FIGURE 4.
FRAP beam-size analysis suggests activator-dependent distinct modes of PLCβ2-GFP interactions with the plasma membrane. FRAP experiments were conducted at 22 °C as in Fig. 3, on COS-7 cells transfected with PLCβ2-GFP, and an excess of empty vector (Control) or vectors encoding the indicated proteins as described under “Experimental Procedures.” Two beam sizes were generated using a 63× and 40× objectives (see “Experimental Procedures”), and the τ values were determined with each. The ratio between the areas illuminated by the two beams, ω2(40×)/ω2(63×), was 2.56 (n = 39). This ratio is expected for FRAP by lateral diffusion, whereas a ratio of 1 is expected for recovery by exchange (37). The Rf values were high in all cases (≥0.93). A, τ values. Bars are means ± S.E. of 40–60 measurements, each conducted on a different cell. Comparing τ values measured with the same beam size, Rac2(G12V) and αq induced significant increases in τ of PLCβ2-GFP relative to the control (***, p < 10−6; **, p < 0.005; Student's t test). β1γ2 had no significant effect on τ(40×), but reduced τ(63×) (*, p < 0.02). B, τ(40×)/τ(63×) ratios. The ratio values (τ ratios and the beam-size ratio) and their S.E. were calculated from the experimentally measured values (τ(40×) and τ(63×) for τ ratio, ω2(40×) and ω2(63×) for the beam-size ratio) using bootstrap analysis. The bootstrap analysis (see “Experimental Procedures”) showed that the τ ratios of PLCβ2 differ significantly from the 2.56 beam-size ratio predicted for FRAP by lateral diffusion in all cases (***, p < 10−6; *, p < 0.02), except for co-expression with β1γ2 (p > 0.3). Comparison of the τ ratios to 1 (the value expected for FRAP by exchange) using bootstrap analysis shows that in the presence of Rac2(G12V) the τ ratio of PLCβ2-GFP is not significantly different from 1 (p > 0.4).