用荧光相关光谱扩散定律定量膜结合和扩散。
Quantifying membrane binding and diffusion with fluorescence correlation spectroscopy diffusion laws.
机构信息
Membrane Domains and Viral Assembly, Montpellier Infectious Disease Research Institute, UMR CNRS 9004, Montpellier, France.
Institute for Glycomics, Griffith University Gold Coast, Southport, QLD, Australia.
出版信息
Biophys J. 2023 Jun 6;122(11):2216-2229. doi: 10.1016/j.bpj.2023.01.006. Epub 2023 Jan 10.
Many transient processes in cells arise from the binding of cytosolic proteins to membranes. Quantifying this membrane binding and its associated diffusion in the living cell is therefore of primary importance. Dynamic photonic microscopies, e.g., single/multiple particle tracking, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy (FCS), enable non-invasive measurement of molecular mobility in living cells and their plasma membranes. However, FCS with a single beam waist is of limited applicability with complex, non-Brownian, motions. Recently, the development of FCS diffusion laws methods has given access to the characterization of these complex motions, although none of them is applicable to the membrane binding case at the moment. In this study, we combined computer simulations and FCS experiments to propose an FCS diffusion law for membrane binding. First, we generated computer simulations of spot-variation FCS (svFCS) measurements for a membrane binding process combined to 2D and 3D diffusion at the membrane and in the bulk/cytosol, respectively. Then, using these simulations as a learning set, we derived an empirical diffusion law with three free parameters: the apparent binding constant K, the diffusion coefficient on the membrane D, and the diffusion coefficient in the cytosol, D. Finally, we monitored, using svFCS, the dynamics of retroviral Gag proteins and associated mutants during their binding to supported lipid bilayers of different lipid composition or at plasma membranes of living cells, and we quantified K and D in these conditions using our empirical diffusion law. Based on these experiments and numerical simulations, we conclude that this new approach enables correct estimation of membrane partitioning and membrane diffusion properties (K and D) for peripheral membrane molecules.
许多细胞内的瞬态过程源于细胞质蛋白与膜的结合。因此,量化这种膜结合及其在活细胞中的相关扩散具有首要重要性。动态光子显微镜技术,如单/多粒子跟踪、光漂白后荧光恢复和荧光相关光谱(FCS),能够非侵入性地测量活细胞及其质膜中分子的流动性。然而,具有单个束腰的 FCS 在处理复杂的、非布朗运动时适用性有限。最近,FCS 扩散定律方法的发展使我们能够对这些复杂运动进行表征,尽管目前还没有一种方法适用于膜结合情况。在这项研究中,我们结合计算机模拟和 FCS 实验,提出了一种用于膜结合的 FCS 扩散定律。首先,我们针对一个膜结合过程生成了点变化 FCS(svFCS)测量的计算机模拟,该过程分别与膜上的 2D 和 3D 扩散以及在细胞质中的扩散相结合。然后,我们使用这些模拟作为学习集,推导出一个具有三个自由参数的经验扩散定律:表观结合常数 K、膜上的扩散系数 D 和细胞质中的扩散系数 D。最后,我们使用 svFCS 监测逆转录病毒 Gag 蛋白及其相关突变体在与不同脂质组成的支撑脂质双层或活细胞质膜结合时的动力学,并在这些条件下使用我们的经验扩散定律量化 K 和 D。基于这些实验和数值模拟,我们得出结论,这种新方法能够正确估计外周膜分子的膜分配和膜扩散特性(K 和 D)。