Motion of VAPB molecules reveals ER-mitochondria contact site subdomains

• 成果类型: Article
• 署名作者: Obara, Christopher J.; Nixon-Abell, Jonathon; Moore, Andrew S.; Riccio, Federica; Hoffman, David P.; Shtengel, Gleb; Xu, C. Shan; Schaefer, Kathy; Pasolli, H. Amalia; Masson, Jean-Baptiste; Hess, Harald F.; Calderon, Christopher P.; Blackstone, Craig; Lippincott-Schwartz, Jennifer
• 署名单位: Howard Hughes Medical Institute; National Institutes of Health (NIH) - USA; NIH National Institute of Neurological Disorders & Stroke (NINDS); Pasteur Network; Universite Paris Cite; Institut Pasteur Paris; Pasteur Network; Universite Paris Cite; Institut Pasteur Paris; University of Colorado System; University of Colorado Boulder; University of London; King's College London; Yale University; Harvard University; Harvard University Medical Affiliates; Massachusetts General Hospital; Harvard University; Harvard University Medical Affiliates; Massachusetts General Hospital; Harvard University; Harvard Medical School
• 刊物名称: Nature
• ISSN/ISSBN: 0028-6316
• DOI: 10.1038/s41586-023-06956-y
• 发表日期: 2024-02-01
• 关键词: freeze-substitution; chemical fixation; micos complex; live-cell; DYNAMICS; mutant; point; fluorophores; ORGANIZATION; trajectories

摘要

To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites3,4. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle5,6. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation7,8, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (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 remodelling. 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-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle 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. High-speed molecular tracking is integrated with three-dimensional electron microscopy to map the diffusion distribution and ultrastructure of endoplasmic reticulum-mitochondria contact sites, revealing the ability of high-speed single-molecule imaging to map contact site interface structures and corresponding diffusion landscapes.