BPS2026 – Integrating molecular dynamics and single-particle tracking across scales to understand the conversations governing mitochondrial regulation

Mitochondria define the cellular metabolic state and respond to cellular needs by dynamically remodeling in response to signals from other organelles. These processes are regulated in space and time by signaling pathways and direct interactions of proteins at membrane contact sites. However, the way the molecular machinery driving these events is coordinated across different spatial scales remains poorly understood. Here, we present a pipeline that integrates computational structural bioinformatics, molecular dynamics simulations, and high-speed single particle tracking to connect protein structure with functional dynamics in living cells. We apply this framework to two key regulators of mitochondrial function. First, we examine VAMP-associated protein B (VAPB), a conserved ER-mitochondria tether that organizes lipid and Ca 2+ exchange. Our results identify evolutionarily conserved features of VAPB, revealed through informatics and modeling, that control the kinetics and stability of ER-mitochondria contact sites. Directed mutagenesis and high-speed single-particle tracking reveal a two-component switch for engagement of these proteins at contact sites in living cells. Second, we analyze protein kinase A (PKA), a central metabolic kinase linked to global regulation of cellular metabolism. We demonstrate specific interactions of single PKA holoenzyme complexes with proteins on the mitochondrial surface and show that this interaction correlates directly with mitochondrial dynamics and function. Thus, local recruitment of the PKA holoenzyme controls signaling nanodomains that regulate fission, fusion, and bioenergetics. Together, this work establishes a broadly applicable strategy for linking protein structure to nanoscale dynamics and provides new mechanistic insight into how tethering and signaling proteins coordinate mitochondrial communication and function.