Summary The ability to visualize endogenous proteins in living neurons provides a powerful means to interrogate neuronal structure and function. Here we generate recombinant antibody-like proteins, termed FingRs (Fibronectin intrabodies generated with mRNA display), that bind endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and which, when fused to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of the FingR incorporates a novel transcriptional regulation system that ties FingR expression to the level of the target and reduces background fluorescence. In dissociated neurons and brain slices FingRs generated against PSD-95 and Gephyrin did not affect the expression patterns of their endogenous target proteins or the number or strength of synapses. Together, our data indicate that PSD-95 and Gephyrin FingRs can report the localization and amount of endogenous synaptic proteins in living neurons and thus may be used to study changes in synaptic strength in vivo.
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.
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