Sari Merve, Schmidt Alina, Dietz Jörn, Steinem Claudia, Janshoff Andreas
Institutes of Physical Chemistry, and Organic and Biomolecular Chemistry, University of Göttingen, Göttingen, Germany.
Institute of Physical Chemistry, University of Göttingen, Göttingen, Germany.
Methods Mol Biol. 2025;2887:207-226. doi: 10.1007/978-1-0716-4314-3_15.
We present two innovative approaches to investigate the dynamics of membrane fusion and the strength of protein-membrane interactions. The first approach employs pore-spanning membranes (PSMs), which allow for the observation of protein-assisted fusion processes. The second approach utilizes colloidal probe microscopy with membrane-coated probes with reconstituted proteins. PSMs enable one to obtain detailed information about the fusion process with particular emphasis on fusion intermediates and fusion pore formation. We demonstrate the potential of the PSM system using SNARE-mediated fusion. Accompanied by colloidal probe microscopy, molecular information can be gathered on how full-length synaptotagmin-1 (syt-1) contributes to the fusion process. We propose that syt-1 engages with anionic bilayers, significantly modifying the adhesion between membranes. The introduction of Ca transforms these interactions, shifting from a state of minimal interaction force between bilayers to one of pronounced strength. This syt-1 interaction facilitates fusion in the presence of Ca with a significant reduction in the occurrence of stalled intermediate fusion states. Moreover, the presence of Ca significantly accelerates the fusion process, an effect that is further amplified by the addition of multivalent anions such as ATP.
我们提出了两种创新方法来研究膜融合的动力学以及蛋白质 - 膜相互作用的强度。第一种方法采用跨孔膜（PSM），它能够观察蛋白质辅助的融合过程。第二种方法利用带有重组蛋白质的膜包被探针的胶体探针显微镜。PSM 使人们能够获得有关融合过程的详细信息，特别关注融合中间体和融合孔的形成。我们使用 SNARE 介导的融合展示了 PSM 系统的潜力。伴随着胶体探针显微镜，可以收集有关全长突触结合蛋白 -1（syt -1）如何促进融合过程的分子信息。我们提出 syt -1 与阴离子双层相互作用，显著改变膜之间的粘附力。Ca 的引入改变了这些相互作用，从双层之间相互作用力最小的状态转变为具有明显强度的状态。这种 syt -1 相互作用在有 Ca 的情况下促进融合，使停滞的中间融合状态的发生率显著降低。此外，Ca 的存在显著加速了融合过程，添加多价阴离子如 ATP 会进一步放大这种效应。
