RESUMO
The regulation of protein synthesis is vital for a host of cell biological processes, but investigating roles for protein synthesis have been hindered by the inability to selectively interfere with it. To inhibit protein synthesis with spatial and temporal control, we have developed a photo-releasable anisomycin compound, N-([6-bromo-7-hydroxycoumarin-4-yl]methyloxycarbonyl)anisomycin (Bhc-Aniso), that can be removed through exposure to UV light. The area of protein synthesis inhibition can be restricted to a small light-exposed region or, potentially, the volume of two-photon excitation if a pulsed IR laser is the light source. We have tested the compound's effectiveness with an in vitro protein-translation system, CHO cells, HEK293 cells, and neurons. The photo-released anisomycin can inhibit protein synthesis in a spatially restricted manner, which will enable the specific inhibition of protein synthesis in subsets of cells with temporal and spatial precision.
Assuntos
Anisomicina/farmacologia , Anisomicina/efeitos da radiação , Luz , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/efeitos da radiação , Animais , Anisomicina/síntese química , Anisomicina/química , Linhagem Celular , Cricetinae , Genes Reporter/genética , Humanos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fotólise/efeitos da radiação , Ratos , Análise Espectral , Raios UltravioletaRESUMO
We examined dendritic protein synthesis after a prolonged blockade of action potentials alone and after a blockade of both action potentials and miniature excitatory synaptic events (minis). Relative to controls, dendrites exposed to a prolonged blockade of action potentials showed diminished protein synthesis. Dendrites in which both action potentials and minis were blocked showed enhanced protein synthesis, suggesting that minis inhibit dendritic translation. When minis were acutely blocked or stimulated, an immediate increase or decrease, respectively, in dendritic translation was observed. Taken together, these results reveal a role for miniature synaptic events in the acute regulation of dendritic protein synthesis in neurons.