Calcium directs actin assembly via allosteric activation of formin INF2

Abstract Calcium signals spatiotemporally orchestrate cytoskeletal dynamics, typically through Rho GTPase-mediated signaling cascades, yet the direct molecular transducers that convert local calcium transients into spatially controlled actin assembly have remained elusive. Here, we uncover a structure-based mechanism by which calcium-bound calmodulin (Ca 2+ -CaM) directly activates formin INF2 to drive actin assembly. We demonstrate that Ca 2+ -CaM binds to the diaphanous inhibitory domain (DID) of INF2 with nanomolar affinity, inducing allosteric conformational changes that sterically disrupt INF2 autoinhibition. The unexpected bipartite Ca 2+ -CaM binding interface on INF2 enables ultrasensitive decoding of local calcium microdomains, such as ER-mitochondrial contacts, where activated INF2 promotes actin polymerization to facilitate mitochondrial fission. We further show that the Charcot-Marie-Tooth disease–associated INF2 R91G mutation enhances Ca 2+ -CaM binding via optimized interfacial dynamics, suggesting a gain-of-function disease mechanism. Our work establishes the Ca 2+ -CaM–INF2 axis as a direct molecular transducer linking spatial calcium signals to actin-dependent organelle dynamics, defining a Rho-GTPase-independent paradigm for calcium–cytoskeleton communication, with broad implications for INF2-linked pathologies.