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Fluorescent and photo-oxidizing TimeSTAMP tags track protein fates in light and electron microscopy
Margaret T Butko1, Jin Yang1, Yang Geng2,3, Hyung Joon Kim4, Noo Li Jeon4, Xiaokun Shu1,5,6, Mason R Mackey7,8, Mark H Ellisman7,8, Roger Y Tsien1 & Michael Z Lin13
npg 2012 Nature America, Inc. All rights reserved.
Protein synthesis is highly regulated throughout nervous system development, plasticity and regeneration. However, tracking the distributions of specific new protein species has not been possible in living neurons or at the ultrastructural level. Previously we created TimeSTAMP epitope tags, drug-controlled tags for immunohistochemical detection of specific new proteins synthesized at defined times. Here we extend TimeSTAMP to label new protein copies by fluorescence or photo-oxidation. Live microscopy of a fluorescent TimeSTAMP tag reveals that copies of the synaptic protein PSD95 are synthesized in response to local activation of growth factor and neurotransmitter receptors, and preferentially localize to stimulated synapses in rat neurons. Electron microscopy of a photo-oxidizing TimeSTAMP tag reveals new PSD95 at developing dendritic structures of immature neurons and at synapses in differentiated neurons. These results demonstrate the versatility of the TimeSTAMP approach for visualizing newly synthesized proteins in neurons.
Spatiotemporal control of protein synthesis is essential for proper development, normal functioning and adaptation of nervous systems. In embryonic neurons, proteins are synthesized in axonal growth cones during migration, and local inhibition of protein synthesis blocks growth-cone responses to axon guidance cues1,2. Later in development, high levels of protein synthesis in dendrites and axons promote synapse formation3,4. In the mature nervous system, protein synthesis is induced by neuronal activity and is required for memory consolidation in animals5,6. Persistence of long-term potentiation (LTP) and long-term depression (LTD), activity-dependent changes in synaptic function believed to underlie learning, also requires new protein synthesis to persist beyond 1 h after induction7,8. The production and targeting of new proteins also appears to be critical, as inhibition of protein synthesis locally at stimulated synapses blocks late-phase LTP at those synapses9.
The intricate regulation of protein synthesis during differentiation and plasticity of subcellular structures such as axons and synapses
suggests that those synthesized proteins are used in these structures. An attractive hypothesis for the function...