Figure 2.

Ca²⁺ wave velocity, rate of rise, decay, and frequency are dependent on temperature. (A) Image sequence shows a Ca²⁺ wave spreading from a single point of origin in an SH-SY5Y cell at 12°C after a 160 ms UV flash. Each frame shows a single 15 ms exposure together with a superimposed outline of the cell periphery; acquisition times are indicated in milliseconds, beginning just before the start of the response and 255 ms after the photolysis flash. The images depict background-subtracted ΔF/F₀ ratios, with the first frame showing a 10 μm scale bar, and the ROI used to generate the kymograph image in B. (B) Kymograph (linescan) images generated by measuring fluorescence ratio changes along 12 μm long lines positioned across cells, as illustrated in A, as a function of time. Data were obtained from representative cells at 12°C (left, the same cell as in A) and at 36°C (right). Time runs from top to bottom, with distance along the line depicted horizontally. Faint artifacts from the initiating UV flash are visible at the top of the image. White lines were superimposed by eye on the leading edge of the waves, and the slopes of these lines (distance/time) were used to calculate wavefront velocities. (C) Mean wavefront velocities estimated as in B, plotted as a function of temperature (n = 20, 15, 15, 15, 15, 15, and 25 cells at 12°C, 15°C, 22°C, 25°C, 30°C, 36°C, and 40°C, respectively). The lines fitted to the linear and Arrhenius plots correspond to a Q₁₀ of ∼1.56. (D) The rate-of-rise of global Ca²⁺ signals (ΔF/F_o s⁻¹) was determined from fluorescence recordings from small (6 × 6 pixel; 2 × 2 μm) ROIs over the site of origin by measuring the slopes of tangents fitted to the rising phase of the signals, as illustrated in the inset. The plots show the rate-of-rise as a function of temperature between 12°C and 40°C (n ≥ 26 cells for all temperatures). Lines are regression fits, with slopes corresponding to Q₁₀ = 1.12. (E) The frequency of Ca²⁺ oscillations in 105 cells showing repetitive Ca²⁺ waves in response to 160 and 320 ms photolysis flashes was determined by measuring the intervals between successive waves, as illustrated in the inset showing a representative trace from a strongly oscillating cell at 22°C. The main graph and Arrhenius graph plot the mean Ca²⁺ spike frequency (1/mean interval) as a function of temperature (n = 15 cells at each temperature). Lines indicate segmental linear regression fits with transition points at 26.2°C. (F) The rate of decay of Ca²⁺ spikes was assessed in cells (n = 20) that demonstrated strong oscillatory responses. First-order exponential curves were fitted to the falling phase of the signals (illustrated by the red curve in the inset), and the resulting mean decay time constants are plotted as a function of temperature. The lines on the main and Arrhenius plots are segmental linear regression fits with transition points at 30.0°C.