Raw Airyscan Images for PTPIP51 experiments for <b>Motion of VAPB molecules reveals ER-mitochondria contact site subdomains</b>

Airyscan images of cells overexpressing PTPIP51 in a variety of tagged and labeled conditions. Expansion of ERMCSs are visible as mitochondria wrapped with ER.Obara + Nixon-Abell et al. 2023, https://www.biorxiv.org/content/10.1101/2022.09.03.505525v1<b>Motion of VAPB molecules reveals ER-mitochondria contact site subdomains</b>To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signaling molecules, lipids, and metabolites. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation, a clear understanding of their nanoscale organization and regulation is still lacking. Here, we combine 3D electron microscopy with high-speed molecular tracking of a model organelle tether, VAPB, to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodeling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis (ALS)-associated mutation in VAPB perturbs these subdomains, likely impairing their remodeling capacity and resulting in impaired inter-organelle communication. These results establish high speed single molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.

本数据集涵盖多种标记与标签条件下过表达蛋白酪氨酸磷酸酶相互作用蛋白51（PTPIP51）的细胞的Airyscan成像图。其中可见内质网-线粒体接触位点（Endoplasmic Reticulum-Mitochondrial Contact Sites, ERMCSs）发生扩张，表现为被内质网包裹的线粒体。本数据来自Obara与Nixon-Abell等人2023年的研究，原文链接：https://www.biorxiv.org/content/10.1101/2022.09.03.505525v1，论文标题为《VAPB分子的运动揭示内质网-线粒体接触位点亚结构域》。为协调细胞生理活动，真核细胞依赖特化的细胞器-细胞器接触位点处的分子快速交换。内质网-线粒体接触位点（ERMCSs）是尤为关键的通讯枢纽，在信号分子、脂质与代谢物的交换过程中发挥核心作用。这类接触位点的维持依赖于两种细胞器表面互补锚定分子之间的相互作用。然而，由于这些膜界面对实验扰动极为敏感，目前学界仍缺乏对其纳米级组织与调控机制的清晰认知。本研究将三维电子显微镜技术与模式细胞器锚定蛋白VAPB的高速分子追踪技术相结合，绘制了ERMCSs的结构与扩散图谱。研究团队发现，VAPB接触位点内存在动态亚结构域，该亚结构域与内质网膜曲率相关且可发生快速重塑。实验结果表明，尽管接触位点本身在更长时间尺度上保持稳定，但VAPB分子可在数秒内进出ERMCSs。这种亚稳态使得ERMCSs能够随生理环境变化发生重塑，以适配细胞的代谢需求。一种与肌萎缩侧索硬化（Amyotrophic Lateral Sclerosis, ALS）相关的VAPB突变会扰乱这些亚结构域，可能削弱其重塑能力，进而导致细胞器间通讯受损。本研究结果证实，高速单分子成像可作为绘制接触位点界面结构的全新工具，并揭示了VAPB在接触位点的扩散图谱是ERMCS稳态的关键组成部分。