dag sensor gfp c1 2 δ 2xnls  (Addgene inc)

Journal: Biophysical Journal

Article Title: Roles for PKC signaling in chromaffin cell exocytosis

doi: 10.1016/j.bpj.2024.12.005

Figure Lengend Snippet: PACAP-stimulated DAG production is impaired in PLCε KO cells. ( A ) A PACAP-triggered signaling cascade regulates Ca 2+ transients in chromaffin cells. PACAP binds to its high-affinity receptor, PAC1, activating Gα s . Gα s stimulates adenylate cyclase, leading to cAMP production. Elevated cAMP activates Epac and, subsequently, PLCε. PLCε hydrolyzes PIP 2 into two key signaling molecules: IP 3 and DAG. IP 3 binds to its receptors on the endoplasmic reticulum, triggering Ca 2+ release into the cytosol. This study investigates the consequences of the DAG signaling axis ( boxed ). ( B ) Representative images obtained by TIRF imaging of WT and PLCε KO cells expressing a DAG sensor during stimulation (begins at time 0) with 500 nM PACAP. The images show changes in fluorescence intensity over time, indicating DAG production. Dotted lines indicate the cell boundaries based on bright-field images. Scale bars, 5 μ m. ( C ) The percentage change in fluorescence (%ΔF/F 0 ) versus time record of the DAG sensor in WT and PLCε KO cells under basal conditions (physiological saline solution [PSS]) and during PACAP stimulation. The graph depicts the time course of DAG production after PACAP stimulation, with bold lines representing the mean response and shaded areas representing the standard error of the mean. Data were collected from two independent experiments. Sample sizes are n = 15 (WT basal), n = 11 (WT PACAP), n = 9 (KO basal), and n = 15 (KO PACAP). ( D ) Scatterplots showing the individual maximum percent change in DAG sensor fluorescence in response to PACAP in both WT and PLCε KO cells, derived from the data shown in ( C ). Data are presented as mean ± SD. Statistical significance: ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons test. Not all comparisons are shown for clarity.

Article Snippet: Chromaffin cells were transduced with a green DAG upward sensor (Montana Molecular, U0300G), following the manufacturer’s protocol.

Techniques: Imaging, Expressing, Fluorescence, Saline, Derivative Assay

Journal: Diabetes

Article Title: Nicotinic Signaling Stimulates Glucagon Secretion in Mouse and Human Pancreatic α-Cells

doi: 10.2337/db23-0809

Figure Lengend Snippet: ACh elevates [DAG] i in α- and β-cells. A : Heat map outlining dynamics of [DAG] i in mouse islet α- and β-cells, in response to 10 μmol/L ACh, imaged using red fluorescent recombinant sensor Downward DAG. α- and β-cells were distinguished according to their positive and negative [cAMP] i response to 10 μmol/L adrenaline, which was imaged concurrently via green reporter of [cAMP] i , Upward cADDis. B : Quantification of [DAG] i data ( A ) for α- ( n = 51; five experiments) and β-cells ( n = 57). C : Representative traces of Ca 2+ dynamics evoked by ACh in pancreatic islet α-cells in presence of 5 mmol/L Ca 2+ chelator EGTA. D : Quantification of [Ca 2+ ] i data ( n = 22; five experiments) ( C ). E and F : Representative current-clamp recordings of plasma membrane electrical potential in α-cells in presence of 10 μmol/L ACh ( n = 6) ( E ) or 10 μmol/L nicotine ( n = 12) ( F ); arrow ( E ) indicates transient repolarization. G : Quantification of V m data ( n = 18, n = 6, n = 12) ( E and F ) at level of action potential amplitude and frequency. #Differences vs. 3 mmol/L glucose alone and *3 mmol/L glucose + 10 μmol/L ACh in α-cells are significant ( P < 0.05). pAUC, partial area under curve.

Article Snippet: Recombinant sensors (Downward Green DAG [diacylglycerol], Upward Red cADDis) were delivered via BacMam (Montana Molecular, Bozeman, MT) vectors at 10 5 international units per islet, followed by 36-h culturing (as above) to express the proteins.

Techniques: Recombinant, Membrane