CaMKII Actomyosin Interactions in Live Cells

Abstract

Synaptic stimulation leads to expansion of the dendritic spine caused by rearrangement of the actin cytoskeleton triggered by phosphorylation of the calcium calmodulin dependent kinase (CaMKII) and by myosin II activity. To study CaMKII-actomyosin interactions we have visualized single GFP-tagged CaMKII holoenzymes bound to tagRFP-tagged cortical actin in human endothelial cells by total internal reflection fluorescence microscopy. CaMKII phosphorylation mutants were used to examine whether release of CaMKII sequestered actin and myosin II induced F-actin fragmentation can explain the timing and extent of spine expansion. Thus far, we have found (i) Single GFP-CaMKII holoenzymes were resolved; in contrast to the less intense and rapidly mobile single GFP molecules. (ii) The holoenzymes have unimodal intensity distributions with < 30% standard deviation. The constitutively-active CaMKII T286D mutant, the sole exception, gave bimodal distributions with the higher mode six times as intense as the lower one. Subunit stoichiometries were determined by comparison against immobilized GFP single molecule intensities. (iii) CaMKII dwell-time distributions on stress fibers and filopodial structures were characterized. Dissociation rates were also measured by evanescent irradiation of photo-activatable GFP-CaMKII constructs bound to cortical actin (iv) The green / red fluorescence ratio of the actin structures relative to background gave an equilibrium measure of CaMKII binding. Affinity differences for different CaMKII isoforms and different phosphorylation states were estimated from the fluorescence ratios. In addition, single particle tracking was used to measure transient binding to the actin cytoskeleton. (v) We are currently measuring spatial co-operativity in the F-actin CaMKII binding sites and extending the methodology to analyse CaMKII diffusion along dendrites and spines in hippocampal neuron cultures. Our single molecule imaging data reveal spatial and macromolecular heterogeneities that may underlie aspects of the signal transduction response and cytoskeletal dynamics that are important for synaptic plasticity.