Arnold Andreas M, Sevcsik Eva, Schütz Gerhard J
Institute of Applied Physics, Technische Universität Wien, Wiedner Hauptstrasse 8-10, 1040 Vienna, Austria.
J Phys D Appl Phys. 2016 Aug 9;49(36). doi: 10.1088/0022-3727/49/36/364002.
Single molecule trajectories of lipids and proteins can yield valuable information about the nanoscopic organization of the plasma membrane itself. The interpretation of such trajectories, however, is complicated, as the mobility of molecules can be affected by the presence of immobile obstacles, and the transient binding of the tracers to these obstacles. We have previously developed a micropatterning approach that allows for immobilizing a plasma membrane protein and probing the diffusional behavior of a putative interaction partner in living cells. Here, we provide guidelines on how this micropatterning approach can be extended to quantify interaction parameters between plasma membrane constituents in their natural environment. We simulated a patterned membrane system and evaluated the effect of different surface densities of patterned immobile obstacles on the relative mobility as well as the surface density of diffusing tracers. In the case of inert obstacles, the size of the obstacle can be assessed from its surface density at the percolation threshold, which in turn can be extracted from the diffusion behavior of the tracer. For sticky obstacles, two-dimensional dissociation constants can be determined from the tracer diffusion or surface density.
脂质和蛋白质的单分子轨迹能够提供有关质膜本身纳米级组织的宝贵信息。然而，由于分子的流动性会受到固定障碍物的存在以及示踪剂与这些障碍物的瞬时结合的影响，对这些轨迹的解释变得复杂。我们之前开发了一种微图案化方法，该方法能够固定质膜蛋白并探测活细胞中假定相互作用伙伴的扩散行为。在此，我们提供有关如何扩展这种微图案化方法以量化天然环境中质膜成分之间相互作用参数的指导方针。我们模拟了一个图案化的膜系统，并评估了图案化固定障碍物的不同表面密度对相对流动性以及扩散示踪剂表面密度的影响。对于惰性障碍物，可以从其在渗流阈值处的表面密度评估障碍物的大小，而这又可以从示踪剂的扩散行为中提取。对于粘性障碍物，可以从示踪剂扩散或表面密度确定二维解离常数。
