Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Nat Chem. 2011 Oct 9;3(11):868-74. doi: 10.1038/nchem.1163.
Membrane lipids are dynamic molecules that play important roles in cell signalling and regulation, but an in situ imaging method for quantitatively tracking lipids in living cells is lacking at present. Here, we report a new chemical method of quantitative lipid imaging using sensors engineered by labelling proteins with an environmentally sensitive fluorophore. A prototype sensor for phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2))--a key signalling lipid in diverse cellular processes--was generated by covalently attaching a single 2-dimethylamino-6-acyl-naphthalene group to the N-terminal α-helix of the engineered epsin1 ENTH domain, a protein that selectively binds PtdIns(4,5)P(2). The sensor allows robust and sensitive in situ quantitative imaging in mammalian cells, providing new insight into the spatiotemporal dynamics and fluctuation of this key signalling lipid. Application of the sensor to immune cells reveals the presence of a local threshold PtdIns(4,5)P(2) concentration required for triggering phagocytosis. This sensor strategy is generally applicable to in situ quantification of other cellular lipids.
膜脂是一类动态分子，在细胞信号转导和调控中发挥着重要作用，但目前缺乏一种能够在活细胞中定量跟踪脂质的原位成像方法。在这里，我们报告了一种使用通过标记蛋白质与环境敏感荧光团来工程化传感器的新型定量脂质成像化学方法。通过将单个 2-二甲氨基-6-酰基-萘基团共价连接到工程化 epsin1 ENTH 结构域的 N 端α螺旋上，生成了一种用于磷脂酰肌醇-4,5-二磷酸（PtdIns(4,5)P(2)）的原型传感器，该结构域是一种选择性结合 PtdIns(4,5)P(2)的蛋白质。该传感器允许在哺乳动物细胞中进行稳健且灵敏的原位定量成像，为研究这种关键信号脂质的时空动力学和波动提供了新的见解。将该传感器应用于免疫细胞揭示了触发吞噬作用所需的局部 PtdIns(4,5)P(2)浓度的存在。该传感器策略通常适用于其他细胞脂质的原位定量。
