Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, Sorbonne Université.
Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, Sorbonne Université; Institute of Biotechnology, Czech Academy of Sciences, BIOCEV.
J Vis Exp. 2022 Aug 17(186). doi: 10.3791/63889.
Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic conditions, protein and lipid compositions, membrane curvature) and the different partners associated with specific membrane reshaping processes, we undertake in vitro bottom-up approaches. In recent years, there has been keen interest in revealing the role of septin proteins associated with major diseases. Septins are essential and ubiquitous cytoskeletal proteins that interact with the plasma membrane. They are implicated in cell division, cell motility, neuro-morphogenesis, and spermiogenesis, among other functions. It is, therefore, important to understand how septins interact and organize at membranes to subsequently induce membrane deformations and how they can be sensitive to specific membrane curvatures. This article aims to decipher the interplay between the ultra-structure of septins at a molecular level and the membrane remodeling occurring at a micron scale. To this end, budding yeast, and mammalian septin complexes were recombinantly expressed and purified. A combination of in vitro assays was then used to analyze the self-assembly of septins at the membrane. Supported lipid bilayers (SLBs), giant unilamellar vesicles (GUVs), large unilamellar vesicles (LUVs), and wavy substrates were used to study the interplay between septin self-assembly, membrane reshaping, and membrane curvature.
膜重塑在质膜和细胞细胞器中不断发生。为了充分剖析环境（离子条件、蛋白质和脂质组成、膜曲率）以及与特定膜重塑过程相关的不同伙伴的作用，我们采用了自下而上的体外方法。近年来，人们对揭示与重大疾病相关的 septin 蛋白的作用产生了浓厚的兴趣。Septins 是与质膜相互作用的必需且普遍存在的细胞骨架蛋白。它们涉及细胞分裂、细胞运动、神经形态发生和精子发生等功能。因此，了解 septin 如何在膜上相互作用和组织，从而随后诱导膜变形，以及它们如何对特定的膜曲率敏感，这一点非常重要。本文旨在解析 septin 在分子水平上的超结构与微米尺度上发生的膜重塑之间的相互作用。为此，我们重组表达和纯化了芽殖酵母和哺乳动物 septin 复合物。然后，使用一系列体外测定法来分析 septin 在膜上的自组装。我们使用支持的脂质双层（SLBs）、巨大的单层囊泡（GUVs）、大单层囊泡（LUVs）和波浪形底物来研究 septin 自组装、膜重塑和膜曲率之间的相互作用。
