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首页> 外文期刊>Optofluidics, Microfluidics and Nanofluidics >Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang
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Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

机译：用eGFP或Dreiklang进行荧光标记后，通过荧光相关光谱（FCS）监测Na，K-ATPase的扩散行为

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Measurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time scale as the diffusion of the labeled complexes through the detection volume (1– 100 ms). Since the origin of this quenching process is currently unclear, care has to be taken when the Dreiklang label is intended to be used in FCS applications.
机译：具有高时空精度的活细胞膜包埋蛋白横向移动性的测量是光流体学的一项艰巨任务。生物膜是复杂的结构，其物理化学性质取决于局部脂质组成，胆固醇含量以及整体或外围膜蛋白的存在，其可能参与超分子复合物中或与细胞基质蛋白或细胞骨架相连。生物膜中高的蛋白脂质比率表明，膜蛋白特别容易受到分子拥挤的影响，因此很难追踪带有荧光标记的单个分子的踪迹。原则上，新型的可切换荧光蛋白（例如Dreiklang [1]）是有前途的工具，由于对ON和OFF切换过程进行了出色的光谱控制，因此它们在分子拥挤情况下研究单个分子的扩散行为。在这项工作中，我们在HEK293T细胞中表达了一种完整的膜转运蛋白，即包含带有N末端eGFP或Dreiklang标签的人α2-亚基和人β1-亚基的Na，K-ATPase，并通过荧光相关光谱法测量了自相关曲线（FCS）。此外，我们通过FCS测量了eGFP和Dreiklang的扩散时间和扩散常数，首先是在纯化后的蛋白质在大肠杆菌中表达后在水溶液中，其次是在可溶蛋白在HEK293T细胞的细胞质中表达后在水溶液中。我们的数据表明，纯化的eGFP和Dreiklang在溶液中的扩散行为以及在细胞质中表达的蛋白质的性质非常相似。但是，在质膜上测得的eGFP和Dreiklang标记的Na，K-ATPase的自相关曲线显示出明显的差异，Dreiklang标记的构建体显示出较短的扩散时间。这可能与另外一个尚未被认识到的淬灭过程有关，该过程与标记的复合物在检测体积中的扩散时间（1-100毫秒）相同。由于目前尚不清楚该淬火过程的起源，因此当打算将Dreiklang标签用于FCS应用时必须格外小心。

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