Pathogenic DRP1 variants reveal a role for biomolecular condensation in mitochondrial fission
Year: 2026
Type: preprint
Abstract: Mitochondrial fission is essential for proper organelle function and for cellular homeostasis. Excessive fission is associated with several neurological disorders, whereas impaired fission leads to the lethal encephalopathy EMPF1, which is caused by heterozygous mutations in DNM1L. DNM1L encodes the mitochondrial fission mechanoenzyme DRP1, which self-assembles to drive membrane scission. Wild-type DRP1 appear as punctate structures thought to be pre-scission complexes of well-ordered oligomeric assemblies. Immunofluorescence imaging of patient-derived EMPF1 fibroblasts carrying assembly-deficient DNM1L variants reveals elongated mitochondrial networks consistent with impaired fission. Despite this loss-of-function phenotype, these cells unexpectedly retain wild-type numbers of DRP1 puncta. We confirmed the previously reported inability of purified pathogenic DRP1 variants p.Gly363Asp and p.Gly401Ser to assemble under conditions in which WT DRP1 forms helical polymers. Under macromolecular crowding conditions, however, both wild-type and mutant DRP1 access condensed states whose formation depends on protein concentration and solution conditions. Acute treatment of EMPF1 fibroblasts with 1,6-hexanediol decreases mitochondrial DRP1 puncta intensity in mutant cells relative to wild type, indicating genotype-dependent differences in puncta. The magnitude of these differences supports a dominant-negative phenotype of the pathogenic variants. Together, these findings support a model in which DRP1 puncta occupy a continuum of states, from liquid-like condensates to ordered, fission-competent assemblies, revealing biomolecular condensation as a previously unrecognized layer of DRP1 regulation. Biasing DRP1 along this continuum may provide a mechanistic basis for impaired fission in EMPF1 and suggest opportunities to restore productive assembly in select pathogenic contexts. Significance Statement DRP1 puncta associated with mitochondrial fission are thought to be well-ordered oligomeric assemblies that precede membrane scission. Yet their dynamic behavior within cells has remained difficult to reconcile as well-ordered assembly. Under prevailing models, cells bearing pathogenic DNM1L variants impaired in assembly would be expected to lack puncta, but we show these cells retain wild-type puncta levels. We demonstrate that both wild-type and pathogenic mutant DRP1 populate multiple condensed states in vitro , and that disease variants are biased toward more fluid, chemically sensitive assemblies. These findings identify biomolecular condensation as a regulatory layer of DRP1 organization and suggest that shifting DRP1 along this assembly continuum may restore productive fission in select pathogenic contexts.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funders: Medical College of Wisconsin, Children’s Hospital of Wisconsin Research Institute, LifeArc, National Institute for Health and Care Research, National Institutes of Health, University of Colorado Boulder, Anschutz Medical Campus, University of Colorado, Medical Research Council, National Institute of General Medical Sciences
Awards: G1775096572, G1902814985, 120 kV Transmission Electron Microscope for CU Anschutz Electron Microscopy Core Facility, Pleiotropic disorders of mitochondrial translation, Fis1 Regulation of Mitochondrial Fission, G3794036287, An Automated Microscale Thermophoresis Instrument for Southeastern Wisconsin: a Shared Resource for Biomolecular Interactions and Drug Discovery
OpenAlex ID: W7167702286