Molecular height measurement by cell surface optical profilometry (CSOP).
Molecular height measurement by cell surface optical profilometry (CSOP).
复制标题
通过细胞表面光学轮廓测定法 (CSOP) 测量分子高度。
DOI：
10.1073/pnas.1922626117
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发表时间：
2020
影响因子：
11.1
通讯作者：
Fletcher,DanielA
中科院分区：
文献类型：
--
作者：
Son,Sungmin;Takatori,ShoC;Belardi,Brian;Podolski,Marija;Bakalar,MatthewH;Fletcher,DanielA
细胞表面蛋白质和聚糖的物理尺寸可以严重影响细胞功能，例如，通过阻止细胞之间的紧密接触和限制受体的可及性。然而，在天然细胞膜上的分子高度的高分辨率测量一直难以获得。在这里，我们提出了一个简单而快速的方法，实现纳米高度分辨率，通过定位在细胞表面分子的尖端和底部的荧光团，并确定它们的分离径向平均跨越许多分子。我们使用这种方法，我们称之为细胞表面光学轮廓术（CSOP），以量化模型细胞上的关键多结构域蛋白质的高度，以及捕获天然细胞膜上的平均蛋白质和聚糖高度。我们发现，蛋白质的平均高度是显着小于其轮廓长度，由于热驱动的弯曲和旋转的膜，高度强烈依赖于当地的表面和解决方案的条件。我们发现，平均高度增加细胞表面分子拥挤，但减少与溶液拥挤的溶质，这两个我们证实与分子动力学模拟。我们还使用实验和模拟来根据抗体的位置确定表位的高度，这使得CSOP能够使用抗体和凝集素来分析天然细胞表面上的各种蛋白质和聚糖。这种用于分析细胞表面的多功能方法有可能促进对细胞分子景观的理解以及分子景观在细胞功能中的作用。
The physical dimensions of proteins and glycans on cell surfaces can critically affect cell function, for example, by preventing close contact between cells and limiting receptor accessibility. However, high-resolution measurements of molecular heights on native cell membranes have been difficult to obtain. Here we present a simple and rapid method that achieves nanometer height resolution by localizing fluorophores at the tip and base of cell surface molecules and determining their separation by radially averaging across many molecules. We use this method, which we call cell surface optical profilometry (CSOP), to quantify the height of key multidomain proteins on a model cell, as well as to capture average protein and glycan heights on native cell membranes. We show that average height of a protein is significantly smaller than its contour length, due to thermally driven bending and rotation on the membrane, and that height strongly depends on local surface and solution conditions. We find that average height increases with cell surface molecular crowding but decreases with solution crowding by solutes, both of which we confirm with molecular dynamics simulations. We also use experiments and simulations to determine the height of an epitope, based on the location of an antibody, which allows CSOP to profile various proteins and glycans on a native cell surface using antibodies and lectins. This versatile method for profiling cell surfaces has the potential to advance understanding of the molecular landscape of cells and the role of the molecular landscape in cell function.