RESUMO
In response to NMDA receptor stimulation, CaMKIIα moves rapidly from a diffuse distribution within the shafts of neuronal dendrites to a clustered postsynaptic distribution. However, less is known about CaMKIIα localization and trafficking within neuronal somata. Here we use a novel recombinant probe capable of labeling endogenous CaMKIIα 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 CaMKIIα at high affinity and specificity following expression in rat cortical neurons in culture. In â¼45% of quiescent cortical neurons, labeled clusters of CaMKIIα 1-4 µm in diameter were present. Upon exposure to glutamate and glycine, CaMKIIα clusters disappeared in a Ca(2+)-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.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Córtex Cerebral/metabolismo , Neurônios/metabolismo , Actinas/metabolismo , Animais , Células COS , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Córtex Cerebral/citologia , Chlorocebus aethiops , Citocalasina D/farmacologia , Ácido Glutâmico/farmacologia , Glicina/farmacologia , Camundongos , Transporte Proteico/efeitos dos fármacos , RatosRESUMO
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 Fibronectin intrabodies generated with mRNA display (FingRs), that bind endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and that, when fused to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of the FingR incorporates a 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.
