Mitochondrial network remodelling is crucial for cells to adapt and respond to metabolic cues and stress signals. Processes collectively known as mitochondrial dynamics, including ongoing cycles of fusion and fission, regulate mitochondrial shape, size and distribution, governing mitochondrial function and cellular homeostasis. Accordingly, abnormal mitochondrial morphology has been described in the pathology of numerous diseases. Nonetheless, the underlying mechanisms of mitochondrial fusion and division are still not fully elucidated. Actin polymerization and actin cytoskeleton machinery, including the actin-related protein 2/3 (Arp2/3)-complex, have been implicated at different stages of the complex multi-step mechanism of mitochondrial division. Recent work has also highlighted the contribution of phosphatidylinositol 4-phosphate (PI4P) trafficking, from trans-Golgi network vesicles (TGNv) and lysosomes, at the mitochondrial fission site during the last steps of the process. However, how the actin machinery is recruited and how PI4P could contribute to mitochondrial division remain elusive.

Family with sequence similarity 21 (FAM21), a retromer- and PI4P-binding protein of the nucleation-promoting factor Wiskott-Aldrich Syndrome protein family member WASH (WASH) complex, recruits WASH and Arp2/3 complexes to endosomal membranes. Coordinated activity of WASH and Arp2/3 complexes drives actin polymerization to facilitate endosomal sorting and potentially fission. To address whether a similar mechanism could be at play in mitochondrial division, the role of FAM21 in regulating mitochondrial morphology and dynamics is here investigated.

The results showed that transient and stable loss of FAM21 in cells induced mitochondrial elongation with hyperconnectivity and increased number of constricted areas at mitochondrial membranes. These morphological effects were accompanied by an increase of the fusion protein mitofusin 2 (Mfn2) and a reduction of the fission machinery components mitochondrial fission factor (MFF) and Dynamin-related protein 1 (Drp1). Mitochondrial elongation induced by FAM21 depletion was sensitive to fragmentation stimulus, suggesting a secondary role in the process of division. Live cell imaging revealed that FAM21 is dynamically recruited to sites of mitochondrial network remodelling and participates in fission events marked by TGNv, lysosomes, Drp1 and PI4P, playing a role at a step downstream of Drp1 and PI4P recruitment.

In conclusion, FAM21 is a non-essential novel regulator of mitochondrial morphology which participates in TGNv and lysosome-mediated mitochondrial division events.