Miro1 functions as an inhibitory regulator of MFN at elevated mitochondrial Ca²⁺ levels

Mitochondria function as an integrated network that moves along the microtubules within cells and changes the morphology through membrane fusion and fission events. Mitofusin (MFN) mediates membrane tethering and subsequent fusion of the mitochondrial outer membrane. Understanding the regulatory mechanisms of MFN function is critical to tackling the pathology related to mitochondrial network imbalance. Here, we reveal a novel inhibitory mechanism of MFN‐mediated fusion by mitochondrial Rho GTPase (Miro1) in response to elevated mitochondrial Ca 2+ concentration ([Ca 2+ ] m ). We showed that elevated [Ca 2+ ] m prevents the fusion between mitochondria forming the outer membrane tether by ectopically expressing MFN. Lowering [Ca 2+ ] m by treating cells with an inhibitor of mitochondrial calcium uniporter or knocking down Miro1/2 induces more fused networks. Miro1 interacts with MFN as supported by co‐immunoprecipitation and protein association identified by proximity labeling proteomics. It suggests that Miro1 functions as a Ca 2+ ‐sensor and inhibits MFN function at elevated [Ca 2+ ] m. Miro1 EF‐hand mutant has a compromised inhibitory effect, which reiterates Ca 2+ ‐modulated regulation. Dysregulated Ca 2+ ‐handling and mitochondrial network imbalance are highly relevant in the pathology of cancers, cardiovascular, and neurodegenerative diseases. Miro1 functions as a coordinated Ca 2+ ‐responder by pausing mitochondrial transport while reducing network fusion and cooperating with Drp1‐mediated fission. It likely prevents the detrimental effect of Ca 2+ m overload and facilitates mitophagy. Our finding reveals a novel regulation of mitochondrial network dynamics responding to [Ca 2+ ] m through the interplay of Miro1 and MFN. Modulation of Miro1 and MFN interaction is a potential intervention to promote network homeostasis.