Abstract

Calcium calmodulin dependent kinase (CaMKII) has an important role in dendritic spine remodelling upon synaptic stimulation. Using fluorescence video microscopy and image analysis, we investigated the architectural dynamics of rhodamine-phalloidin stabilized F-actin networks cross-linked by CaMKII. We used automated image analysis to identify F-actin bundles and cross-over junctions and developed a dimensionless metric to characterize network architecture. Similar networks were formed by three different CaMKII species with ten-fold length difference in the linker region between the kinase domain and holoenzyme hub; implying linker length is not a primary determinant of F-actin binding. Electron micrographs showed that, at physiological molar ratios, single CaMKII holoenzymes cross-linked multiple F-actin filaments in random networks, whereas at higher CaMKII / F-actin ratios filaments bundled. Light microscopy established that random networks resisted macromolecular crowding, with polyethylene glycol mimicking cytoplasmic osmolarity, and blocked ATP-powered compaction by myosin-2 mini-filaments. Importantly, the networks disassembled following addition of calcium calmodulin and were then rapidly spaced into compacted foci by myosin motors or, more slowly, aggregated by crowding. Single molecule TIRF microscopy showed CaMKII dissociation from surface-immobilized G-actin exhibited a mono-exponential dwell-time distribution, whereas CaMKII bound to F-actin networks had a long-lived fraction, trapped at cross-over junctions. Release of CaMKII from F-actin, triggered by calcium calmodulin did not require ATP (hence phosphorylation) and was too rapid to measure with video-rate imaging. The residual bound-fraction was reduced substantially upon addition of an NMDA receptor peptide analogue. These results provide mechanistic insights to CaMKII-actin interactions at the collective network and single molecule level. Our findings argue that CaMKII-actin networks in dendritic spines are stable enough to protect the basal network architecture against physical stress but once CaMKII is disengaged by calcium calmodulin and sequestered by receptors at the synapse; F-actin compaction by myosin motors stabilizes the expanded spine compatible with the recorded times.