FIGURE 3.

Spatiotemporal patterns of intracellular Ca²⁺ puffs stimulated by an LPA gradient. A, Fluo-4 AM fluorescence images of [Ca²⁺]_i responses in DKO-LPA₂ MEFs exposed to an LPA gradient emitted by a micropipette containing 200 nm LPA. Scale bar, 50 μm. B, TIRF imaging of Fluo-4 AM-stained DKO-LPA₂ MEFs exposed to an LPA gradient produced by a micropipette containing 200 nm LPA. Scale bar, 10 μm. White stars, sites of the micropipette. C, schematic showing the method used to define the distribution of the percentage of Ca²⁺ puffs at the regions exposed to higher LPA concentration (a) and the regions exposed to lower LPA concentration (b) in relation to the site of the micropipette. D, the quantitative analysis of the percentage of Ca²⁺ puffs localized in regions a and b. Data are presented as mean ± S.E. (error bars). E, LPA gradient-induced differential intracellular Ca²⁺ mobilization in DKO-LPA₂ MEFs, not in DKO MEFs. F, plot showing the probability of triggering a Ca²⁺ wave by LPA-induced Ca²⁺ puffs in DKO-LPA₂ MEFs (n = 42). G, low frequency Ca²⁺ puffs (<3 Hz) failed to trigger a Ca²⁺ wave. Scale bar, 10 μm. H, high frequency Ca²⁺ puffs (>5 Hz) triggered a Ca²⁺ wave. Scale bar, 10 μm. In G and H, the circles show the locations of the active Ca²⁺ puff sites. The Ca²⁺ signals recorded at marked regions are depicted by the corresponding traces. The amplitude is expressed as F/F₀. F is the fluorescence intensity in the marked regions where the spark appeared. F₀ is fluorescence intensity of the same area in the absence of Ca²⁺ puffs. The red arrows indicate the site with the highest LPA concentration. IB, immunoblot.