A perilysosomal feedforward mechanism regulates starvation-induced calcium signaling

Nutrient depletion triggers a starvation-induced calcium (Ca²⁺) signal (SICS) that promotes Ca²⁺-dependent responses. However, the components and regulations of SICS are unclear. Here, we explored SICS components and their regulation by the Ca²⁺ sensor calmodulin (CaM). Overexpression of the stromal interaction molecule 1 (STIM1), a key switcher of store-operated Ca²⁺ entry (SOCE), enhances SICS by fourfold. This effect is abolished by the truncation of the Ca²⁺-binding loop within STIM1. Consistently, SOCE inhibition strongly suppresses SICS. Nutrient removal or stimulation of the transient receptor potential mucolipin-1 (TRPML1, encoded by the Mcoln1 gene) triggers intracellular Ca²⁺ release that is prevented by pre-emptying of endoplasmic reticulum (ER) Ca²⁺. In the presence of extracellular Ca²⁺, inhibition and silencing of Mcoln1 reduces SICS by 35-40%. We identified a CaM-binding site in the second cytoplasmic loop of TRPML1 that interacts with Ca²⁺-bound CaM. Mcoln1 overexpression enhances the upstroke and peak of SICS, effects that are absent with a mutated CaM-binding domain. Further, we generated a genetically encoded biosensor for TRPML1 (BS-ML1). BS-ML1 produces a robust signal upon nutrient deprivation, which is substantially reduced with the Mcoln1 mutant. The response of BS-ML1 to nutrient depletion is equally reduced by extracellular Ca²⁺ removal or SOCE inhibition. Reduced CaM availability significantly prolongs SICS, consistent with an ~40% reduction in cytoplasmic Ca²⁺ removal rate. Our data thus indicate that (1) SICS comprises multiple components, including linked Ca²⁺ release from the lysosome and ER and subsequent SOCE; and (2) CaM regulates the kinetics and magnitude of SICS by controlling cytoplasmic Ca²⁺ removal and a perilysosomal feedforward mechanism that promotes TRPML1 activity. The dynamics of this feedforward mechanism likely regulate subsequent tissue responses to nutrient starvation.