Phase separation of OPTN initiates mitophagy to orchestrate craniofacial bone mineralization

Recently, mitophagy-mediated bone mineralization of mesenchymal stem cells has emerged as another bone formation pattern, but whether mitophagy-mediated bone mineralization shapes craniofacial development remains unknown. Here, we demonstrate that loss of OPTN, a keystone macroautophagy/autophagy receptor, impairs mitophagy and acidic calcium phosphate (ACP) transport in orofacial bone mesenchymal stem cells (OMSCs), leading to craniofacial bone mineralization defects. We substantiate that OPTN undergoes LLPS both <i>in vitro</i> and <i>in vivo</i>, driven by S173 phosphorylation within its intrinsically disordered N-terminal domain (NTD), facilitating the association of OPTN complexes with phagophore membranes. Additionally, the ubiquitin-binding domain (UBD) in OPTN's C-terminal domain (CTD) also promotes LLPS to recruit ubiquitin-modified mitochondria. Physiochemically, mutations at the conserved sites in human OPTN (S173A and D474N) disrupt the OPTN LLPS, as validated in mouse and zebrafish, thereby inhibiting mitophagy and impairing bone mineralization. Together, our findings reveal a new mechanism through which OPTN LLPS couples mitophagy-mediated mineralization to craniofacial bone development, highlighting its potential as a therapeutic target for treating orofacial malformations via modulation of mitophagy.<b>Abbreviations</b>: 1, 6HD: 1, 6-hexanediol; ACP: acidic calcium phosphate; ALP: alkaline phosphatase; ARS: Alizarin Red staining; BFR/BS: bone formation rate per bone surface; Baf-A1: bafilomycin A₁; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CTD: C-terminal domain; dpf: days post-fertilization; EDS: energy dispersive spectroscopy; FL: full length; FRAP: fluorescence recovery after photobleaching; hpf: 24h post-fertilization; IDR: intrinsically disordered region; IHC: immunohistochemistry; LLPS: liquid-liquid phase separation; LC-MS/MS: liquid chromatography-tandem mass spectrometry; MAR: mineral apposition rate; MS/BS: mineralizing surface per bone surface; NTD: N-terminal domain; ODM: osteogenic differentiation medium; OMSCs: orofacial bone mesenchymal stem cells; OPTN: optineurin; P1: postnatal day 1; P21: postnatal day 21; PDB: Paget disease of bone; PTMs: post-translational modifications; qRT-PCR: quantitative real-time PCR; S173: serine 173; STK4: serine/threonine kinase 4; SEM: scanning electron microscopy; TMD: tissue mineral density; TEM: transmission electron microscopy; UBD: ubiquitin-binding domain; Ub: ubiquitin.