Pathogenic DRP1 variants reveal a role for biomolecular condensation in mitochondrial fission

Fission is essential for proper mitochondrial function and for cellular homeostasis. Dysfunction in mitochondrial fission is associated with several neurological disorders, including the rare and lethal encephalopathy EMPF1, which is caused by de novo heterozygous DNM1L variants. DNM1L encodes the mitochondrial fission mechanoenzyme DRP1, which can intrinsically self-assemble and induce membrane scission. Wild-type DRP1 puncta that appear throughout the cytoplasm are 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 retain essentially 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 preferentially alters DRP1 puncta fluorescence intensity and distribution in mutant cells relative to wild type, indicating genotype-dependent differences in puncta material properties. Together, these findings support a model in which DRP1 puncta occupy a continuum of condensed states, only a subset of which mature into 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.

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.