Figure 1.
(A) To create potential DAG sensors, we inserted the cpGFP from G-GECO into the region interconnecting the pseudo substrate and the C1 domain, or in the hinge that connects the C1 domain with the enzyme (red). Some constructs were created with the entire PKCδ, in others the C2 domain was removed. (B) To pair the Upward or Downward DAG sensors with R-GECO, we connected the two coding regions, in frame, with an intervening 2A peptide sequence of 17 amino acids.
Table 1.
Summary of constructs created and tested.
Figure 2.
The responses of Green Downward DAG and Upward DAG sensors.
(A) Carbachol stimulation of the M1 receptor on cells expressing the Downward DAG sensor produces a 40% loss in fluorescence that occurs over ∼15 seconds (mean fluorescence over time of 4 cells). (B) The Upward DAG sensor shows a fluorescence increase of 45% over a similar time scale. (C) The signals generated by either sensor return to baseline quite slowly. (D) The apparent EC50 for carbacol-stimulated Upward DAG response is 3.5 uM. (E) The carbachol stimulation does not appear to activate all of the sensor pool in the cell since direct activation of the sensors with a subsequent application of PDBu produces an additional increase in fluorescence.
Figure 3.
Pairing the Green Upward and Downward DAG sensors with R-GECO makes it possible to simultaneously measure DAG and Ca2+ signaling in single cells.
(A) The Green Upward DAG sensor response is considerably slower than the red Ca2+ response in response to carbachol stimulation of the M1 receptor. (B) Similar kinetics occur with the Downward DAG sensor. (C) The two sensors can be activated independently: ionomycin, which should raise intracellular Ca2+ without affecting DAG levels produces a change in R-GECO but not Downward DAG, while the subsequent addition of PDBu activates Downward DAG (arrows indicate stimulus artifact).