Single-cell measurements of two-dimensional binding affinity across cell contacts.
Single-cell measurements of two-dimensional binding affinity across cell contacts.
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DOI：
10.1016/j.bpj.2021.10.010
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发表时间：
2021-11-16
影响因子：
3.4
通讯作者：
Jönsson P
中科院分区：
文献类型：
--
作者：
Chouliara M;Junghans V;Dam T;Santos AM;Davis SJ;Jönsson P
接触细胞之间的蛋白质分子之间的二维 (2D) 亲和力是调节和启动多个细胞过程的关键参数。然而，测量 2D 亲和力可能具有挑战性，并且实验数据有限。此外，获得的 2D 亲和力通常是细胞群的平均值。我们在这里提出了一种测量单细胞与锚定在支持的脂质双层 (SLB) 上的多组氨酸标记荧光配体结合的 2D 亲和力的方法。通过使用咪唑降低 SLB 中配体的密度，可以获得接触中新的稳态积累，并且根据这种变化，可以确定细胞上的 2D 亲和力和受体数量。该方法在含有与 Jurkat T 细胞上表达的大鼠 CD48 突变体 T92A 结合的大鼠 CD2 的 SLB 上进行了验证。咪唑的添加不会影响平均 2D 亲和力 (1/Kd)，并且细胞群内亲和力的分布较低，Kd = 4.9 ± 0.9 分子/μm2（平均值 ± 标准差），尽管由于受体密度的差异，配体积累存在一个数量级的分布。还发现，细胞接触尺寸随着配体密度和每个细胞的受体数量的增加而增加，但当配体密度降低时，接触尺寸保持大致恒定，在接触首次形成后，高于约10个大鼠CD2分子/μm2的密度，这表明存在异质过程。综上所述，该方法不仅可以测量单细胞亲和力，还可以减少测量和分析时间，提高测量精度。由于细胞群内 2D Kd 的扩散程度较低，因此分析可以进一步限制于显示最强结合的细胞，从而为使用该方法研究弱结合事件铺平了道路。
The two-dimensional (2D) affinity between protein molecules across contacting cells is a key parameter regulating and initiating several cellular processes. However, measuring 2D affinity can be challenging, and experimental data are limited. In addition, the obtained 2D affinities are typically averaged over the cell population. We here present a method to measure 2D affinity on single cells binding to polyhistidine-tagged fluorescent ligands anchored to a supported lipid bilayer (SLB). By decreasing the density of ligands in the SLB using imidazole, a new steady-state accumulation in the contact is obtained, and from this change, both the 2D affinity and the number of receptors on the cell can be determined. The method was validated on an SLB containing rat CD2 binding to the rat CD48 mutant T92A expressed on Jurkat T cells. The addition of imidazole did not influence the average 2D affinity (1/Kd), and the spread in affinities within the cell population was low, Kd = 4.9 ± 0.9 molecules/μm2 (mean ± SD), despite an order of magnitude spread in ligand accumulation because of differences in receptor density. It was also found that cell contact size increased both with ligand density and with the number of receptors per cell but that the contact size stayed approximately constant when lowering the ligand density, above a density of around 10 rat CD2 molecules/μm2, after the contact first had formed, indicative of a heterogeneous process. In summary, this method not only allows for single-cell affinities to be measured, but it can also reduce measurement and analysis time and improve measurement accuracy. Because of the low spread in 2D Kd within the cell population, the analysis can further be restricted to the cells showing the strongest binding, paving the way for using this method to study weak binding events.
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影响因子：
4.8
作者：
Dustin, ML;Golan, DE;van der Merwe, PA
通讯作者：
van der Merwe, PA
影响因子：
30.5
作者：
Chang VT;Fernandes RA;Ganzinger KA;Lee SF;Siebold C;McColl J;Jönsson P;Palayret M;Harlos K;Coles CH;Jones EY;Lui Y;Huang E;Gilbert RJC;Klenerman D;Aricescu AR;Davis SJ
通讯作者：
Davis SJ
DOI：
10.1073/pnas.1513918113
发表时间：
2016-05-17
影响因子：
11.1
作者：
Jonsson, Peter;Southcombe, Jennifer H.;Klenerman, David
通讯作者：
Klenerman, David
影响因子：
3.4
作者：
Chesla, SE;Selvaraj, P;Zhu, C
通讯作者：
Zhu, C
DOI：
10.1073/pnas.1305766110
发表时间：
2013-09-17
影响因子：
11.1
作者：
Hu, Jinglei;Lipowsky, Reinhard;Weikl, Thomas R.
通讯作者：
Weikl, Thomas R.