Drebrin regulates sub-spine localization of Calcium/calmodulin-dependent kinase II β

Abstract

Dendritic spines are actin-rich small protrusions that contain postsynaptic components of excitatory synapse. Many actin-binding proteins have been identified as spine-resident protein, and they regulate actin-cytoskeleton through diverse processes. Drebrin is a major F-actin binding protein in neurons, and is localized in the center of dendritic spines. In this study, we isolated CaMKIIβ as a drebrin-binding protein by yeast two-hybrid screen and investigated drebrin-CaMKIIβ relationship in dendritic spines. CaMKIIβ is localized in dendritic spines more than in dendritic shaft. However, drebrin knockdown (KD) caused diffuse localization of CaMKIIβ in dendrites, suggesting that drebrin anchors CaMKIIβ in dendritic spines. To analyze drebrin-dependence of CaMKIIβ stability in dendritic spine more directly, we performed fluorescence recovery after photobleaching (FRAP) experiments on individual dendritic spines. We found that the stable fraction of CaMKIIβ in drebrin-KD neurons was greater than that of control neurons. This result seems to be inconsistent with the results that CaMKIIβ diffused in dendrites of drebrin-KD neurons. We consequently think that drebrin-independent stable pool became dominant in drebrin-KD neurons. To test the hypothesis, we used stochastic optical reconstruction microscopy (STORM) to elucidate the localization of drebrin and CaMKIIβ in a dendritic spine on the nanometer scale. Our super resolution data showed that CaMKIIβ is partially co-localize with drebrin in the inner side of dendritic spines. In addition, the co-localization of CaMKIIβ and drebrin was separated by NMDA receptor activation, suggesting that active form of CaMKIIβ is free from drebrin. Furthermore, FRAP analysis showed that NMDA receptor activation markedly increased the stable fraction of CaMKIIβ. Together, these results suggest that CaMKIIβ is localized in dendritic spines as both drebrin-dependent pool and drebrin-independent more stable pool.