Journal of Neuroscience 2014-12-10

Live imaging of endogenous PSD-95 using ENABLED: a conditional strategy to fluorescently label endogenous proteins.

Dale A Fortin, Shane E Tillo, Guang Yang, Jong-Cheol Rah, Joshua B Melander, Suxia Bai, Omar Soler-Cedeño, Maozhen Qin, Boris V Zemelman, Caiying Guo, Tianyi Mao, Haining Zhong

文献索引：J. Neurosci. 34(50) , 16698-712, (2014)

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摘要

Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo.Copyright © 2014 the authors 0270-6474/14/3416698-15$15.00/0.