Figure 3.

CaMKIIβ decoration of the actin cytoskeleton visualized by two-color TIRFM. (A) Averaged images of β (left panel, green, 400 frames) and tRFP-actin (right panel, red, 100 frames). Mean tRFP intensity = 119 ± 9 counts/pixel. The bottom panels (arrow) show the two frames superimposed (P_pix = 0.27, P_rand = 0.09 ± 0.07) (left) and the single-particle tracks (right) accumulated over 10 s of video (Movie S1). (B) MSD-versus-time interval (Δt) for the total population of tracks (white circles) and short-lived (yellow circles) and long-lived (blue circles) track subpopulations (± standard deviation (σ)). The initial gradient of the short-lived track data gives D_lat = 0.28 μm²/s, whereas that of the long-lived tracks gives 0.04 μm²/s. Total number of tracks, n = 12,723. (C) Intensity histograms for the short-lived track subpopulation (yellow bars) and long-lived track subpopulation (blue bars). The asterisk (black) marks the region of the histogram that was used to analyze photobleaching. (D) Sample intensity-versus-time plots for some of the objects from the asterisk-marked region. Stepwise intensity changes as detected by Student’s t-test (Fig. S1) are marked immediately below each trace to indicate sudden intensity transitions. The starting intensity for each spot was >170 counts/pixel, which is ∼8-fold greater than the unitary GFP intensity. The green line (in the lowest panel), is the two-step immobilized GFP photobleaching, redrawn from Fig. 2B(v), shown for reference.