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<p>Neuromodulators impose powerful control over brain function via their regulation of intracellular signaling through G-protein coupled receptors. In contrast to those of Gs and Gi pathways, in vivo imaging of the signaling events downstream of Gq-coupled receptors remains challenging. Here, we introduce CKAR3, a genetically encoded fluorescence lifetime sensor that reports the activity of protein kinase C (PKC), a major downstream effector of the Gq pathway. CKAR3 exhibits a lifetime dynamic range 5-fold larger than any existing PKC sensor. It specifically detects PKC phosphorylation with seconds kinetics without perturbing neuronal functions. In vivo two-photon lifetime imaging of CKAR3 reveals tonic PKC activity in cortical neurons. Animal locomotion elicits robust PKC activity in sparse neuronal ensembles in the motor cortex. Both basal and locomotion-elicited PKC activities are in part mediated by muscarinic acetylcholine receptors. Overall, CKAR3 enables interrogation of Gq signaling dynamics mediated by PKC in behaving animals.</p>

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A high-performance genetically encoded sensor for cellular imaging of PKC activity in vivo

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月10日

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  • Takaki Yahiro,
  • Landon Bayless-Edwards,
  • James A. Jones,
  • Yizhou Zhuo,
  • Lei Ma,
  • Maozhen Qin,
  • Tianyi Mao,
  • Haining Zhong

摘要

Neuromodulators impose powerful control over brain function via their regulation of intracellular signaling through G-protein coupled receptors. In contrast to those of Gs and Gi pathways, in vivo imaging of the signaling events downstream of Gq-coupled receptors remains challenging. Here, we introduce CKAR3, a genetically encoded fluorescence lifetime sensor that reports the activity of protein kinase C (PKC), a major downstream effector of the Gq pathway. CKAR3 exhibits a lifetime dynamic range 5-fold larger than any existing PKC sensor. It specifically detects PKC phosphorylation with seconds kinetics without perturbing neuronal functions. In vivo two-photon lifetime imaging of CKAR3 reveals tonic PKC activity in cortical neurons. Animal locomotion elicits robust PKC activity in sparse neuronal ensembles in the motor cortex. Both basal and locomotion-elicited PKC activities are in part mediated by muscarinic acetylcholine receptors. Overall, CKAR3 enables interrogation of Gq signaling dynamics mediated by PKC in behaving animals.