Department of Biological Sciences and NUS Centre for Bio-Imaging Sciences, National University of Singapore, 14 Science Drive 4, 117557 Singapore, Singapore.
Department of Biological Sciences and NUS Centre for Bio-Imaging Sciences, National University of Singapore, 14 Science Drive 4, 117557 Singapore, Singapore; Department of Chemistry, National University of Singapore, 3 Science Drive 3, 117543 Singapore, Singapore.
Methods. 2018 May 1;140-141:140-150. doi: 10.1016/j.ymeth.2017.11.016. Epub 2017 Dec 5.
The cellular plasma membrane is the barrier over which cells exchange materials and communicate with their surroundings, and thus plays the central role in cellular sensing and metabolism. Therefore, the investigation of plasma membrane organization and dynamics is required for understanding of cellular functions. The plasma membrane is a heterogeneous matrix. The presence of structures such as lipid and protein domains and the cytoskeleton meshwork poses a hindrance to the free diffusion of membrane associated biomolecules. However, these domains and the cytoskeleton meshwork barriers are below the optical diffraction limit with potentially short lifetimes and are not easily detected even in super-resolution microscopy. Therefore, dynamic measurements are often used to indirectly prove the existence of domains and barriers by analyzing the mode of diffusion of probe molecules. One of these tools is the Fluorescence Correlation Spectroscopy (FCS) diffusion law. The FCS diffusion law is a plot of diffusion time (τ) versus observation area. For at least three different diffusive modes - free, domain confined, and meshwork hindered hop diffusion - the expected plots have been characterized, typically by its y-intercept (τ) when fit with a linear model, and have been verified in many cases. However, a description of τ has only been given for pure diffusive modes. But in many experimental cases it is not evident that a protein will undergo only one kind of diffusion, and thus the interpretation of the τ value is problematic. Here, we therefore address the question about the absolute value of τ in the case of complex diffusive modes, i.e. when either one molecule is domain confined and cytoskeleton hindered or when two molecules exhibit different diffusive behavior at the same position in a sample. In addition, we investigate how τ changes when the diffusive mode of a probe alters upon disruption of domains or the cytoskeleton by drug treatments. By a combination of experimental studies and simulations, we show that τ is not influenced equally by the different diffusive modes as typically found in cellular environments, and that it is the relative change of τ rather than its absolute value that provides information on the mode of diffusion.
细胞质膜是细胞内外物质交换和与周围环境进行通讯的屏障，因此在细胞感应和代谢中起着核心作用。因此，为了理解细胞功能，需要研究质膜的组织和动力学。质膜是一种不均匀的基质。脂质和蛋白质域以及细胞骨架网格的存在构成了膜相关生物分子自由扩散的障碍。然而，这些域和细胞骨架网格障碍低于光学衍射极限，具有潜在的短寿命，即使在超分辨率显微镜下也不容易检测到。因此，通过分析探针分子的扩散模式，动态测量通常用于间接证明域和障碍的存在。其中一种工具是荧光相关光谱（FCS）扩散定律。FCS 扩散定律是扩散时间（τ）与观察面积的关系图。对于至少三种不同的扩散模式——自由扩散、域限制扩散和网格阻碍跳跃扩散——已经对预期的图谱进行了特征描述，通常以线性模型拟合时的 y 截距（τ）来表示，并且在许多情况下已经得到了验证。然而，τ 的描述仅适用于纯扩散模式。但是在许多实验情况下，不能确定蛋白质只经历一种扩散，因此τ 值的解释存在问题。在这里，我们因此解决了复杂扩散模式下τ 的绝对值问题，即在一种分子被域限制和细胞骨架阻碍，或者在样品中同一位置的两个分子表现出不同的扩散行为的情况下。此外，我们研究了当通过药物处理破坏域或细胞骨架时，探针的扩散模式改变时，τ 如何变化。通过实验研究和模拟的结合，我们表明，τ 受到不同扩散模式的影响并不像在细胞环境中通常发现的那样均匀，并且τ 的相对变化而不是其绝对值提供了有关扩散模式的信息。
