The study investigates the dynamics and structure of endoplasmic reticulum (ER)-mitochondria contact sites (ERMCSSs) using high-speed molecular tracking of Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB) and three-dimensional electron microscopy (3D-SEM). VAPB molecules, which are key tethers at ERMCSSs, were tracked to reveal dynamic subdomains within these sites that correlate with ER membrane curvature and undergo rapid remodeling. VAPB molecules enter and leave ERMCSSs within seconds, despite the contact site's stability over longer time scales. This metastability allows ERMCSSs to adapt to changes in the physiological environment, facilitating metabolic needs. An amyotrophic lateral sclerosis-associated mutation in VAPB disrupts these subdomains, impairing their remodeling capacity and interorganelle communication. The findings establish high-speed single-molecule imaging as a tool for mapping contact site interfaces and highlight the diffusion landscape of VAPB as a crucial component of ERMCSSs.The study investigates the dynamics and structure of endoplasmic reticulum (ER)-mitochondria contact sites (ERMCSSs) using high-speed molecular tracking of Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB) and three-dimensional electron microscopy (3D-SEM). VAPB molecules, which are key tethers at ERMCSSs, were tracked to reveal dynamic subdomains within these sites that correlate with ER membrane curvature and undergo rapid remodeling. VAPB molecules enter and leave ERMCSSs within seconds, despite the contact site's stability over longer time scales. This metastability allows ERMCSSs to adapt to changes in the physiological environment, facilitating metabolic needs. An amyotrophic lateral sclerosis-associated mutation in VAPB disrupts these subdomains, impairing their remodeling capacity and interorganelle communication. The findings establish high-speed single-molecule imaging as a tool for mapping contact site interfaces and highlight the diffusion landscape of VAPB as a crucial component of ERMCSSs.