Abstract：
Biomolecular condensates play a major role in numerous cellular processes, including several that occur on the surface of lipid bilayer membranes. There is increasing evidence that cellular membrane trafficking phenomena, including the internalization of the plasma membrane through endocytosis, are mediated by multivalent protein-protein interactions that can lead to phase separation. We have recently found that proteins involved in the clathrin-independent endocytic pathway named Fast Endophilin Mediated Endocytosis can undergo liquid-liquid phase separation (LLPS) in solution and on lipid bilayer membranes. Here, the protein solution concentrations required for phase separation to be observed are significantly smaller compared to those required for phase separation in solution. LLPS is challenging to systematically characterize in cellular systems in general, and on biological membranes in particular. Model membrane approaches are more suitable for this purpose as they allow for precise control over the nature and amount of the components present in a mixture. Here we describe a method that enables the imaging of LLPS domain formation on solid supported lipid bilayers. These allow for facile imaging, provide long-term stability, and avoid clustering of vesicles and vesicle-attached features (such as buds and tethers) in the presence of multi-valent membrane interacting proteins.
摘要：
生物分子缩合物在许多细胞过程中起着重要作用，包括一些发生在脂质双层膜表面的。越来越多的证据表明，细胞膜贩运现象，包括通过胞吞作用使质膜内化,是由多价蛋白质-蛋白质相互作用介导的，可以导致相分离。我们最近发现，与网格蛋白无关的内吞途径有关的蛋白质称为FastEndophilin介导的内吞，可以在溶液中和脂质双层膜上进行液-液相分离（LLPS）。这里,与溶液中的相分离所需的蛋白质溶液浓度相比,观察到的相分离所需的蛋白质溶液浓度显著较小。通常，LLPS在蜂窝系统中系统地表征是具有挑战性的，特别是在生物膜上。模型膜方法更适合于该目的,因为它们允许精确控制存在于混合物中的组分的性质和量。在这里，我们描述了一种能够在固体支持的脂质双层上成像LLPS结构域形成的方法。这些可以方便地成像，提供长期稳定性，并避免在多价膜相互作用蛋白的存在下聚集囊泡和囊泡附着的特征（例如芽和系链）。
