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The identification of Drp1 patient mutations that limit interactions with Mff
Yuli Buckley1, Anelise N Hutson1, Brianna L Bauer1
1Department of Pharmacology and Center for Mitochondrial Research and Therapeutics, Case Western Reserve University School of Medicine, Cleveland, OH, 44016.
Abstract:
Patient mutations within Drp1, the master regulator of mitochondrial fission, lead to severe neurological defects and poor patient outcomes. Many of these mutations have been characterized as causing functional or assembly defects in Drp1, but our study highlights three mutations (G362S, E379K, and E410K) that do not have an apparent defect in core Drp1 functions. We investigated the possibility that these mutations impact interactions with Mff, a pro-fission partner protein of Drp1. Negative stain electron microscopy and mass photometry were used to visualize and quantify assembly properties, while GTPase assays assessed the enzymatic activities of distinct proteins and protein complexes. In parallel, confocal microscopy highlighted the effects of overexpressing each mutation on mitochondrial morphology in cells. We discovered that G362S and E410K Drp1 mutations limit interactions with Mff, as co-assembly into larger filaments and the associated stimulation of GTPase activity was inhibited. Conversely, the E379K mutation is able to form functional complexes with Mff, and no apparent defect was observed, warranting additional studies focused on unique mitochondrial fission attributes. Overall, our data highlight the complex nature of disease-associated mutations in Drp1 and emphasize the importance of Drp1-Mff interactions in sustaining mitochondrial and cellular health.
Insights
Mutations in Drp1 (dynamin-related protein 1) can cause neurological issues. This study found specific Drp1 mutations impair interactions with Mff, a key partner protein, impacting mitochondrial fission and cellular health.
Area of Science:
- Cell Biology
- Biochemistry
- Genetics
Background:
- Drp1 (dynamin-related protein 1) is crucial for mitochondrial fission, a process vital for cellular health.
- Mutations in Drp1 are linked to severe neurological disorders.
- While some mutations affect Drp1's core functions, others may impact its interactions with regulatory partners.
Purpose of the Study:
- To investigate whether specific Drp1 mutations (G362S, E379K, E410K), which do not show apparent defects in core Drp1 functions, affect interactions with the pro-fission partner Mff.
- To understand the impact of these mutations on Drp1 assembly, GTPase activity, and mitochondrial morphology.
Main Methods:
- Negative stain electron microscopy and mass photometry were used to analyze Drp1 assembly properties.
- GTPase assays were performed to assess the enzymatic activity of Drp1 proteins and complexes.
- Confocal microscopy was employed to observe the effects of Drp1 mutations on mitochondrial morphology in cells.
Main Results:
- The G362S and E410K Drp1 mutations were found to limit interactions with Mff, inhibiting co-assembly into filaments and reducing GTPase activity stimulation.
- The E379K Drp1 mutation did not impair functional complex formation with Mff, showing no apparent defect in this interaction.
- Overexpression of G362S and E410K Drp1 mutations led to altered mitochondrial morphology in cells.
Conclusions:
- Disease-associated Drp1 mutations can disrupt interactions with partner proteins like Mff, even without affecting core Drp1 functions.
- Proper Drp1-Mff interaction is essential for regulating mitochondrial fission and maintaining cellular health.
- These findings underscore the complexity of Drp1 mutations in neurological diseases and highlight the importance of protein-protein interactions in Drp1 regulation.
