Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides
Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides
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DOI：
10.1101/2023.10.18.562882
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发表时间：
2023-10
期刊：
影响因子：
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通讯作者：
Hannes Maib;Petia Adarska;Robert Hunton;James H Vines;David Strutt;Francesca Bottanelli;David H. Murray
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文献类型：
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作者：
Hannes Maib;Petia Adarska;Robert Hunton;James H Vines;David Strutt;Francesca Bottanelli;David H. Murray
磷脂酰肌醇是磷脂的一个小家族，作为信号中枢和细胞功能的关键调节剂。检测它们的亚细胞分布对于深入了解膜组织至关重要，并且最常见的是通过生物传感器的过度表达来完成。然而，这导致磷酸肌醇信号转导的扰动，并且在不能被转染的系统中具有挑战性。在这里，我们提出了一个工具包的可靠，快速，多重和超分辨率检测所有8个磷酸肌醇使用一个统一的染色方法固定的细胞和组织，基于重组生物传感器与自标记SNAP标签。这些重组生物传感器是高度特异性的，并且可靠地可视化了从2D或3D细胞培养到果蝇组织的磷酸肌醇的亚细胞分布。使用受激发射损耗（STED）显微镜，我们揭示了纳米级组织的PI（3）P的内涵体和PI（4）P的高尔基体，并确认保存的亚细胞膜。多重染色使得能够研究磷酸肌醇转化，并揭示了在PIKfyve抑制后肿胀的溶酶体中意外存在残留的PI（3，5）P2阳性膜。这种方法能够以前所未有的方式实现多种磷酸肌醇种类的多功能，高分辨率可视化。
Phosphoinositides are a small family of phospholipids, acting as signalling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organisation and is most commonly done by over-expression of biosensors. However, this leads to perturbations of phosphoinositide signalling and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of all 8 phosphoinositides using a unifying staining approach for fixed cells and tissue, based on recombinant biosensors with self-labelling SNAP tags. These recombinant biosensors are highly specific, and reliably visualise the subcellular distributions of phosphoinositides across scales, ranging from 2D or 3D cell culture to Drosophila tissue. Using stimulated emission depletion (STED) microscopy, we reveal the nanoscale organisation of PI(3)P on endosomes and PI(4)P on the Golgi and confirm the preservation of subcellular membranes. Multiplex staining enables the investigation of phosphoinositide conversions and reveals an unexpected presence of residual PI(3,5)P2 positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualisation of multiple phosphoinositide species in an unprecedented manner.