Biology subjects

Harwig, M. C.

Publications and source records attributed to Harwig, M. C..

4 recordsLinked to original sources

bioRxiv2026

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. Significance StatementDRP1 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.

biophysics↗

bioRxiv2023

The variable domain from the mitochondrial fission mechanoenzyme Drp1 promotes liquid-liquid phase separation

Dynamins are an essential superfamily of mechanoenzymes that remodel membranes and often contain a "variable domain" (VD) important for regulation. For the mitochondrial fission dynamin, Drp1, a regulatory role for the VD is demonstrated by mutations that can elongate, or fragment, mitochondria. How the VD encodes inhibitory and stimulatory activity is unclear. Here, isolated VD is shown to be intrinsically disordered (ID) yet undergoes a cooperative transition in the stabilizing osmolyte TMAO. However, the TMAO stabilized state is not folded and surprisingly appears as a condensed state. Other co-solutes including known molecular crowder Ficoll PM 70, also induce a condensed state. Fluorescence recovery after photobleaching experiments reveal this state to be liquid-like indicating the VD undergoes a liquid-liquid phase separation under crowding conditions. These crowding conditions also enhance binding to cardiolipin, a mitochondrial lipid, raising the possibility that phase separation may enable rapid tuning of Drp1 assembly necessary for fission.

biophysics↗

bioRxiv2023

Human Fis1 directly interacts with Drp1 in an evolutionarily conserved manner to promote mitochondrial fission

Mitochondrial Fission Protein 1 (Fis1) and Dynamin Related Protein 1 (Drp1) are the only two proteins evolutionarily conserved for mitochondrial fission, and directly interact in S. cerevisiae to facilitate membrane scission. However, it remains unclear if a direct interaction is conserved in higher eukaryotes as other Drp1 recruiters, not present in yeast, are known. Using NMR, differential scanning fluorimetry, and microscale thermophoresis, we determined that human Fis1 directly interacts with human Drp1 (KD = 12-68 {micro}M), and appears to prevent Drp1 assembly, but not GTP hydrolysis. Similar to yeast, the Fis1-Drp1 interaction appears governed by two structural features of Fis1: its N-terminal arm and a conserved surface. Alanine scanning mutagenesis of the arm identified both loss- and gain-of-function alleles with mitochondrial morphologies ranging from highly elongated (N6A) to highly fragmented (E7A) demonstrating a profound ability of Fis1 to govern morphology in human cells. An integrated analysis identified a conserved Fis1 residue, Y76, that upon substitution to alanine, but not phenylalanine, also caused highly fragmented mitochondria. The similar phenotypic effects of the E7A and Y76A substitutions, along with NMR data, support that intramolecular interactions occur between the arm and a conserved surface on Fis1 to promote Drp1-mediated fission as in S. cerevisiae. These findings indicate that some aspects of Drp1-mediated fission in humans derive from direct Fis1-Drp1 interactions that are conserved across eukaryotes.

biochemistry↗

bioRxiv2020

Inhibition of Fis1 by Novel Peptide Pep213 or Molecular Suppression Reverses Diabetic- and High Glucose-Induced Endothelial Dysfunction in Human Resistance Arteries

Mitochondrial dysfunction is one of several factors that drive development of vascular endothelial dysfunction in type 2 diabetes (T2DM). In endothelial cells from T2DM patients, mitochondrial networks are highly fragmentated with increased expression of mitochondrial fission protein 1 (Fis1). However, whether manipulation of Fis1 expression and activity in endothelial vessels from T2DM patients alters endothelial function remains unknown. Here, molecular suppression of Fis1 reversed impaired endothelium-dependent vasodilation of vessels from T2DM patients, as well as healthy human vessels exposed to high (33 mM) or low (2.5 mM) glucose, while preserving NO bioavailability and improving endothelial cell layer integrity. Conversely, overexpression of Fis1 in healthy vessels impaired vasodilation and increased mitochondrial superoxide, suggesting a causative role. Application of a novel and specific Fis1 inhibitor, pep213, improved endothelium-dependent vasodilation of vessels from T2DM patients, as well as healthy vessels exposed to high glucose or Fis1 overexpression, by improving NO bioavailability and decreasing excess mitochondrial ROS generation. The specificity of pep213 was determined through multiple biophysical techniques and a 1.85 [A] crystal structure of pep213 in complex with Fis1. These data support that excessive mitochondrial fragmentation drives endothelial vessel dysfunction and supports a potential novel therapeutic route for treating diabetic microvascular disease through pharmacological inhibition of Fis1.

physiology↗