Characterization of Cytoplasmic Gag-Gag Interactions by Dual-Color Z-Scan Fluorescence Fluctuation Spectroscopy
Characterization of Cytoplasmic Gag-Gag Interactions by Dual-Color Z-Scan Fluorescence Fluctuation Spectroscopy
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DOI：
10.1016/j.bpj.2011.02.008
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发表时间：
2011-03-16
影响因子：
3.4
通讯作者：
Mueller, Joachim D.
中科院分区：
文献类型：
--
作者：
Fogarty, Keir H.;Chen, Yan;Mueller, Joachim D.
荧光涨落光谱（FFS）通过亮度分析定量活细胞内荧光标记蛋白质的相互作用。然而，与质膜相互作用的细胞质蛋白质的研究是具有挑战性的FFS。如果细胞质切片比观察体积的轴向尺寸薄，则细胞质和膜结合蛋白质被共激发，这导致亮度伪影。这种亮度偏差，如果没有认识到，导致错误的数据解释。我们通过引入双色z扫描FFS和添加明显着色的参考蛋白克服了这一挑战。在这里，我们应用这种技术来研究人类免疫缺陷病毒1型（HIV-1）和人类嗜T淋巴细胞病毒1型（HTLV-1）的Gag蛋白的细胞质相互作用。Gag蛋白在逆转录病毒的组装中起着至关重要的作用，并且在细胞膜和细胞质中都有发现。双色z扫描表明，亮度伪影是由一个暗淡的nonpunctate膜结合部分的加格。我们通过避免膜结合部分来执行细胞质Gag的无偏亮度表征，并揭示了两种逆转录病毒Gag物种的行为中先前未知的差异。HIV-1 Gag在细胞质中表现出浓度依赖性寡聚化，而HTLV-1 Gag缺乏显著的细胞质Gag-Gag相互作用。
Fluorescence fluctuation spectroscopy (FFS) quantifies the interactions of fluorescently-labeled proteins inside living cells by brightness analysis. However, the study of cytoplasmic proteins that interact with the plasma membrane is challenging with FFS. If the cytoplasmic section is thinner than the axial size of the observation volume, cytoplasmic and membrane-bound proteins are coexcited, which leads to brightness artifacts. This brightness bias, if not recognized, leads to erroneous interpretation of the data. We have overcome this challenge by introducing dual-color z-scan FFS and the addition of a distinctly colored reference protein. Here, we apply this technique to study the cytoplasmic interactions of the Gag proteins from human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type 1 (HTLV-1). The Gag protein plays a crucial role in the assembly of retroviruses and is found in both membrane and cytoplasm. Dual-color z-scans demonstrate that brightness artifacts are caused by a dim nonpunctate membrane-bound fraction of Gag. We perform an unbiased brightness characterization of cytoplasmic Gag by avoiding the membrane-bound fraction and reveal previously unknown differences in the behavior of the two retroviral Gag species. HIV-1 Gag exhibits concentration-dependent oligomerization in the cytoplasm, whereas HTLV-1 Gag lacks significant cytoplasmic Gag-Gag interactions.