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<p>The phosphatase and tensin homolog (PTEN) is a vital protein that maintains an inhibitory brake for cellular proliferation and growth. Accordingly, <i>PTEN</i> loss-of-function mutations are associated with a broad spectrum of human pathologies. Despite its importance, there is currently no method to directly monitor PTEN activity with cellular specificity within intact biological systems. Here we describe the development of a FRET-based biosensor using PTEN conformation as a proxy for the PTEN activity state, for two-photon fluorescence lifetime imaging microscopy. We identify a point mutation that allows the monitoring of PTEN activity with minimal interference to endogenous PTEN signaling. We demonstrate imaging of PTEN activity in cell lines, intact <i>Caenorhabditis</i> <i>elegans</i> and in the mouse brain. Finally, we develop a red-shifted sensor variant that allows us to identify cell-type-specific PTEN activity in excitatory and inhibitory cortical cells. In summary, our approach enables dynamic imaging of PTEN activity in vivo with unprecedented spatial and temporal resolution.</p>

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Genetically encoded biosensor for fluorescence lifetime imaging of PTEN dynamics in the intact brain

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年2月20日

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  • Tomer Kagan,
  • Matan Gabay,
  • Aasha Meenakshisundaram,
  • Yossi Levi,
  • Sharbel Eid,
  • Nikol Malchenko,
  • Maya Maman,
  • Anat Nitzan,
  • Luca Ravotto,
  • Ronen Zaidel-Bar,
  • Britta Johanna Eickholt,
  • Maayan Gal,
  • Tal Laviv

摘要

The phosphatase and tensin homolog (PTEN) is a vital protein that maintains an inhibitory brake for cellular proliferation and growth. Accordingly, PTEN loss-of-function mutations are associated with a broad spectrum of human pathologies. Despite its importance, there is currently no method to directly monitor PTEN activity with cellular specificity within intact biological systems. Here we describe the development of a FRET-based biosensor using PTEN conformation as a proxy for the PTEN activity state, for two-photon fluorescence lifetime imaging microscopy. We identify a point mutation that allows the monitoring of PTEN activity with minimal interference to endogenous PTEN signaling. We demonstrate imaging of PTEN activity in cell lines, intact Caenorhabditis elegans and in the mouse brain. Finally, we develop a red-shifted sensor variant that allows us to identify cell-type-specific PTEN activity in excitatory and inhibitory cortical cells. In summary, our approach enables dynamic imaging of PTEN activity in vivo with unprecedented spatial and temporal resolution.