Fast spatiotemporal correlation spectroscopy to determine protein lateral diffusion laws in live cell membranes
Fast spatiotemporal correlation spectroscopy to determine protein lateral diffusion laws in live cell membranes
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DOI：
10.1073/pnas.1222097110
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发表时间：
2013-07-23
影响因子：
11.1
通讯作者：
Cardarelli, Francesco
中科院分区：
文献类型：
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作者：
Di Rienzo, Carmine;Gratton, Enrico;Cardarelli, Francesco
质膜蛋白的空间分布和动态被认为是由脂质组成和底层的细胞骨架，形成短暂的扩散障碍调制。到目前为止，这一想法是通过膜成分的单粒子跟踪来探索的，其中使用金粒子或抗体来单独监测感兴趣的分子。不幸的是，单粒子跟踪所需的相对较大的粒子原则上可以改变正在研究的动力学。在这里，我们使用的方法，使它能够通过小分子标记，特别是单一的绿色荧光蛋白结构的手段，调查质膜蛋白。首先，对膜上的感兴趣区域进行快速成像。对于所得到的图像堆叠中的每个时间延迟，计算平均空间相关函数。我们表明，通过拟合一系列的相关函数，实际的蛋白质“扩散法”可以直接从成像中获得，在一个均方位移与时间延迟图的形式，而不需要解释模型。这种方法进行了测试，几个模拟的二维扩散条件和活的中国仓鼠卵巢细胞与GFP标记的跨膜转铁蛋白受体，一个众所周知的基准膜骨架依赖性瞬时限制扩散。这种方法不需要提取单独的轨迹，也可以用于暗淡和密集的分子。我们认为，它是一个强大的工具，在很宽的空间和时间尺度上的动力学和热力学参数的测定。
Spatial distribution and dynamics of plasma-membrane proteins are thought to be modulated by lipid composition and by the underlying cytoskeleton, which forms transient barriers to diffusion. So far this idea was probed by single-particle tracking of membrane components in which gold particles or antibodies were used to individually monitor the molecules of interest. Unfortunately, the relatively large particles needed for single-particle tracking can in principle alter the very dynamics under study. Here, we use a method that makes it possible to investigate plasma-membrane proteins by means of small molecular labels, specifically single GFP constructs. First, fast imaging of the region of interest on the membrane is performed. For each time delay in the resulting stack of images the average spatial correlation function is calculated. We show that by fitting the series of correlation functions, the actual protein "diffusion law" can be obtained directly from imaging, in the form of a mean-square displacement vs. time-delay plot, with no need for interpretative models. This approach is tested with several simulated 2D diffusion conditions and in live Chinese hamster ovary cells with a GFP-tagged transmembrane transferrin receptor, a well-known benchmark of membrane-skeleton-dependent transiently confined diffusion. This approach does not require extraction of the individual trajectories and can be used also with dim and dense molecules. We argue that it represents a powerful tool for the determination of kinetic and thermodynamic parameters over very wide spatial and temporal scales.