Quantitative measurement of F-actin accumulation at the NK cell immunological synapse
Quantitative measurement of F-actin accumulation at the NK cell immunological synapse
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DOI：
10.1016/j.jim.2010.02.003
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发表时间：
2010-04-15
影响因子：
2.2
通讯作者：
Orange, Jordan S.
中科院分区：
文献类型：
--
作者：
Banerjee, Pinaki P.;Orange, Jordan S.
NK细胞是先天免疫系统的淋巴细胞，在激活信号诱导下定向分泌溶解性颗粒内容物，杀死靶细胞。这一过程依赖于NK细胞免疫突触（NKIS）上的f -肌动蛋白聚合，NKIS是NK细胞和靶细胞之间界面上分子的动态组织。虽然f -肌动蛋白在NKIS的积累很容易可视化，但要了解f -肌动蛋白重组和积累如何使NK细胞发挥功能，需要量化NKIS上的f -肌动蛋白的能力。在这里，我们展示了几种新的算法来测量NKIS积累的f -肌动蛋白含量，特别强调NK细胞贡献的肌动蛋白。这些算法不依赖于过表达荧光蛋白或预先培养细胞与重要的荧光染料。使用激活和抑制NKIS以及表达荧光蛋白-细胞表面受体融合蛋白的NK细胞模型，对这些算法进行了测试，并用于定量证明F-actin在激活NKIS处积累，而不是在抑制NKIS处积累。通过这些方法，我们还建立了数学公式，这些公式在NKIS的全面定量评估中应该是有价值的，并且更广泛地适用于测量细胞间连接处任何荧光团的积累。(C) 2010 Elsevier B.V.版权所有
NK cells are lymphocytes of the innate immune system that can kill target cells after activation signal-induced directional secretion of lytic granule contents. This process depends upon F-actin polymerization at the NK cell immunological synapse (NKIS), which is the dynamic organization of molecules at the interface between the NK cell and target cell. Although F-actin accumulation at the NKIS is easily visualized, the ability to quantify F-actin at the NKIS is required to understand how F-actin reorganization and accumulation enable NK cell function. Here, we demonstrate several novel algorithms for measuring the content of F-actin accumulated at the NKIS with special emphasis upon actin contributed by the NK cell. These algorithms do not rely upon overexpressing fluorescent proteins or preincubating cells with vital fluorescent dyes. Using models of the activating and inhibitory NKIS as well as NK cells expressing fluorescent protein - cell surface receptor fusion proteins, these algorithms were tested and were used to quantitatively demonstrate that F-actin accumulates at the activating, but not at the inhibitory NKIS. With these approaches, we have also established mathematical formulas that should prove valuable in the comprehensive quantitative evaluation of the NKIS and be more broadly applicable in the measurement of the accumulation of any fluorophore at an intercellular junction. (C) 2010 Elsevier B.V. All rights reserved.