JournalNeural Excitability, Synapses, and Glia

Imaging ERK and PKA Activation in Single Dendritic Spines during Structural Plasticity

Top: Representative fluorescence lifetime images of ERK activation during glutamate uncaging. Stimulated spine was marked with the white arrowhead. Bottom: ERK activation during sLTP along the stimulated dendrite as a function of the distance from the base of the stimulated spine.

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  • Highly sensitive ERK and PKA FLIM sensors with novel fluorophore pair
  • Image ERK and PKA activation in single dendritic spines during structural plasticity
  • Mobile and immobilized sensors resolve spatiotemporal pattern of kinase activity

Summary

Extracellular signal-regulated kinase (ERK) and protein kinase A (PKA) play important roles in LTP and spine structural plasticity. While fluorescence resonance energy transfer (FRET)-based sensors for these kinases had previously been developed, they did not provide sufficient sensitivity for imaging small neuronal compartments, such as single dendritic spines in brain slices. Here we improved the sensitivity of FRET-based kinase sensors for monitoring kinase activity under two-photon fluorescence lifetime imaging microscopy (2pFLIM). Using these improved sensors, we succeeded in imaging ERK and PKA activation in single dendritic spines during structural long-term potentiation (sLTP) in hippocampal CA1 pyramidal neurons, revealing that the activation of these kinases spreads widely with length constants of more than 10 μm. The strategy for improvement of sensors used here should be applicable for developing highly sensitive biosensors for various protein kinases.


Tang, S., and Yasuda, R. Imaging ERK and PKA Activation in Single Dendritic Spines during Structural Plasticity. Neuron.
DOI: http://dx.doi.org/10.1016/j.neuron.2017.02.032

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