Kulenkampff Klara, Lippert Anna H, McColl James, Santos Ana Mafalda, Ponjavic Aleks, Jenkins Edward, Humphrey Jane, Winkel Alexander, Franze Kristian, Lee Steven F, Davis Simon J, Klenerman David
Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
Radcliffe Department of Medicine and MRC Human Immunology Unit, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom.
Biophys J. 2020 Mar 24;118(6):1261-1269. doi: 10.1016/j.bpj.2020.01.019. Epub 2020 Jan 28.
Cell-cell contacts often underpin signaling between cells. For immunology, the binding of a T cell receptor to an antigen-presenting pMHC initiates downstream signaling and an immune response. Although this contact is mediated by proteins on both cells creating interfaces with gap sizes typically around 14 nm, many, often contradictory observations have been made regarding the influence of the contact on parameters such as the binding kinetics, spatial distribution, and diffusion of signaling proteins within the contact. Understanding the basic physical constraints on probes inside this crowded environment will help inform studies on binding kinetics and dynamics of signaling of relevant proteins in the synapse. By tracking quantum dots of different dimensions for extended periods of time, we have shown that it is possible to obtain the probability of a molecule entering the contact, the change in its diffusion upon entry, and the impact of spatial heterogeneity of adhesion protein density in the contact. By analyzing the contacts formed by a T cell interacting with adhesion proteins anchored to a supported lipid bilayer, we find that probes are excluded from contact entry in a size-dependent manner for gap-to-probe differences of 4.1 nm. We also observed probes being trapped inside the contact and a decrease in diffusion of up to 85% in dense adhesion protein contacts. This approach provides new, to our knowledge, insights into the nature of cell-cell contacts, revealing that cell contacts are highly heterogeneous because of topography- and protein-density-related processes. These effects are likely to profoundly influence signaling between cells.
细胞间接触常常是细胞间信号传导的基础。对于免疫学而言，T细胞受体与抗原呈递的肽 - 主要组织相容性复合体（pMHC）的结合会启动下游信号传导和免疫反应。尽管这种接触是由两个细胞上的蛋白质介导的，形成的界面间隙大小通常约为14纳米，但关于这种接触对诸如结合动力学、空间分布以及信号蛋白在接触区域内的扩散等参数的影响，已经有了许多常常相互矛盾的观察结果。了解在这个拥挤环境中探针所面临的基本物理限制，将有助于为突触中相关蛋白的结合动力学和信号传导动力学研究提供信息。通过长时间追踪不同尺寸的量子点，我们已经表明，有可能获得分子进入接触区域的概率、进入时其扩散的变化以及接触区域内黏附蛋白密度的空间异质性的影响。通过分析T细胞与锚定在支持脂质双分子层上的黏附蛋白形成的接触，我们发现，对于间隙与探针的差异为4.1纳米的情况，探针以尺寸依赖的方式被排除在接触进入之外。我们还观察到探针被困在接触区域内，并且在密集黏附蛋白接触区域扩散减少高达85%。据我们所知，这种方法为细胞间接触的性质提供了新的见解，揭示了由于地形和蛋白质密度相关过程，细胞接触具有高度异质性。这些效应可能会深刻影响细胞间的信号传导。
