Mitochondria are double-membrane organelles with varying shapes influenced by metabolic conditions, developmental stage, and environmental stimuli1–4. Their dynamic morphology is realized through regulated and balanced fusion and fission processes5, 6. Fusion is crucial for the health and physiological functions of mitochondria, including complementation of damaged mitochondrial DNAs and maintenance of membrane potential6–8. Mitofusins (Mfns) are dynamin-related GTPases essential for mitochondrial fusion9, 10. They are embedded in the mitochondrial outer membrane and thought to fuse adjacent mitochondria via concerted oligomerization and GTP hydrolysis11–13. However, the molecular mechanisms behind this process remains elusive. Here we present crystal structures of engineered human Mfn1 containing the GTPase domain and a helical domain in different stages of GTP hydrolysis. The helical domain is composed of elements from widely dispersed sequence regions of Mfn1 and resembles the Neck of the bacterial dynamin-like protein. The structures reveal unique features of its catalytic machinery and explain how GTP binding induces conformational changes to promote G domain dimerization in the transition state. Disruption of G domain dimerization abolishes the fusogenic activity of Mfn1. Moreover, a conserved aspartate trigger was found in Mfn1 to affect mitochondrial elongation, likely through a GTP-loading-dependent domain rearrangement. Based on these results, we propose a mechanistic model for Mfn1-mediated mitochondrial tethering. Our study provides important insights in the molecular basis of mitochondrial fusion and mitofusin-related human neuromuscular disorders14.

Genetic studies demonstrate that ␥-protocadherins (PCDH-␥) are required for the survival and synaptic connectivity in neuronal subpopulations of the central nervous system. However, the intracellular signaling mechanisms for PCDH-␥ are poorly understood. Here, we show that PCDH-␥ binds two tyrosine kinases, PYK2 and focal adhesion kinase (FAK), and interaction with PCDH-␥ inhibits kinase activity. Consistent with this, PYK2 activity is abnormally up-regulated in the Pcdh-␥-deficient neurons. Overexpression of PYK2 induces apoptosis in the chicken spinal cord. Thus, negative regulation of PYK2 activity by PCDH could contribute to the survival of subsets of neurons. Surprisingly, we found that PCDH-␣ interacts similarly with PYK2 and FAK despite containing a distinct cytoplasmic domain. In neural tissue, PCDH-␥, together with PCDH-␣, forms functional complexes with PYK2 and/or FAK. Therefore, the identification of common intracellular effectors for PCDH-␥ and PCDH-␣ suggests that dozens of protocadherins generated by Pcdh-␣ and Pcdh-␥ gene clusters can converge different extracellular signals into common intracellular pathways.

Mitofusin-2 (MFN2) is a dynamin-like GTPase that plays a central role in regulating mitochondrial fusion and cell metabolism. Mutations in MFN2 cause the neurodegenerative disease Charcot-Marie-Tooth type 2A (CMT2A). The molecular basis underlying the physiological and pathological relevance of MFN2 is unclear. Here, we present crystal structures of truncated human MFN2 in different nucleotide-loading states. Unlike other dynamin superfamily members including MFN1, MFN2 forms sustained dimers even after GTP hydrolysis via the GTPase domain (G) interface, which accounts for its high membrane-tethering efficiency. The biochemical discrepancy between human MFN2 and MFN1 largely derives from a primate-only single amino acid variance. MFN2 and MFN1 can form heterodimers via the G interface in a nucleotide-dependent manner. CMT2A-related mutations, mapping to different functional zones of MFN2, lead to changes in GTP hydrolysis and homo/hetero-association ability. Our study provides fundamental insight into how mitofusins mediate mitochondrial fusion and the ways their disruptions cause disease.

Mitochondria continually undergo fusion and fission, and these dynamic processes play a major role in regulating mitochondrial function. Studies of several genes associated with familial Parkinson's disease (PD) have implicated aberrant mitochondrial dynamics in the disease pathology, but the importance of these processes in dopaminergic neurons remains poorly understood. Because the mitofusins Mfn1 and Mfn2 are essential for mitochondrial fusion, we deleted these genes from a subset of dopaminergic neurons in mice. Loss of Mfn2 results in a movement defect characterized by reduced activity and rearing. In open field tests, Mfn2 mutants show severe, age-dependent motor deficits that can be rescued with L-3,4 dihydroxyphenylalanine. These motor deficits are preceded by the loss of dopaminergic terminals in the striatum. However, the loss of dopaminergic neurons in the midbrain occurs weeks after the onset of these motor and striatal deficits, suggesting a retrograde mode of neurodegeneration. In our conditional knockout strategy, we incorporated a mitochondrially targeted fluorescent reporter to facilitate tracking of mitochondria in the affected neurons. Using an organotypic slice culture system, we detected fragmented mitochondria in the soma and proximal processes of these neurons. In addition, we found markedly reduced mitochondrial mass and transport, which may contribute to the neuronal loss. These effects are specific for Mfn2, as the loss of Mfn1 yielded no corresponding defects in the nigrostriatal circuit. Our findings indicate that perturbations of mitochondrial dynamics can cause nigrostriatal defects and may be a risk factor for the neurodegeneration in PD.

SUMMARY Mitochondrial fission requires recruitment of dynamin-related protein 1 (Drp1) to the mitochondrial surface and activation of its GTP-dependent scission function. The Drp1 receptors MiD49 and MiD51 recruit Drp1 to facilitate mitochondrial fission, but their mechanism of action is poorly understood. Using X-ray crystallography, we demonstrate that MiD51 contains a nucleotidyl transferase domain that binds ADP with high affinity. MiD51 recruits Drp1 via a surface loop that functions independently of ADP binding. However, in the absence of nucleotide binding, the recruited Drp1 cannot be activated for fission. Purified MiD51 strongly inhibits Drp1 assembly and GTP hydrolysis in the absence of ADP. Addition of ADP relieves this inhibition and promotes Drp1 assembly into spirals with enhanced GTP hydrolysis. Our results reveal ADP as an essential cofactor for MiD51 during mitochondrial fission.