2. Academic Validation
  3. Phase separation of OPTN initiates mitophagy to orchestrate craniofacial bone mineralization

Phase separation of OPTN initiates mitophagy to orchestrate craniofacial bone mineralization

  • Autophagy. 2026 May;22(5):1021-1043. doi: 10.1080/15548627.2026.2624745.
Haojie Liu 1 2 3,  Zhenyi Lu 1 2 3,  Xinyu Zhang 1 2 3,  Yan Wang 1 2 3,  Xiao Ge 1 3 4,  Simai Chen 1 2 3,  Yumeng Shi 1 2 3,  Jingjing Yan 1 2 3,  Rongyao Xu 1 3 4,  Junqing Ma 5 6,  Shuyu Guo 1 2 3
Affiliations

Affiliations

  • 1 Department of Orthodontics, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, China.
  • 2 State Key Laboratory Cultivation Base of Research, Prevention and Treatment for Oral Diseases, Nanjing, China.
  • 3 Jiangsu Province Engineering Research Center of Stomatological Translational Medicine, Nanjing, China.
  • 4 Department of Oral and Maxillofacial Surgery, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, China.
  • 5 Savaid Stomatology School, Hangzhou Medical College, Hangzhou, China.
  • 6 Hangzhou Stomatological Hospital (Zijingang Campus), Hangzhou, China.
PMID: 41692957 DOI: 10.1080/15548627.2026.2624745
Abstract

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 in vitro and in vivo, 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.Abbreviations: 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.

Keywords

Bone mineralization; OPTN; craniofacial development; mitophagy; phase separation.

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