Figure 4.

CaMKII variants differ in their motility within cells. FRAP of GFP-CaMKII was assessed in Cos-7 cells at 30°C. (A) Top, examples of GFP-CaMKII bleach and recovery in Cos-7 cells. Two examples are shown for βe: one bleach of its typical dispersed localization and one bleach is of an F-actin–like structure. Bottom, quantitative assessment of bleach recovery over time, with fluorescence before and immediately after bleach rescaled to 1 and 0, respectively. FRAP differed between cytosolic (α and βe) and actin-binding (β and β′) variants (p < 0.005 in two tailed t tests; n = 10 bleaches in 5 cells for each variant). (B) Apparent bleach efficiencies differed between cytosolic and actin-binding variants in live but not in fixed cells. *, not different from each other (p > 0.4), but different from all other measurements (p < 0.005); other data points do not differ from each other (p > 0.4; ANOVA) (n = 10). This suggests partial recovery even before the first picture could be taken (∼2 s after bleach). This indicates that the recoverable pool for the cytosolic variants is even greater than indicated by the rescaled measurements in A. Error bars show SEM.