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

IF 36.1 1区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Nature Methods Pub Date : 2025-04-01 Epub Date: 2025-02-20 DOI:10.1038/s41592-025-02610-9
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
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引用次数: 0

Abstract

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.

完整脑内PTEN动态荧光寿命成像的基因编码生物传感器。
磷酸酶和紧张素同源物(PTEN)是维持细胞增殖和生长抑制刹车的重要蛋白质。因此,PTEN功能丧失突变与广泛的人类病理有关。尽管它很重要,但目前还没有方法直接监测完整生物系统中具有细胞特异性的PTEN活性。在这里,我们描述了一种基于fret的生物传感器的发展,使用PTEN构象作为PTEN活性状态的代理,用于双光子荧光寿命成像显微镜。我们确定了一个点突变,可以监测PTEN活性,对内源性PTEN信号的干扰最小。我们展示了PTEN在细胞系、完整秀丽隐杆线虫和小鼠大脑中的活性成像。最后,我们开发了一种红移传感器变体,使我们能够识别兴奋性和抑制性皮质细胞中细胞类型特异性PTEN活性。总之,我们的方法能够以前所未有的空间和时间分辨率动态成像体内PTEN活动。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Nature Methods
Nature Methods 生物-生化研究方法
CiteScore
58.70
自引率
1.70%
发文量
326
审稿时长
1 months
期刊介绍: Nature Methods is a monthly journal that focuses on publishing innovative methods and substantial enhancements to fundamental life sciences research techniques. Geared towards a diverse, interdisciplinary readership of researchers in academia and industry engaged in laboratory work, the journal offers new tools for research and emphasizes the immediate practical significance of the featured work. It publishes primary research papers and reviews recent technical and methodological advancements, with a particular interest in primary methods papers relevant to the biological and biomedical sciences. This includes methods rooted in chemistry with practical applications for studying biological problems.
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