Vannier Christian, Galli Thierry
Université Paris Cité, Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM U1266, Paris, France.
GHU-Paris Psychiatrie et Neurosciences, Hôpital Sainte Anne, Paris, France.
Methods Mol Biol. 2025;2887:263-280. doi: 10.1007/978-1-0716-4314-3_19.
Studies on the mechanisms and regulation of functional assemblies of SNARE proteins mediating membrane fusion essentially make use of recombinant proteins and artificial phospholipid bilayers. We have developed an easy-to-use in vivo system reconstituting membrane fusion in living bacteria. It relies on the formation of caveolin-dependent intracytoplasmic cisternae followed by the controlled synthesis of members of the synaptic SNARE machinery. Only when a SNARE complex is formed with its intact components does the docking and subsequent fusion occur between the cisternae and the plasma membrane that is accompanied by the disappearance of the former. The phenotypic response of the bacterial cell to fusion events is a remarkable increase in cell body length due to an expansion of the plasma membrane. Therefore, such an easy-to-observe phenotype makes this system amenable to structure-function studies of SNAREs. We describe here the specific ways to produce caveolin and the SNARE proteins from compatible plasmids upon bacterial transformation and to obtain the elongated cell phenotype. We also provide protocols to carry out the preparation of cell culture samples suitable for biochemical and light microscopy analysis.
对介导膜融合的SNARE蛋白功能组装机制及调控的研究主要利用重组蛋白和人工磷脂双层。我们开发了一种易于使用的体内系统，可在活细菌中重建膜融合。它依赖于小窝蛋白依赖性胞质内池的形成，随后是突触SNARE机制成员的可控合成。只有当SNARE复合体与其完整成分形成时，池与质膜之间才会发生对接及随后的融合，同时前者消失。细菌细胞对融合事件的表型反应是由于质膜扩张导致细胞体长显著增加。因此，这种易于观察的表型使该系统适用于SNARE的结构-功能研究。我们在此描述了细菌转化后从兼容质粒产生小窝蛋白和SNARE蛋白以及获得伸长细胞表型的具体方法。我们还提供了用于制备适合生化和光学显微镜分析的细胞培养样品的方案。
