Fig. 3.

Simulation and experimental measurement of cell responses to two-step stimuli of variable durations separated by a wash period of variable length. (A) Top: input stimulation profile of variable initial stimulation times followed by fixed wash and restimulation periods and corresponding LEGI responses. Bottom: simulated aLEGI responses for the population average (black) and for single cells with values of θ ranging from 0.55 to 0.85 (orange to brown curves). (B) Experimental verification of the model predictions in (A). Cells (n = 26) were stimulated with an initial 100 nM cAMP step input of variable duration (30 s or 2.5 min) and then washed for a short time interval (23 s) followed by a 5-min prolonged restimulation. (C) Model predictions for responses of cells stimulated with 100 nM cAMP for 5 min, washed with buffer for the indicated time periods, and restimulated with 100 nM cAMP for 5 min. (D) Percentage of the cells (n = 116) responding to the second step stimulation as a function of washing period in the model simulations (red line) and experiment (black circles). (E) Experimentally measured recovery of the average population response to the second stimulation step [cells stimulated as in (C)]. The pink curve denotes the cell response to the initial stimulation, and the green curves represent responses to the restimulation. Error bars denote SEM. (F) Sample images of PHcrac-GFP–expressing cells before and after their exposure to the first and second step stimulations with the indicated intermediate wash times. Scale bar, 10 µm.