A study using three-dimensional electron microscopy and high-speed single-molecule tracking reveals dynamic subdomains within ER–mitochondria contact sites (ERMCSs), where VAMP-associated protein B (VAPB) molecules exhibit rapid diffusion and exchange. These subdomains correlate with ER membrane curvature and undergo rapid remodelling, allowing ERMCSs to adapt to physiological changes. VAPB molecules enter and exit ERMCSs within seconds, despite the contact site remaining stable over longer time scales. This metastability enables ERMCSs to remodel in response to metabolic needs. An ALS-associated mutation in VAPB disrupts these subdomains, impairing interorganelle communication. The study also shows that VAPB tethers interact with mitochondrial binding partners, facilitating calcium and lipid transfer. In cells with ALS-linked VAPB mutations, VAPB molecules become trapped in subdomains, disrupting normal dynamics and potentially leading to impaired ERMCS function. The findings highlight the importance of dynamic regulation of ERMCSs and suggest that high-speed single-molecule imaging is a powerful tool for studying contact site interfaces. The results provide new insights into the structure and function of ERMCSs, emphasizing the role of VAPB diffusion in maintaining their homeostasis. The study also demonstrates that ERMCSs can dynamically reorganize in response to metabolic demands, such as during nutrient deprivation, and that VAPB availability and interactions regulate contact site size and function. The research underscores the significance of ERMCSs in cellular metabolism and their potential role in disease.A study using three-dimensional electron microscopy and high-speed single-molecule tracking reveals dynamic subdomains within ER–mitochondria contact sites (ERMCSs), where VAMP-associated protein B (VAPB) molecules exhibit rapid diffusion and exchange. These subdomains correlate with ER membrane curvature and undergo rapid remodelling, allowing ERMCSs to adapt to physiological changes. VAPB molecules enter and exit ERMCSs within seconds, despite the contact site remaining stable over longer time scales. This metastability enables ERMCSs to remodel in response to metabolic needs. An ALS-associated mutation in VAPB disrupts these subdomains, impairing interorganelle communication. The study also shows that VAPB tethers interact with mitochondrial binding partners, facilitating calcium and lipid transfer. In cells with ALS-linked VAPB mutations, VAPB molecules become trapped in subdomains, disrupting normal dynamics and potentially leading to impaired ERMCS function. The findings highlight the importance of dynamic regulation of ERMCSs and suggest that high-speed single-molecule imaging is a powerful tool for studying contact site interfaces. The results provide new insights into the structure and function of ERMCSs, emphasizing the role of VAPB diffusion in maintaining their homeostasis. The study also demonstrates that ERMCSs can dynamically reorganize in response to metabolic demands, such as during nutrient deprivation, and that VAPB availability and interactions regulate contact site size and function. The research underscores the significance of ERMCSs in cellular metabolism and their potential role in disease.