Ca2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites Lysosomes are essential in maintaining cellular health. Endocytic lysosome reformation (ELR) regenerates functional lysosomes following degradation of endocytic cargo, yet the mechanisms driving this process remain largely unknown. Here, we define the molecular machinery underlying ELR. We find that unlike autophagic lysosome reformation (ALR), ELR proceeds independently of mTOR and dynamin 2, but requires the mitochondrial fission GTPase DRP1. DRP1 mediates scission of endolysosomal tubules at contact sites with the endoplasmic reticulum (ER) and mitochondria. Disruption of DRP1 function or ER endolysosome contact results in elongated tubules, indicating defective lysosome reformation. Moreover, mitochondrial activity is essential for tubule initiation, and Ca2+ transfer from endolysosomes to mitochondria is crucial for ELR onset. Our findings reveal a dual role for mitochondria in ELR: first in ELR initiation and second in DRP1-dependent tubule fission at ER-mitochondria-endolysosome tripartite contact sites, uncovering the previously unappreciated role of mitochondria in endolysosome remodeling and fission. ### Competing Interest Statement The authors have declared no competing interest. Swiss National Science Foundation, 320030-231859, 310030-197779 University of Basel, https://ror.org/02s6k3f65

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