FIGURE 5.

Binding of A633(C2)CaM and eGFP-CaM-kinase II under varying conditions in living cells in culture. (Top) Confocal images of stably transfected eGFP-CaM-kinase II HEK293 cells electroporated with A633(C2)CaM are shown under 10 mM Ca²⁺ (left) followed by 10 mM Ca²⁺/1 mM MgATP (middle) and 200 mM EGTA (right) in the presence of 15 mg/ml α-hemolysin in the same dish (scale bar = 10 μm). (Middle) Intracellular auto- and cross-correlation curves were measured simultaneously under each of the conditions but at lower intracellular protein concentrations than used for the LSM images above. Plotted are the six subsequent 10-s acquisitions (thin lines) for both the auto- and cross-correlation measurements and their respective average curves with data fits (corresponding thick lines). (Note that the y axis for the green autocorrelation in A, right side in green, has a different scale than the other curves.) The amplitude of the cross-correlation curve (black) increases under elevated Ca²⁺ conditions (A, B) and almost disappears in the absence of Ca²⁺ (C). In comparison to the in vitro case, the cross-correlation amplitudes for both elevated Ca²⁺ conditions (A, B) are reduced due to the additional presence of unlabeled endogenous CaM in the cell. Distribution plots are shown at the bottom as seen in Fig. 4. The hatched shaded bars represent the distribution of CaM irrespective of the label, and the red bars denote the distribution of Alexa 633-labeled CaM molecules only. In A and B, an assumption of a high binding affinity yields virtually full binding (hatched shaded bar) and the indicated values for the labeled fractions r. In C the hatched bars exemplify a CaM distribution for r = 0.3 based on A and B. Distributions for conditions with other r fractions are also denoted (shaded lines).