揭示酵母质子泵Pma1的扩散状态，以及标记方法如何改变扩散行为
Uncovering diffusive states of the yeast proton pump, Pma1, and how labeling method can change diffusive behavior
摘要：
我们提出并分析了基于视频显微镜的单粒子跟踪测量的芽殖酵母（酿酒酵母）膜蛋白，Pma 1，荧光标记的直接融合到可切换的荧光蛋白，mEos3.2，或通过一种新的，轻触，标记方案，其中5个氨基酸的标签直接融合到Pma 1的C-末端，然后结合mEos3.2。这两个群体的单粒子径迹的扩散率分布显着不同，表明标记方法可以是一个重要的决定因素的扩散行为。我们还应用了扰动期望最大化（pEMv 2）[Physical Review E 94，052412（2016）]，它将轨迹分类为扩散状态的最佳数量。对于TRAP标记的Pma 1和Pma 1-mEos3.2，pEMv 2将轨迹分为两种扩散状态：基本上不移动的状态和更移动的状态。然而，Pma 1-mEos3.2轨迹的可动部分（0.1）比TRAP标记的Pma 1轨迹的可动部分（0.5）小得多。此外，Pma 1-mEos3.2的移动状态的扩散率比TRAP标记的Pma 1的移动状态的扩散率小几倍。为了严格评估pEMv 2的性能，我们比较了实验pEMv 2分选种群的扩散率和协方差分布与相应的理论分布，假设Pma 1位移实现了高斯随机过程。TRAP标记的Pma 1和Pma 1-mEos3.2的实验-理论比较显示出良好的一致性，支持pEMv 2方法。
英文摘要：
We present and analyze video-microscopy-based single-particle-tracking measurements of the budding yeast (Saccharomyces cerevisiae) membrane protein, Pma1, fluorescently-labeled either by direct fusion to the switchable fluorescent protein, mEos3.2, or by a novel, light-touch, labeling scheme, in which a 5 amino acid tag is directly fused to the C-terminus of Pma1, which then binds mEos3.2. The diffusivity distributions of these two populations of single particle tracks differ significantly, demonstrating that labeling method can be an important determinant of diffusive behavior. We also applied perturbation expectation maximization (pEMv2) [Physical Review E 94, 052412 (2016)], which sorts trajectories into the statistically-optimum number of diffusive states. For both TRAP-labeled Pma1 and Pma1-mEos3.2, pEMv2 sorts the tracks into two diffusive states: an essentially immobile state and a more mobile state. However, the mobile fraction of Pma1-mEos3.2 tracks is much smaller (0.1) than the mobile fraction of TRAP-labeled Pma1 tracks (0.5). In addition, the diffusivity of Pma1-mEos3.2's mobile state is several times smaller than the diffusivity of TRAP-labeled Pma1's mobile state. To critically assess pEMv2's performance, we compare the diffusivity and covariance distributions of the experimental pEMv2-sorted populations to corresponding theoretical distributions, assuming that Pma1 displacements realize a Gaussian random process. The experiment-theory comparisons for both the TRAP-labeled Pma1 and Pma1-mEos3.2 reveal good agreement, bolstering the pEMv2 approach.
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