Fluorescence lifetime imaging of biosensor peptide phosphorylation in single live cells.
Fluorescence lifetime imaging of biosensor peptide phosphorylation in single live cells.
复制标题
单个活细胞中生物传感器肽磷酸化的荧光寿命成像。
DOI：
10.1002/anie.201209303
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发表时间：
2013-04-02
影响因子：
16.6
通讯作者：
Irudayaraj, Joseph M. K.
中科院分区：
文献类型：
--
作者：
Damayanti, Nur P.;Parker, Laurie L.;Irudayaraj, Joseph M. K.
许多癌症表现出蛋白激酶活性失调，但并非所有的抑制剂药物敏感，主要是因为复杂组织中的磷酸化动力学还没有很好地理解。[1]活的亚细胞分析可以揭示混合细胞群中激酶信号的细节。[2]目前用于原位成像激酶活性的工具依赖于基于强度的测量（如荧光和Förster共振能量转移），这可能受到光谱渗透和光漂白的限制。[3]我们报道了一种基于荧光寿命成像显微镜（FLIM）的动态监测Abl激酶磷酸化的细胞穿透肽生物传感器。[4]FLIM不受光漂白或细胞自发荧光的干扰，用于检测完整活细胞中磷酸化依赖性荧光团寿命偏移（1-2 ns）（图1）。我们建立了依赖性的荧光团寿命偏移的磷酸化特异性由Abl激酶，映射的荧光团强度和寿命组件，以量化亚细胞磷酸化，并监测激酶抑制在真实的时间。这种方法应该是generalizable其他激酶，并提供了一种新的方法，用于查询实时，亚细胞信号转导活动的细胞群体，不服从表达的基因工程生物传感器proteins.Measuring在活细胞中的亚细胞激酶活性仍然是一个重大挑战。遗传编码的Förster共振能量转移（FRET）生物传感器可用于简单的基于细胞的测定和基础研究应用。[3，5]这些传感器利用磷酸化序列和磷酸肽结合结构域之间的结合，使两种荧光蛋白足够接近，以便发生能量转移。然而，在细胞中表达基因工程蛋白具有挑战性，包括a）蛋白荧光团的均匀转染和表达（在原代患者来源的细胞或组织中应用的障碍）和B）可影响底物功能和与激酶相互作用的大标记。[5-6]小分子荧光团原则上能够
Many cancers exhibit deregulated activity of protein kinase enzymes, but not all are sensitive to inhibitor drugs, largely because phosphorylation dynamics in complex tissues are not well understood.[1] Live, subcellular analysis can reveal the details of kinase signaling in mixed populations of cells.[2] Current tools to image kinase activity in situ depend on intensity-based measurements (such as fluorescence and Förster resonance energy transfer) that can be limited by spectral bleed-through and photobleaching.[3] We report a cell-penetrating peptide biosensor for dynamic monitoring of phosphorylation by Abl kinase based on fluorescence lifetime imaging microscopy (FLIM).[4] FLIM, which is not confounded by photobleaching or cellular autofluorescence, was applied to detect phosphorylation-dependent fluorophore lifetime shifts (1–2 ns) in intact, living cells (Fig. 1). We established the dependence of the fluorophore lifetime shift on phosphorylation specifically by Abl kinase, mapped the fluorophore intensity and lifetime components to quantify subcellular phosphorylation, and monitored kinase inhibition in real time. This approach should be generalizable to other kinases and provides a new method for interrogating real-time, subcellular signaling activities in cell populations that are not amenable to expression of genetically engineered biosensor proteins.Measuring subcellular kinase activity in living cells remains a major challenge. Genetically encoded Förster resonance energy transfer (FRET) biosensors can be used for this purpose in simple cell-based assays and basic research applications.[3, 5] These sensors take advantage of binding between phosphorylated sequences and phosphopeptide binding domains to bring two fluorescent proteins close enough for energy transfer to occur. However, expressing genetically engineered proteins in cells has challenges, including a) uniform transfection and expression of protein fluorophores (a roadblock for applications in primary patient-derived cells or tissues) and b) the large labels which can affect substrate function and interaction with a kinase.[5–6] Small molecule fluorophores are able in principle
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影响因子：
7.3
作者：
Colicelli J
通讯作者：
Colicelli J
影响因子：
4
作者：
Chen, Jiji;Miller, Andrew;Irudayaraj, Joseph M. K.
通讯作者：
Irudayaraj, Joseph M. K.
影响因子：
2.9
作者：
Wu, Ding;Sylvester, Juliesta E.;Parker, Laurie L.;Zhou, Guangchang;Kron, Stephen J.
通讯作者：
Kron, Stephen J.
影响因子：
2.9
作者：
Placzek, Ekaterina A.;Plebanek, Michael P.;Parker, Laurie L.
通讯作者：
Parker, Laurie L.
影响因子：
64.8
作者：
SONGYANG, Z;CARRAWAY, KL;CANTLEY, LC
通讯作者：
CANTLEY, LC