GFP fluorescence tagging alters dynamin-related protein 1 oligomerization dynamics and creates disassembly-refractory puncta to mediate mitochondrial fission

Montecinos-Franjola, Felipe; Bauer, Brianna L.; Mears, Jason A.; Ramachandran, Rajesh

doi:10.1038/s41598-020-71655-x

Cited by 33 publications

(25 citation statements)

References 67 publications

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“…While our results regarding the lack of fission by Drp1 alone contradict the conclusions of ref. 44, they are otherwise consistent with multiple previous investigations that employed either tagless or N-terminally His 6 -tagged Drp1 to report robust GTP hydrolysis-dependent local high membrane curvature generation (or stabilization) without any accompanying fission or membrane vesiculation (12,29,45,78,79). We surmise that the altered conformational and assembly-disassembly dynamics of the C-terminally tagged Drp1 construct employed in ref.…”

Section: Discussionsupporting

confidence: 90%

NMR identification of a conserved Drp1 cardiolipin-binding motif essential for stress-induced mitochondrial fission

Mahajan

Bharambe

Shang

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Mitochondria form tubular networks that undergo coordinated cycles of fission and fusion. Emerging evidence suggests that a direct yet unresolved interaction of the mechanoenzymatic GTPase dynamin-related protein 1 (Drp1) with mitochondrial outer membrane–localized cardiolipin (CL), externalized under stress conditions including mitophagy, catalyzes essential mitochondrial hyperfragmentation. Here, using a comprehensive set of structural, biophysical, and cell biological tools, we have uncovered a CL-binding motif (CBM) conserved between the Drp1 variable domain (VD) and the unrelated ADP/ATP carrier (AAC/ANT) that intercalates into the membrane core to effect specific CL interactions. CBM mutations that weaken VD–CL interactions manifestly impair Drp1-dependent fission under stress conditions and induce “donut” mitochondria formation. Importantly, VD membrane insertion and GTP-dependent conformational rearrangements mediate only transient CL nonbilayer topological forays and high local membrane constriction, indicating that Drp1–CL interactions alone are insufficient for fission. Our studies establish the structural and mechanistic bases of Drp1–CL interactions in stress-induced mitochondrial fission.

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Section: Discussionsupporting

confidence: 90%

NMR identification of a conserved Drp1 cardiolipin-binding motif essential for stress-induced mitochondrial fission

Mahajan

Bharambe

Shang

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…8 A ) but is not able to achieve fission (abnormal mitochondrial network in homozygous animals in Fig. 8 B ; Montecinos-Franjola et al, 2020 ). Therefore, drp-1(or1941) , which phenocopies a drp-1 mutant, was used to visualize the presumptive mitochondrial fission sites and was compared with a wild-type drp-1 as a positive control.…”

Section: Resultsmentioning

confidence: 99%

Autophagy facilitates mitochondrial rebuilding after acute heat stress via a DRP-1–dependent process

Chen

Leboutet

Largeau

et al. 2021

Journal of Cell Biology

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Acute heat stress (aHS) can induce strong developmental defects in Caenorhabditis elegans larva but not lethality or sterility. This stress results in transitory fragmentation of mitochondria, formation of aggregates in the matrix, and decrease of mitochondrial respiration. Moreover, active autophagic flux associated with mitophagy events enables the rebuilding of the mitochondrial network and developmental recovery, showing that the autophagic response is protective. This adaptation to aHS does not require Pink1/Parkin or the mitophagy receptors DCT-1/NIX and FUNDC1. We also find that mitochondria are a major site for autophagosome biogenesis in the epidermis in both standard and heat stress conditions. In addition, we report that the depletion of the dynamin-related protein 1 (DRP-1) affects autophagic processes and the adaptation to aHS. In drp-1 animals, the abnormal mitochondria tend to modify their shape upon aHS but are unable to achieve fragmentation. Autophagy is induced, but autophagosomes are abnormally elongated and clustered on mitochondria. Our data support a role for DRP-1 in coordinating mitochondrial fission and autophagosome biogenesis in stress conditions.

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“…To assess the effects of these Drp1 mutations on mitochondrial recruitment and recruitment receptor preference, we used Drp1 −/− 293T cells as a model (Yu et al, 2019), to avoid interference of endogenous Drp1. Meanwhile, in order to exclude any effect of immunotags on Drp1 structure and function (Montecinos-Franjola et al, 2020), untagged Drp1-WT, Drp1-K668E, Drp1-A395D, Drp1-4A, and Drp1-M482D mutants were expressed in the Drp1 −/− 293T cells. In vivo chemical crosslinking revealed that exogenous Drp1-WT in Drp1 −/− 293T cells as well as endogenous Drp1 in WT 293T cells formed multiple assembly subunits with a minimal species at ∼280 kDa (Figure 3B, lanes 2 and 3).…”

Section: The Self-assembly State Of Drp1 Regulates Its Binding To the Mitochondrial Receptors Miefs And Mffmentioning

confidence: 99%

The Molecular Assembly State of Drp1 Controls its Association With the Mitochondrial Recruitment Receptors Mff and MIEF1/2

Jin

Ankarcrona

et al. 2021

Front. Cell Dev. Biol.

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Drp1 is a central player in mitochondrial fission and is recruited to mitochondria by Mff and MIEFs (MIEF1 and MIEF2), but little is known about how its assembly state affects Drp1 mitochondrial recruitment and fission. Here, we used in vivo chemical crosslinking to explore the self-assembly state of Drp1 and how it regulates the association of Drp1 with MIEFs and Mff. We show that in intact mammalian cells Drp1 exists as a mixture of multiple self-assembly forms ranging from the minimal, probably tetrameric, self-assembly subunit to several higher order oligomers. Precluding mitochondria-bound Drp1 in Mff/MIEF1/2-deficient cells does not affect the oligomerization state of Drp1, while conversely forced recruitment of Drp1 to mitochondria by MIEFs or Mff facilitates Drp1 oligomerization. Mff preferentially binds to higher order oligomers of Drp1, whereas MIEFs bind to a wider-range of Drp1 assembly subunits, including both lower and higher oligomeric states. Mff only recruits active forms of Drp1, while MIEFs are less selective and recruit both active and inactive Drp1 as well as oligomerization- or GTPase-deficient Drp1 mutants to mitochondria. Moreover, all the fission-incompetent Drp1 mutants tested (except the monomeric mutant K668E) affect Drp1-driven mitochondrial dynamics via incorporation of the mutants into the native oligomers to form function-deficient Drp1 assemblies. We here confirm that MIEFs also serve as a platform facilitating the binding of Drp1 to Mff and loss of MIEFs severely impairs the interaction between Drp1 and Mff. Collectively, our findings suggest that Mff and MIEFs respond differently to the molecular assembly state of Drp1 and that the extent of Drp1 oligomerization regulates mitochondrial dynamics.

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GFP fluorescence tagging alters dynamin-related protein 1 oligomerization dynamics and creates disassembly-refractory puncta to mediate mitochondrial fission

Cited by 33 publications

References 67 publications

NMR identification of a conserved Drp1 cardiolipin-binding motif essential for stress-induced mitochondrial fission

NMR identification of a conserved Drp1 cardiolipin-binding motif essential for stress-induced mitochondrial fission

Autophagy facilitates mitochondrial rebuilding after acute heat stress via a DRP-1–dependent process

The Molecular Assembly State of Drp1 Controls its Association With the Mitochondrial Recruitment Receptors Mff and MIEF1/2

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