Department of Chemistry, University of Pennsylvania, Philadelphia, PA.
Department of Chemistry, University of Pennsylvania, Philadelphia, PA.
Methods Enzymol. 2024;700:33-48. doi: 10.1016/bs.mie.2024.04.007. Epub 2024 Apr 23.
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.
生物分子凝聚物在许多细胞过程中发挥着重要作用，包括发生在脂质双层膜表面的几个过程。越来越多的证据表明，细胞膜运输现象，包括通过胞吞作用内化质膜，是由多价蛋白质-蛋白质相互作用介导的，这些相互作用可能导致相分离。我们最近发现，参与网格蛋白非依赖性胞吞作用途径的蛋白质，即快速内吞素介导的胞吞作用，在溶液中和脂质双层膜上可以发生液-液相分离（LLPS）。在这里，与在溶液中发生相分离所需的浓度相比，观察到相分离所需的蛋白质溶液浓度要小得多。一般来说，LLPS 在细胞系统中很难进行系统地表征，特别是在生物膜上。模型膜方法更适合于这一目的，因为它们可以精确控制混合物中存在的成分的性质和数量。在这里，我们描述了一种能够在固体支撑脂质双层上成像 LLPS 域形成的方法。这些方法允许轻松成像，提供长期稳定性，并避免多价膜相互作用蛋白存在时囊泡和囊泡附着特征（如芽和系绳）的聚集。
