Recombinant Probes Reveal Dynamic Localization of CaMKIIα within Somata of Cortical Neurons

Abstract

In response to NMDA receptor stimulation, CaMKII␣ moves rapidly from a diffuse distribution within the shafts of neuronal dendrites to a clustered postsynaptic distribution. However, less is known about CaMKII␣ localization and trafficking within neuronal somata. Here we use a novel recombinant probe capable of labeling endogenous CaMKII␣ in living rat neurons to examine its localization and trafficking within the somata of cortical neurons. This probe, which was generated using an mRNA display selection, binds to endogenous CaMKII␣ at high affinity and specificity following expression in rat cortical neurons in culture. In ϳ45% of quiescent cortical neurons, labeled clusters of CaMKII␣ 1-4 m in diameter were present. Upon exposure to glutamate and glycine, CaMKII␣ clusters disappeared in a Ca 2ϩ -dependent manner within seconds. Moreover, minutes after the removal of glutamate and glycine, the clusters returned to their original configuration. The clusters, which also appear in cortical neurons in sections taken from mouse brains, contain actin and disperse upon exposure to cytochalasin D, an actin depolymerizer. In conclusion, within the soma, CaMKII localizes and traffics in a manner that is distinct from its localization and trafficking within the dendrites.

Simultaneous Live Imaging of Multiple Endogenous Proteins Reveals a Mechanism for Alzheimer’s-Related Plasticity Impairment

“…A third intrabody was directed against CaMKIIα and was labeled with YFP2. Individual expression of the corresponding originally GFP-labeled intrabodies does not alter synaptic functions or localization of the detected endogenous proteins (Barcomb et al, 2015; Gross et al, 2013; Mora et al, 2013). Individual expression of the modified intrabodies also resulted in faithful labeling of the subcellular localization of their respective endogenous protein (Figures S1A and S1B).…”

“…The expression vectors for the GFP-labeled FingR intrabodies targeting CaMKIIα, PSD-95, and gephyrin were kindly provided by Dr. Donald Arnold (University of Southern California, Los Angeles, CA, USA) as described previously (Gross et al, 2013; Mora et al, 2013). The FingRs against PSD95 and gephyrin contained the CCR5TC repressor (Gross et al, 2013); the FingR against CaMKIIα did not contain a repressor (Mora et al, 2013). The fluorophore label was exchanged to contain the following tags in place of GFP: CaMKIIa-FingR-YFP2, PSD-95-FingR-mCh, and gephyrin-FingR-mTurquois.…”

DAPK1 Mediates LTD by Making CaMKII/GluN2B Binding LTP Specific

“…Then, inhibition of either CaN or DAPK1 should allow synaptic CaMKII accumulation also during LTD stimuli. Here, we expressed a GFP-labelled intrabody against CaMKII (Mora et al, 2013) in hippocampal neurons in order to enable live-imaging of the movement of endogenous CaMKII during LTD stimuli (Figure 5A; Movies 1–3). The monitoring of endogenous rather than overexpressed CaMKII is important in LTD: While endogenous CaMKII accumulates in dendritic spines only during LTP, overexpressed GFP-CaMKII shows such accumulation also after LTD (Marsden et al, 2010).…”

An E3-ligase-based method for ablating inhibitory synapses

“…FingRs can target fluorescent proteins to endogenous target proteins, allowing the targets and the macromolecular structures where they reside to be visualized in vivo 11,27 . Here we show that an E3 ligase can also be targeted specifically using a FingR, causing expression of its target protein to be quickly and reversibly eliminated.…”