Evaluating integrin activation with time-resolved flow cytometry.
Evaluating integrin activation with time-resolved flow cytometry.
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DOI：
10.1117/1.jbo.23.7.075004
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发表时间：
2018-07
影响因子：
3.5
通讯作者：
Houston JP
中科院分区：
文献类型：
--
作者：
Sambrano J;Chigaev A;Nichani KS;Smagley Y;Sklar LA;Houston JP
福斯特共振能量转移（FRET）仍然是研究活细胞内蛋白质之间的运动和相互作用的有用工具。当使用流式细胞术测量作为一种光学技术的FRET时，可以逐个细胞快速测量蛋白质的构象变化，这有利于筛选和分析。我们利用FRET来研究白细胞表面表达的整合素二聚体的活化程度。当不活跃时，茎状跨膜异二聚体呈弯曲状，活化时则向外伸展。整合素的伸展是由分别结合在整合素头部和细胞膜上的FRET供体和受体之间的最近距离的变化所决定的。时间分辨流式细胞术分析显示，在活化过程中供体发射增加高达17%，荧光寿命移动超过1.0纳秒，对应于整合素的非活化和活化状态的FRET效率分别为37%和26%。最后，图形相量分析，包括群体聚类、设门以及FRET轨迹的形成，提高了对经历FRET、部分供体恢复和完全供体恢复的群体进行比较分析的精度。这项工作建立了一种定量的细胞计数方法，用于在单细胞水平上分析整合素构象变化过程中荧光供体的衰减动力学。
Förster resonance energy transfer (FRET) continues to be a useful tool to study movement and interaction between proteins within living cells. When FRET as an optical technique is measured with flow cytometry, conformational changes of proteins can be rapidly measured cell-by-cell for the benefit of screening and profiling. We exploit FRET to study the extent of activation of integrin dimers expressed on the surface of leukocytes. The stalk-like transmembrane heterodimers when not active lay bent and upon activation extend outward. Integrin extension is determined by changes in the distance of closest approach between an FRET donor and acceptor, bound at the integrin head and cell membrane, respectively. Time-resolved flow cytometry analysis revealed donor emission increases up to 17%, fluorescence lifetime shifts over 1.0 ns during activation, and FRET efficiencies of 37% and 26% corresponding to the inactive and active integrin state, respectively. Last, a graphical phasor analysis, including population clustering, gating, and formation of an FRET trajectory, added precision to a comparative analysis of populations undergoing FRET, partial donor recovery, and complete donor recovery. This work establishes a quantitative cytometric approach for profiling fluorescence donor decay kinetics during integrin conformational changes on a single-cell level.