PMC9538651_01
DNM1L/DRP1 immunostaining shows the loss of DNM1L normal forms in patient fibroblasts. (A1) Comparison of the GTPase domain structure of nucleotide-free, wild-type (orange, pdb code 4H1U) and mutated THR59ASN DNM1L (gray) in ribbon and Gaussian volume representation, using RCSB pairwise structure alignment tool. The mutation site is indicated by a red circle. Modeling indicates a disorganization of the switch 1 in which the mutation is located, but also in the unique 80-loop, alphaE1G helix and the loop connecting alphaE1G and the beta2A sheet (indicated by black arrow). (A2) Close-up view of the switch1 in the nucleotide-free structure of the WT (orange) and mutated (gray) DNM1L. ASN59 is underlined in green. Water molecules are represented in yellow. The catalytic water molecule connecting switch I (THR59) with switch II (GLY149) by hydrogen bonds is circled in red. This interaction is lost with the amino acid switch THR59ASN. Amino acids interacting with THR59 or ASN59 are indicated. Detailed view of amino acid interactions in nucleotide-free wild-type (A3) and mutated (A4) protein structures. Hydrophobic interaction is in green, hydrogen bonds in red, and VDW in blue. Analysis was performed using Dynamut2. (B) OPA1 and DNM1L/DRP1 protein quantification and alpha-tubulin (TUB) as loading control in control (CTL) and patient (P.) fibroblasts. Control and DRP1 knock out MEFs: Mouse embryonic Fibroblasts models. On the right, overlay yellow staining of 2 DNM1L/DRP1 antibodies Ab1 and 2 indicates specific staining. <<  >> and <<  >>: controls were charged at 1/2 and  to control the linearity range of DNM1L signal detection.
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F1
The 32-year-old female patient is the only child of healthy non-consanguineous Caucasian parents (Supplementary Figure 1). She had a long and asymmetric face with deep-set eyes, divergent strabismus, high nasal bridge, and high arched palate (Supplementary Figure 1). In silico structural modeling of DNM1L Thr59Asn and comparison with the wild-type, nucleotide-free structure (PDB:4H1U) indicated conformational changes in the switch s1, alphaE1G helix, and two disorders loop, the 80-loop and the loop connecting alphaE1G and the beta2A sheet (Figure 1A). In addition, bioinformatics tests predicted that the Thr59Asn variant affects the stability of DNM1L (Supplementary Figure 3). Unexpectedly, by Western blot, we showed a strong decrease of DNM1L protein expression (Figure 1B), with the very faint of signal corresponding to the two 81-83Kda bands generally revealed by DNM1L immunostaining.
Patient fibroblasts show highly connected mitochondrial network and elongation of peroxisome compare to control. (A) Representative fluorescent images of mitochondrial network structure (in purple) in control (CTL) and patient (P.) fibroblasts. Mitochondrial volume was assessed using the MitoTracker Green fluorescent signal which was analyzed using Imaris software. The color code highlights the different lengths of the mitochondrial tubules with short isolated mitochondria in violet and connected network in red. To present the changes in mitochondrial morphology in patient's cells, types of mitochondria were classified into six groups according to mitochondrial volume, for example, red color represents mitochondria > 20 mum3. Oligomycin 4 mug/ml coupled to antimycin 2 mug/ml (O/A) treatment leads to mitochondrial fission within 4 h in CTL cells. 6 h nocodazole (Noc), 10 muM disrupts microtubules. (B) Immunostaining against mitochondrial Mff DRP1 receptor. Representative TIRF image of mitochondria (left, in purple) shows the hyper-connectivity of the network. Single-molecule localization microscopy dSTORM was used to analyze mitochondrial MFF distribution. The white dots are the fiducial signals used to control drift during acquisition. Scale bar: 2 mum. (C) Immunostaining against peroxisomal PMP70 protein. Peroxisome (PO) volume was assessed using fluorescent signal by Imaris software and color-coded. To present the changes in peroxisome morphology in patient's cells, types of Peroxisomes were classified into five groups according to peroxisome volume, for example, red color represents peroxisomes > 20 mum3. Scale bar = 10 mum; Scale bar in details 1 mum.
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F2
Paternity and maternity were molecularly confirmed, and the variant was not found in the parents suggesting that it arose de novo (paternal coverage 146X; maternal coverage 99X) (Supplementary Figure 2). In the proband, cDNA short- and long-range amplification and sequencing revealed that the mutant and the wide-type DNML1 alleles are both expressed, in similar proportion (50%-50%), in blood extracted lymphocytes, in lymphoblastoid cells, and in fibroblasts (Supplementary Figure 2 and Supplementary material and method). In the mother, this mitochondrial variant was found at 1.3% heteroplasmy in urine but was not significantly detected in blood (Supplementary Figure 2). Normal skin fibroblasts show a set of dynamic tubular networks with some isolated mitochondria that we could fragment by bioenergetic stress (Figure 2A). 3D analysis and morphometric measurements revealed a hyper-connected mitochondrial network (Figure 2A) without any isolated mitochondria. We performed immunostaining of Mff, the mitochondrial outer membrane DRP1 receptor, by super-resolution microscopy and confirmed normal Mff distribution on mitochondrial hyper-connected network of the mutated fibroblasts (Figure 2B). Compared to control fibroblasts, disclosing small and abundant peroxisomes, mutated fibroblasts showed a decreased number of peroxisomes, which appeared significantly elongated (Figure 2C).
Electron microscopy imaging of the patient fibroblasts and muscle. (A) Skin fibroblasts: M, mitochondria; PO, peroxisomes; N, nucleus; AV, autophagic vacuole. (B) Muscle biopsy: a: concentric laminated bodies; b: mitochondrial nano-tunnel; c multiple degenerative myelinoid intramitochondrial foci.
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F3
In silico structural modeling of DNM1L Thr59Asn and comparison with the wild-type, nucleotide-free structure (PDB:4H1U) indicated conformational changes in the switch s1, alphaE1G helix, and two disorders loop, the 80-loop and the loop connecting alphaE1G and the beta2A sheet (Figure 1A). In addition, bioinformatics tests predicted that the Thr59Asn variant affects the stability of DNM1L (Supplementary Figure 3). While contradictory results were obtained according to the predictive tools used for the nucleotide-free structure, the Thr59Asn substitution is predicted to destabilize the protein in its nucleotide-bound state, particularly in the GDP-bound one (Supplementary Figure 3B). Transmission electron microscopy (TEM) pictures of patient fibroblasts confirmed the presence of enlarged peroxisomes with many peroxisomes in autophagic vacuoles and longer mitochondria than in control cells (Figure 3A). Subsarcolemmal concentric laminated bodies and several multiple degenerative myelinoid intramitochondrial foci were identified (Figure 3B).