressing PIEZO1 (left) and P1-A2094W (center) assessed by GCamp8 imaging together with comparison of maximum responses (right). (C) Modulation of PIEZO1 (black, top) and P1.A2094W (yellow, bottom) stretch-evoked currents by 30µM Yoda1. Example traces evoked by incrementing pressure stimuli (left), comparison of peak/sustained ratio (middle) using Mann-Whitney test (PIEZO1: CTL = 4.02, N=14 vs Yoda1 = 1.38, N=15, P=0.0000131) and Student’s t-test(P1.A2094W: CTL = 8.35, N=10 vs Yoda1 = 1.19, N=10, P=0.0033), pressure-response curves (i.e. peak current amplitude at indicated pressure normalized to maximal response, bottom right) and comparison of P 50 values in the absence and presence of Yoda1 using Student’s t-test (PIEZO1: CTL = 26.1 ± 8.9 mmHg, N=14 vs Yoda1 = -16.9 ± 6.65 mmHg, N=13, P=0.006) and Mann-Whitney test (P1.A2094W: CTL = -43.7 ± 10.04 mmHg, N=10 vs Yoda1 = -27.4 ± 6.7 mmHg, N=10, P=0.0005). (D) Modulation of PIEZO1 (black, top) and P1.A2094W (yellow, bottom) poking-evoked currents in whole-cell recordings by 30µM Yoda1. Example traces evoked by incrementing (Δ 800nm, left), comparison of inactivation time constants obtained with exponential decay fit (middle) using Mann-Whitney test (PIEZO1: CTL = 15.9 ± 4.3 ms, N=14 vs Yoda1 = 41.1 ± 21.6, N=12, P=0.000258) and Student’s t-test (P1.A2094W: CTL = 6.39 ± 2.8 ms, N=18 vs Yoda1 = 8.3 ± 2.7, N=20, P=0.0405), displacement-response curves (i.e. peak current amplitude vs. indicated stimulus magnitude; bottom, right) and comparison of mechanical activation thresholds using Mann-Whitney test (PIEZO1: CTL = 4.1 ± 1.2 µm, N=14 vs Yoda1 = 2.8 ± 1.4 µm, N=12, P=0.029; P1.A2094W: CTL = 4.8 ± 1.04 µm, N=18 vs Yoda1 = 3.8 ± 1.1 µm, N=20, P=0.0069).

Article Snippet: To independently test if Yoda1 could potentially bind to these cavities, we next calculated possible docking poses of Yoda1 using the AutoDock Vina , .

Techniques: Binding Assay, Expressing, Imaging, Comparison, MANN-WHITNEY, Activation Assay

Journal: bioRxiv

Article Title: 3D-MINFLUX nanoscopy reveals distinct allosteric mechanisms for activation and modulation of PIEZO1 by Yoda1

doi: 10.1101/2025.07.10.664100

Figure Lengend Snippet: (A) Side view of curved (PDB:7WTL, top left) and flattened (PDB:7WTU, bottom left) PIEZO1 cryo-EM structure with close-up side (middle) and top (right) view of THU8–9 interfaces with the putative binding pockets detected by DoGSite3 algorithm shown in isomesh representation. Amino acid side chains that line the pockets are shown in stick representation. (B) , Close-up views of Yoda1 binding poses detected by AutoDock Vina in the curved (top) and flattened (bottom) PIEZO1 conformation. Note, only the binding poses with the highest scores that align with the binding pockets detected in (A) are shown. Residues that are within a distance of 3.5 Å of Yoda1 are shown in stick representation. Note, F1715 appears to be involved in Yoda1 binding in pocket-2 and pocket-3 in the flat conformation. (C) , AutoDock Vina detects multiple possible docking poses for Yoda1. The bar graph shows the percentage of docking poses in which the indicated amino acids are located within 3.5 Å of Yoda1. Note, F1715 is in close proximity of Yoda1 in 78% of all possible binding poses indicating an important role of F1715 in Yoda1 binding in the flat state.

Article Snippet: To independently test if Yoda1 could potentially bind to these cavities, we next calculated possible docking poses of Yoda1 using the AutoDock Vina , .

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: bioRxiv

Article Title: 3D-MINFLUX nanoscopy reveals distinct allosteric mechanisms for activation and modulation of PIEZO1 by Yoda1

doi: 10.1101/2025.07.10.664100

Figure Lengend Snippet: (A) Cartoon depicting the overall strategy to resolve Yoda1 induced conformational changes (flattening) measured through changes in interblade distance. Insets illustrate the labelling of PIEZO1 with ALFA tag inserted after H86, and the DNA-PAINT method (left). Individual bound fluorophore is located with high precision in 3 dimensions via 3D-MINFLUX and its iterative process (middle), leading to multiple localisations of the same molecule. (B) Confocal image of a PIEZO1-ALFA-mGL expressing N2a-P1KO cell (left) and corresponding 3D-MINFLUX localizations (right), colored by Z range. Inset shows a triple-labelled PIEZO1, with the 3D scatter plots of the raw localizations and a superimposed cryo-EM structure. The 2D in-plane projections of the 3D data were fitted with a bivariate Gaussian distribution, with their probability densities, enabling determination of the average interblade distance (right). (C) Time-course of the average ± s.e.m. (from N=3-4 independent experiments) of the maximal Yoda1 (50µM) effect on the normalized fluorescence (F/F0) for PIEZO1 (top), A2094W (middle) and V1714A_F1715A (bottom). (D-F) In-plane projections of representative trimers examples of PIEZO1 ( D ), A2094W ( E ) and V1714A_1715A ( F ) from cells treated with cytochalasin-D (CTL) or with cytochalasin-D and Yoda1 (50µM) (left). Comparison of the mean ± s.e.m. interblade distance of the identified trimers after addition of Yoda1 for PIEZO1 ( D , N=93 and 110), A2094W ( E , N=61 and 64) and V1714A_1715A ( F , N=87 and 59), with unpaired t-test (right).

Article Snippet: To independently test if Yoda1 could potentially bind to these cavities, we next calculated possible docking poses of Yoda1 using the AutoDock Vina , .

Techniques: Expressing, Cryo-EM Sample Prep, Fluorescence, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Activation of Piezo1 Channels Enhances Astrocyte–Neuron Communication via NMDA Receptors in the Murine Neocortex

doi: 10.3390/ijms25073994

Figure Lengend Snippet: The Piezo1 opener Yoda1 increases SIC frequency. ( A – D ). Representative current traces under control conditions (in magnesium-free aCSF) and after addition of 10 µM Yoda1. ( A ). Current trace under control conditions. Gray arrows: SICs, asterisks: examples of EPSCs. Gray square: area magnified on panel ( B ). ( C ) Current trace with Yoda1. The area indicated with the gray square is magnified on panel ( D ) . ( E ) Statistical comparison of SIC parameters. The charge transfer is the area of the individual SICs, SIC activity is the area of all SICs in a minute. The columns and error bars represent average ± SEM, the gray dots are the individual datapoints. *: p < 0.05.

Article Snippet: Yoda1 (2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine; ProbeChem Biochemicals Ltd., Shanghai, China) [ , , , ] and D-AP5 (nonspecific inhibitor, Tocris Cookson Ltd., Bristol, UK) [ ] were used in 10 µM concentration.

Techniques: Comparison, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Activation of Piezo1 Channels Enhances Astrocyte–Neuron Communication via NMDA Receptors in the Murine Neocortex

doi: 10.3390/ijms25073994

Figure Lengend Snippet: The NMDA receptor blocker D-AP5 prevents increase of SIC activity by Yoda1. ( A – C ). Representative current traces under control conditions (magnesium-free aCSF), 10 µM D-AP5 and additional 10 µM Yoda1, respectively (gray arrows: SICs, asterisks: examples of EPSCs). ( D ). Statistical comparison of SIC parameters with different treatments. The columns and error bars represent average ± SEM, the gray dots are the individual datapoints. *: p < 0.05.

Article Snippet: Yoda1 (2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine; ProbeChem Biochemicals Ltd., Shanghai, China) [ , , , ] and D-AP5 (nonspecific inhibitor, Tocris Cookson Ltd., Bristol, UK) [ ] were used in 10 µM concentration.

Techniques: Activity Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Activation of Piezo1 Channels Enhances Astrocyte–Neuron Communication via NMDA Receptors in the Murine Neocortex

doi: 10.3390/ijms25073994

Figure Lengend Snippet: The Piezo1 inhibitor Dooku1 reverted the actions of Yoda1 on SICs. ( A – C ). Representative current traces under control conditions where no SICs appeared (magnesium-free aCSF, ( A )), with addition of 10 µM Yoda1 ( B ) and supplementation with 10 µM Dooku1 ( C ). Gray arrows: SICs, asterisks: examples of EPSCs. ( D ). Statistical comparison of SIC parameters when 10 µM Dooku1 was used for reverting Yoda1 actions. The columns and error bars represent average ± SEM, the gray dots are the individual datapoints. ***: p < 0.001.

Article Snippet: Yoda1 (2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine; ProbeChem Biochemicals Ltd., Shanghai, China) [ , , , ] and D-AP5 (nonspecific inhibitor, Tocris Cookson Ltd., Bristol, UK) [ ] were used in 10 µM concentration.

Techniques: Comparison

Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Activation of Piezo1 Channels Enhances Astrocyte–Neuron Communication via NMDA Receptors in the Murine Neocortex

doi: 10.3390/ijms25073994

Figure Lengend Snippet: The Piezo1 inhibitor Dooku1 fully reverted the actions of Yoda1 on SICs in higher concentration. ( A – C ) . Representative current traces under control conditions (magnesium-free aCSF, ( A )), with addition of 10 µM Yoda1 ( B ) and further addition of 20 µM Dooku1 ( C ). Gray arrows: SICs, asterisks: examples of EPSCs. ( D ). Statistical comparison of SIC parameters when 20 µM Dooku1 was used for reverting Yoda1 actions. The columns and error bars represent average ± SEM, the gray dots are the individual datapoints. *: p < 0.05; **: p < 0.01 (Tukey’s multiple comparisons test).

Article Snippet: Yoda1 (2-[5-[[(2,6-dichlorophenyl)methyl]thio]-1,3,4-thiadiazol-2-yl]-pyrazine; ProbeChem Biochemicals Ltd., Shanghai, China) [ , , , ] and D-AP5 (nonspecific inhibitor, Tocris Cookson Ltd., Bristol, UK) [ ] were used in 10 µM concentration.

Techniques: Concentration Assay, Comparison