Journal: The EMBO Journal

Article Title: Absolute proteomic quantification reveals design principles of sperm flagellar chemosensation

doi: 10.15252/embj.2019102723

Figure Lengend Snippet: A, B Ca 2+ signals elicited by cGMP release and recorded with FluoForte. The UV flash released 28 (A) and 215 B) cGMP molecules. Light flashes are indicated by dashed magenta lines. C pH i signals, elicited by the release of approximately 1,400 cGMP molecules, were recorded with pHrodo Red. An increase in pH i is indicated by a decrease in ∆ F / F . Exemplary recording from a single cell. D Average of pH i signals recorded from n = 25 cells. Error bars represent SD See Appendix Fig S2 for a longer time course.

Article Snippet: Sperm cells were suspended 1:100 (v/v) in ASW supplemented with 0.5% Pluronic F127 (Sigma‐Aldrich) and either 30 μM GFP‐certified FluoForte‐AM (Enzo Life Sciences; Ca 2+ imaging) or 20 μM pHrodo Red (Molecular Probes; pH imaging).

Techniques:

Journal: Nature chemical biology

Article Title: Signaling Diversity of PKA Achieved Via a Ca 2+ -cAMP-PKA Oscillatory Circuit

doi: 10.1038/nchembio.478

Figure Lengend Snippet: Oscillatory changes in PKA activity, cAMP and Ca 2+ dynamics are highly coordinated in MIN6 cells. (A) Domain structures of ICUE-YR, AKAR-GR, and a single-chain dual-specificity biosensor, ICUEPID, for PKA activity and cAMP dynamics. (B) (Top panel) Fluorescence images of MIN6 cells expressing AKAR-GR loaded with Fura-2. (Bottom Panel) Representative time courses showing coordinated oscillations in PKA activity (monitored by AKAR-GR, red) and Ca 2+ (monitored by Fura-2, black) in single MIN6 cells. Scale bar = 10 μm. (C) (Top panel) Fluorescence images of a MIN6 cell expressing ICUE-YR loaded with Fura-2. (Bottom Panel) Representative time courses showing coordinated oscillations in cAMP (monitored by ICUE-YR, red) and Ca 2+ (monitored by Fura-2, black) in single MIN6 cells. Scale bar = 10 μm. (D) (Top panel) Fluorescence images of a MIN6 cell expressing ICUEPID (bottom Panel). Representative time courses showing coordinated oscillations in PKA activity (red) and cAMP (black) monitored by ICUEPID in single MIN6 cells. Scale bar = 10 μm. (E) Simulation of the model showing Ca 2+ (black) and active PKA (PKA*, red) oscillations. (F) Simulation of the mathematical model showing Ca 2+ (black) and cAMP (red) oscillations. (G) Simulation of the model showing cAMP (black) and active PKA (PKA*, red) oscillations. Norm. emission and Norm. conc. refer to normalized emission and normalized concentration respectively, with normalization in simulations made with respect to the maximal value in the corresponding time course.

Article Snippet: For Ca 2+ imaging experiments, cells were preincubated with 1 μM Fura-2/AM (Molecular Probes) for 10–20 min at 37 °C before imaging

Techniques: Activity Assay, Fluorescence, Expressing, Concentration Assay

Journal: Nature chemical biology

Article Title: Signaling Diversity of PKA Achieved Via a Ca 2+ -cAMP-PKA Oscillatory Circuit

doi: 10.1038/nchembio.478

Figure Lengend Snippet: PKA activity is required for Ca 2+ oscillation and tunes its frequency. (A) Simulation of the model in the presence or absence of PKA (shaded region). (B) The effect of inhibiting PKA by H89 (10 μM) on Ca 2+ oscillation. (C) Simulation of the model with increased feedback achieved when PDE activity is decreased (shaded region). (D) Effect of adding a PDE inhibitor IBMX (100 μM) on Ca 2+ oscillations (n = 15). (E) Simulation of the model with increased feedback achieved when PP2B activity is decreased (shaded region). (F) Effect of adding a PP2B inhibitor cyclosporine A (CsA) (3 μM) on Ca 2+ oscillations (n = 7). (G) Effect of PKA activation and activity parameters on the frequency of oscillations, simulated by the simultaneous variation of a parameter relating to the extent of PKA phosphorylation of channels (k PKA,V ) and a parameter controlling the maximal activity of PDE (k PDE ). (H) Effect of PKA activation and activity parameters on the amplitude of oscillations, simulated by the simultaneous variation of k PKA,V and k PDE . Note the scale of the amplitude changes. Norm. [Ca 2+ ] i refers to intracellular Ca 2+ concentration normalized to the maximal level and Amp. refers to amplitude of oscillations. See related analysis in the .

Article Snippet: For Ca 2+ imaging experiments, cells were preincubated with 1 μM Fura-2/AM (Molecular Probes) for 10–20 min at 37 °C before imaging

Techniques: Activity Assay, Activation Assay, Phospho-proteomics, Concentration Assay

Journal: Nature chemical biology

Article Title: Signaling Diversity of PKA Achieved Via a Ca 2+ -cAMP-PKA Oscillatory Circuit

doi: 10.1038/nchembio.478

Figure Lengend Snippet: Oscillatory PKA activity confers spatial control of substrates. (A) Simulation of the model showing the indirect activities of local (normalized mean [Ca 2+ ]) and global (using normalized mean PKA C-subunit concentration as a proxy) targets of PKA activation upon increase in the input AC activity and hence frequency of oscillations. The expected “local-activation” regime, defined by the AC activity at which the difference between log (Normalized mean PKAactivity) and log (Normalized peak PKAactivity) is maximal, is shaded in orange. The area shaded in green is bounded by the nominal AC activity, reflecting the expected physiological scenario. (B) Representative time courses of nuclear localized AKAR (NLS-AKAR) showing the absence and presence of nuclear PKA activity upon stimulation with low (1–3 μM) and high (10–20 μM) doses of a PKA-specific cAMP analog, respectively (n = 7 and 4, respectively). (C) Phospho-immunoblot analysis using antiphospho-CREB (pS133) shows no changes in CREB phosphorylation upon stimulation with a low dose (LD) of the cAMP analog (2 μM), while increased phosphorylation of CREB is observed upon stimulation with a high dose (HD) of the same cAMP analog (10 μM) or 50 μM forskolin (FSK). (D) Densitometric analysis of phosphorylated CREB (pS133) (n =3) normalized to CREB expression shows a significant difference between the levels of CREB phosphorylation stimulated by the low and high doses of the cAMP analog.

Article Snippet: For Ca 2+ imaging experiments, cells were preincubated with 1 μM Fura-2/AM (Molecular Probes) for 10–20 min at 37 °C before imaging

Techniques: Activity Assay, Control, Concentration Assay, Activation Assay, Western Blot, Phospho-proteomics, Expressing

Journal: Stem Cell Reports

Article Title: Characterization of N-terminal RYR2 variants outside CPVT1 hotspot regions using patient iPSCs reveal pathogenesis and therapeutic potential

doi: 10.1016/j.stemcr.2022.07.002

Figure Lengend Snippet: RYR2-L14P iPSC-CMs display changes in Ca 2+ transient measurements and sparking activity compared with isogenic control (A) Calcium transient amplitude normalized by (ΔF/F0). (B) Percentage of area of 40× microscopic field displaying calcium sparking activity using Fluo-4 Ca 2+ imaging at baseline and following treatment with 1 μM ISO. (C) Representative calcium transient tracings in isogenic control (black) and RYR2-L14P (orange) iPSC-CMs at BL and after ISO. (D) Representative splice-view images of calcium transients after ISO treatment. Data presented as mean ± SEM. n = 5–72 per group ( Table S1 ). 3–8 independent experiments were conducted. A two-way ANOVA was performed with post hoc Tukey-Kramer testing. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. iPSC-CMs used were 30 to 50 days old. See also Figure S4 .

Article Snippet: Imaging of Ca 2+ transients was taken under a 40× objective using a Nikon Eclipse Ti light microscope under BL conditions, and then the same dish was stimulated with ISO for 1 to 10 min. Time-lapse videos of multiple, individual beating isogenic control, RYR2-L14P, RYR2-F13L, RYR2-R15P, and RYR2-R176Q iPSC-CMs, paced at 0.5 Hz, were recorded at a speed of 20 ms per frame for 20 s at 10% LED power.

Techniques: Activity Assay, Control, Imaging

Journal: Stem Cell Reports

Article Title: Characterization of N-terminal RYR2 variants outside CPVT1 hotspot regions using patient iPSCs reveal pathogenesis and therapeutic potential

doi: 10.1016/j.stemcr.2022.07.002

Figure Lengend Snippet: Altered Ca 2+ handling kinetics and sparking activity in RYR2-F13L, -R15P, and -R176Q iPSC-CMs (A) Calcium transient amplitude normalized by (ΔF/F0). (B) Percentage of area of 40× microscopic field displaying calcium sparking activity using Fluo-4 Ca 2+ imaging at baseline and following treatment with 1 μM ISO. (C) Representative calcium transient tracings in control (black), F13L (red), R15P (blue), and R176Q (green) iPSC-CMs at BL and after ISO. (D) Representative splice-view images of calcium transients after ISO treatment. Data presented as mean ± SEM. n = 2–67 per group ( Table S1 ). 3–7 independent experiments were conducted. A two-way ANOVA was performed with post hoc Tukey-Kramer testing. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. iPSC-CMs used were 30 to 50 days old. See also Figure S5 .

Article Snippet: Imaging of Ca 2+ transients was taken under a 40× objective using a Nikon Eclipse Ti light microscope under BL conditions, and then the same dish was stimulated with ISO for 1 to 10 min. Time-lapse videos of multiple, individual beating isogenic control, RYR2-L14P, RYR2-F13L, RYR2-R15P, and RYR2-R176Q iPSC-CMs, paced at 0.5 Hz, were recorded at a speed of 20 ms per frame for 20 s at 10% LED power.

Techniques: Activity Assay, Imaging, Control

Journal: Stem Cell Reports

Article Title: Characterization of N-terminal RYR2 variants outside CPVT1 hotspot regions using patient iPSCs reveal pathogenesis and therapeutic potential

doi: 10.1016/j.stemcr.2022.07.002

Figure Lengend Snippet: Ca 2+ transient amplitude and sparking activity in RYR2-L14P compared with its isogenic control after CPVT1 pharmacotherapy (A–G) Calcium transient amplitude normalized by (ΔF/F0), percentage of area of 40× microscopic field displaying calcium sparking activity, and representative tracings in isogenic control (black); RYR2-L14P (orange); RYR2-L14P + 10 μM Nad (purple); RYR2-L14P + 10 μM Flec (blue); RYR2-L14P + 25 μM Flec (green); and RYR2-L14P + 10 μM Nad +25 μM Flec (red) iPSC-CMs at BL (A, C, and E), and following 1 μM ISO (B, D, F, and G). Data presented as mean ± SEM. n = 3–97 per group ( Table S2 ). independent experiments were conducted. A one-way ANOVA was performed with post-hoc Tukey-Kramer testing. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. iPSC-CMs used were 30–50 days old.

Article Snippet: Imaging of Ca 2+ transients was taken under a 40× objective using a Nikon Eclipse Ti light microscope under BL conditions, and then the same dish was stimulated with ISO for 1 to 10 min. Time-lapse videos of multiple, individual beating isogenic control, RYR2-L14P, RYR2-F13L, RYR2-R15P, and RYR2-R176Q iPSC-CMs, paced at 0.5 Hz, were recorded at a speed of 20 ms per frame for 20 s at 10% LED power.

Techniques: Activity Assay, Control

Journal: Nature Cardiovascular Research

Article Title: Piezo1 is the cardiac mechanosensor that initiates the cardiomyocyte hypertrophic response to pressure overload in adult mice

doi: 10.1038/s44161-022-00082-0

Figure Lengend Snippet: ( a ) Exemplary Ca 2+ images of single mouse LV cardiomyocyte. Scale bar = 50 µm. ( b ) Representative normalized intensity traces of the cardiomyocytes during experiment. Gray arrows indicating Yoda1 addition (final concentration 30 μM). Top: peanut oil-treated P1 fl/fl MCM +/- control mice without Yoda1 addition; middle: peanut oil-treated P1 fl/fl MCM +/- mice with Yoda1 addition; bottom: tamoxifen-treated P1 fl/fl MCM +/- mice with Yoda1 addition, in both ( a ) and ( b ). ( c ) The ratio of cardiomyocytes responding to Yoda1, compared between peanut oil-treated and tamoxifen-treated P1 fl/fl MCM +/- mice. n = 5 wells in a 96-well plate in each group. In total 50 cells from 2 hearts in peanut oil-treated group; 72 cells from 2 hearts in tamoxifen-treated group. Results are presented as mean ± SEM with scatter plot, unpaired two-tailed student’s T-test, *** p < 0.001 vs. peanut oil-treated cardiomyocytes.

Article Snippet: Ca 2+ imaging and data recording were carried out on a Nikon Eclipse Ti2-E Inverted epifluorescence microscope (Nikon Instruments), using a ×20 objective lens, with 2 × 2 binning imaging at 50 frames per s. The data were continuously recorded for 160 s in total.

Techniques: Concentration Assay, Control, Two Tailed Test