Figure 6. Reversible activity-dependent colocalization of CaMKII holoenzymes.

(A) Schematic representation of CaMKII in vitro enzymatic labeling with maleimide dyes and biotin, and TIRF experimental set-up. (B) Representative TIRF images of unactivated (basal) CaMKII^WT sample (left), activated CaMKII^WT sample (middle), and CaMKII^WT sample first activated and then quenched with EDTA (right). Raw, unprocessed, images are shown. (C) Summary of colocalization analysis (Pearson coefficient) for TIRF images for CaMKII under different conditions. Statistical significance was calculated using a multi-comparison test (Dunnett test) with α=0.05. Mean and standard deviation of the mean are indicated. Each condition was done at least two times on different days, with different protein preps. Five ROIs coming from different slides were selected for analysis for each condition. Scale bar: 10 µm.

Figure 6—figure supplement 1. Activation stimuli are necessary for CaMKII^WT holoenzyme colocalization.

(A) Absence of colocalization between differently labeled CaMKII^WT holoenzymes in the absence of Mg²⁺:ATP. (B) Absence of colocalization between differently labeled CaMKII^WT holoenzymes upon chelation of Ca²⁺ by BAPTA. (C) Absence of colocalization between differently labeled CaMKII^WT holoenzymes in the absence of CaM. (D) Absence of colocalization between differently labeled CaMKII^WT holoenzymes incubated first in activating conditions, and following later chelation of Mg²⁺ by EDTA. Scale bar: 10 µm.

Figure 6—figure supplement 2. CaMKII^KD holoenzymes cluster only in the presence of activated CaMKII^WT holoenzymes.

(A) Absence of colocalization between CaMKII^KD and CaMKII^WT in basal conditions. (B) Colocalization of CaMKII^KD and CaMKII^WT upon activation. (C) Absence of colocalization between differently labeled CaMKII^KD in basal conditions. (D) Absence of colocalization between differently labeled CaMKII^KD in activating conditions. (E) Summary of colocalization analysis. Probability of no difference from Dunnett’s multiple comparison test. Scale bar: 10 µm.