Data_Sheet_1_Variations in Plasma Membrane Topography Can Explain Heterogenous Diffusion Coefficients Obtained by Fluorescence Correlation Spectroscopy.PDF

Fluorescence correlation spectroscopy (FCS) is frequently used to study diffusion in cell membranes, primarily the plasma membrane. The diffusion coefficients reported in the plasma membrane of the same cell type and even within single cells typically display a large spread. We have investigated whether this spread can be explained by variations in membrane topography throughout the cell surface, that changes the amount of membrane in the FCS focal volume at different locations. Using FCS, we found that diffusion of the membrane dye DiI in the apical plasma membrane was consistently faster above the nucleus than above the cytoplasm. Using live cell scanning ion conductance microscopy (SICM) to obtain a topography map of the cell surface, we demonstrate that cell surface roughness is unevenly distributed with the plasma membrane above the nucleus being the smoothest, suggesting that the difference in diffusion observed in FCS is related to membrane topography. FCS modeled on simulated diffusion in cell surfaces obtained by SICM was consistent with the FCS data from live cells and demonstrated that topography variations can cause the appearance of anomalous diffusion in FCS measurements. Furthermore, we found that variations in the amount of the membrane marker DiD, a proxy for the membrane, but not the transmembrane protein TCRζ or the lipid-anchored protein Lck, in the FCS focal volume were related to variations in diffusion times at different positions in the plasma membrane. This relationship was seen at different positions both at the apical cell and basal cell sides. We conclude that it is crucial to consider variations in topography in the interpretation of FCS results from membranes.

荧光相关光谱（Fluorescence correlation spectroscopy, FCS）常被用于研究细胞膜（主要是质膜）中的扩散过程。同一细胞类型乃至单个细胞的质膜中已报道的扩散系数，通常存在较大的离散度。我们探究了该离散度是否可由整个细胞表面的膜形貌变化所解释——这类变化会改变不同位置处FCS焦体积内的膜含量。借助FCS实验，我们发现膜染料DiI在顶质膜中的扩散速率在细胞核上方区域始终快于细胞质上方区域。通过活细胞扫描离子电导显微镜（SICM）获取细胞表面形貌图，我们证实细胞表面粗糙度分布不均，细胞核上方的质膜最为平滑，这表明FCS实验中观测到的扩散速率差异与膜形貌相关。基于SICM获取的细胞表面形貌构建的模拟扩散模型所得的FCS结果，与活细胞的FCS实验数据相符，并证明形貌变化可导致FCS测量中呈现反常扩散现象。此外，我们发现FCS焦体积内的膜标志物DiD（作为膜含量的替代指标）的变化，与质膜不同位置的扩散时间变化相关；而跨膜蛋白TCRζ或脂锚定蛋白Lck的信号变化则无此关联。这一现象在细胞顶侧和基底侧的不同位置均有观测到。综上，在解读膜相关FCS实验结果时，考虑形貌变化的影响至关重要。