Correction: New interfaces on MiD51 for Drp1 recruitment and regulation.
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ABSTRACT:
SUBMITTER: Ma J
PROVIDER: S-EPMC6538172 | biostudies-literature | 2019
REPOSITORIES: biostudies-literature
Publications
Correction: New interfaces on MiD51 for Drp1 recruitment and regulation.
Ma Jun J Zhai Yujia Y Chen Ming M Zhang Kai K Chen Quan Q Pang Xiaoyun X Sun Fei F
PloS one 20190528 5
[This corrects the article DOI: 10.1371/journal.pone.0211459.]. ...[more]
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Project description:Mitochondrial fission is facilitated by dynamin-related protein Drp1 and a variety of its receptors. However, the molecular mechanism of how Drp1 is recruited to the mitochondrial surface by receptors MiD49 and MiD51 remains elusive. Here, we showed that the interaction between Drp1 and MiD51 is regulated by GTP binding and depends on the polymerization of Drp1. We identified two regions on MiD51 that directly bind to Drp1, and found that dimerization of MiD51, relevant to residue C452, is required for mitochondrial dynamics regulation. Our Results have suggested a multi-faceted regulatory mechanism for the interaction between Drp1 and MiD51 that illustrates the potentially complicated and tight regulation of mitochondrial fission.
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Project description:Several mitochondrial outer membrane proteins-mitochondrial fission protein 1 (Fis1), mitochondrial fission factor (Mff), mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively)-have been proposed to promote mitochondrial fission by recruiting the GTPase dynamin-related protein 1 (Drp1), but fundamental issues remain concerning their function. A recent study supported such a role for Mff but not for Fis1. In addition, it is unclear whether MiD49 and MiD51 activate or inhibit fission, because their overexpression causes extensive mitochondrial elongation. It is also unknown whether these proteins can act in the absence of one another to mediate fission. Using Fis1-null, Mff-null, and Fis1/Mff-null cells, we show that both Fis1 and Mff have roles in mitochondrial fission. Moreover, immunofluorescence analysis of Drp1 suggests that Fis1 and Mff are important for the number and size of Drp1 puncta on mitochondria. Finally, we find that either MiD49 or MiD51 can mediate Drp1 recruitment and mitochondrial fission in the absence of Fis1 and Mff. These results demonstrate that multiple receptors can recruit Drp1 to mediate mitochondrial fission.
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Project description:Drp1 (dynamin-related protein 1) is recruited to both mitochondrial and peroxisomal membranes to execute fission. Fis1 and Mff are Drp1 receptor/effector proteins of mitochondria and peroxisomes. Recently, MiD49 and MiD51 were also shown to recruit Drp1 to the mitochondrial surface; however, different reports have ascribed opposing roles in fission and fusion. Here, we show that MiD49 or MiD51 overexpression blocked fission by acting in a dominant-negative manner by sequestering Drp1 specifically at mitochondria, causing unopposed fusion events at mitochondria along with elongation of peroxisomes. Mitochondrial elongation caused by MiD49/51 overexpression required the action of fusion mediators mitofusins 1 and 2. Furthermore, at low level overexpression when MiD49 and MiD51 form discrete foci at mitochondria, mitochondrial fission events still occurred. Unlike Fis1 and Mff, MiD49 and MiD51 were not targeted to the peroxisomal surface, suggesting that they specifically act to facilitate Drp1-directed fission at mitochondria. Moreover, when MiD49 or MiD51 was targeted to the surface of peroxisomes or lysosomes, Drp1 was specifically recruited to these organelles. Moreover, the Drp1 recruitment activity of MiD49/51 appeared stronger than that of Mff or Fis1. We conclude that MiD49 and MiD51 can act independently of Mff and Fis1 in Drp1 recruitment and suggest that they provide specificity to the division of mitochondria.
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Project description:Cytosolic dynamin-related protein 1 (Drp1, also known as DNM1L) is required for both mitochondrial and peroxisomal fission. Drp1-dependent division of these organelles is facilitated by a number of adaptor proteins at mitochondrial and peroxisomal surfaces. To investigate the interplay of these adaptor proteins, we used gene-editing technology to create a suite of cell lines lacking the adaptors MiD49 (also known as MIEF2), MiD51 (also known as MIEF1), Mff and Fis1. Increased mitochondrial connectivity was observed following loss of individual adaptors, and this was further enhanced following the combined loss of MiD51 and Mff. Moreover, loss of adaptors also conferred increased resistance of cells to intrinsic apoptotic stimuli, with MiD49 and MiD51 showing the more prominent role. Using a proximity-based biotin labeling approach, we found close associations between MiD51, Mff and Drp1, but not Fis1. Furthermore, we found that MiD51 can suppress Mff-dependent enhancement of Drp1 GTPase activity. Our data indicates that Mff and MiD51 regulate Drp1 in specific ways to promote mitochondrial fission.
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Project description:Drp1 is a dynamin-family GTPase recruited to mitochondria and peroxisomes, where it oligomerizes and drives membrane fission. Regulation of mitochondrial Drp1 recruitment is not fully understood. We previously showed that Drp1 binds actin filaments directly, and actin polymerization is necessary for mitochondrial Drp1 oligomerization in mammals. Here we show the Drp1/actin interaction displays unusual properties that are influenced by several factors. At saturation, only a fraction Drp1 binds actin filaments, and the off-rate of actin-bound Drp1 is significantly increased by unbound Drp1. GDP and GTP accelerate and decelerate Drp1/actin binding dynamics, respectively. Actin has a biphasic effect on Drp1 GTP hydrolysis, increasing at low actin:Drp1 ratio but returning to baseline at high ratio. Drp1 also bundles filaments. Bundles have reduced dynamics but follow the same trends as single filaments. Drp1 preferentially incorporates into bundles at higher ionic strength. We measure Drp1 concentration to be ∼0.5 μM in U2OS cell cytosol, suggesting the actin-binding affinity measured here (Kd = 0.6 μM) is in the physiologically relevant range. The ability of Drp1 to bind actin filaments in a highly dynamic manner provides potential for actin filaments to serve as reservoirs of oligomerization-competent Drp1 that can be accessed for mitochondrial fission.
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Project description:Mitochondria form intricate networks through fission and fusion events. Here, we identify mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively) anchored in the mitochondrial outer membrane. MiD49/51 form foci and rings around mitochondria similar to the fission mediator dynamin-related protein 1 (Drp1). MiD49/51 directly recruit Drp1 to the mitochondrial surface, whereas their knockdown reduces Drp1 association, leading to unopposed fusion. Overexpression of MiD49/51 seems to sequester Drp1 from functioning at mitochondria and cause fused tubules to associate with actin. Thus, MiD49/51 are new mediators of mitochondrial division affecting Drp1 action at mitochondria.
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Project description:Recruitment of the GTPase dynamin-related protein 1 (Drp1) to mitochondria is a central step required for mitochondrial fission. Reversible Drp1 phosphorylation has been implicated in the regulation of this process, but whether Drp1 phosphorylation at Ser-637 determines its subcellular localization and fission activity remains to be fully elucidated. Here, using HEK 293T cells and immunofluorescence, immunoblotting, RNAi, subcellular fractionation, co-immunoprecipitation assays, and CRISPR/Cas9 genome editing, we show that Drp1 phosphorylated at Ser-637 (Drp1pS637) resides both in the cytosol and on mitochondria. We found that the receptors mitochondrial fission factor (Mff) and mitochondrial elongation factor 1/2 (MIEF1/2) interact with and recruit Drp1pS637 to mitochondria and that elevated Mff or MIEF levels promote Drp1pS637 accumulation on mitochondria. We also noted that protein kinase A (PKA), which mediates phosphorylation of Drp1 on Ser-637, is partially present on mitochondria and interacts with both MIEFs and Mff. PKA knockdown did not affect the Drp1-Mff interaction, but slightly enhanced the interaction between Drp1 and MIEFs. In Drp1-deficient HEK 293T cells, both phosphomimetic Drp1-S637D and phospho-deficient Drp1-S637A variants, like wild-type Drp1, located to the cytosol and to mitochondria and rescued a Drp1 deficiency-induced mitochondrial hyperfusion phenotype. However, Drp1-S637D was less efficient than Drp1-WT and Drp1-S637A in inducing mitochondrial fission. In conclusion, the Ser-637 phosphorylation status in Drp1 is not a determinant that controls Drp1 recruitment to mitochondria.
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Project description:Mitochondrial fission is critically important for controlling mitochondrial morphology, function, quality and transport. Drp1 is the master regulator driving mitochondrial fission, but exactly how Drp1 is regulated remains unclear. Here, we identified Drosophila Clueless and its mammalian orthologue CLUH as key regulators of Drp1. As with loss of drp1, depletion of clueless or CLUH results in mitochondrial elongation, while as with drp1 overexpression, clueless or CLUH overexpression leads to mitochondrial fragmentation. Importantly, drp1 overexpression rescues adult lethality, tissue disintegration and mitochondrial defects of clueless null mutants in Drosophila. Mechanistically, Clueless and CLUH promote recruitment of Drp1 to mitochondria from the cytosol. This involves CLUH binding to mRNAs encoding Drp1 receptors MiD49 and Mff, and regulation of their translation. Our findings identify a crucial role of Clueless and CLUH in controlling mitochondrial fission through regulation of Drp1.
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Project description:Mitotic fission is increased in hyperproliferative, apoptosis-resistant diseases, such as pulmonary arterial hypertension (PAH). PAH’s fissogenic phenotype includes activation of the fission mediator, dynamin related protein 1 (Drp1), which must complex with its adaptor proteins to cause fission. Drp1-induced fission has been therapeutically targeted in experimental PAH. Here we examine the role of two recently discovered, poorly understood, Drp1 adapter proteins, mitochondrial dynamics protein of 49 and 51 kDa (MiD49 and MiD51) in normal vascular cells and explore the role of their dysregulation in the pathogenesis of PAH. MiDs are increased in PAH PASMC. This accelerates Drp1-mediated mitotic fission, which increases cell proliferation and decreases apoptosis. Silencing MiDs (but not Fis1 or MFF) promotes mitochondrial fusion and G1-phase cell cycle arrest through an ERK1/2 and CDK4-dependent mechanism. Augmenting MiDs in normal cells causes fission and recapitulates the PAH phenotype. MiD upregulation results from decreased microRNA-34a-3p (miR-34a-3p) expression. Circulatory miR34a-3p expression is decreased in PAH patients as well as in preclinical models of PAH. Silencing MiDs or augmenting miR-34a-3p regresses experimental PAH. We used microarrays to identify differences in miR expression in pulmonary artery smooth muscle cells (PASMC) taken from either pulmonary arterial hypertension patients or healthy controls
2018-01-31 | GSE108707 | GEO
Project description:Mitochondrial fission protein 1 (FIS1) is conserved in all eukaryotes, yet its function in metazoans is thought divergent. Structure-based sequence alignments of FIS1 revealed a conserved, but noncanonical, three-residue insert in its first tetratricopeptide repeat (TPR) suggesting a conserved function. In vertebrates, this insert is serine (S45), lysine (K46), and tyrosine (Y47). To determine the biological role of the "SKY insert," three variants were tested in HCT116 cells for altered mitochondrial morphology and recruitment of fission mechanoenzyme DRP1 and mitophagic adaptor TBC1D15. Similar to ectopically expressed wildtype FIS1, substitution of the SKY insert with alanine (AAA) fragmented mitochondria into perinuclear clumps associated with increased mitochondrial DRP1. In contrast, deletion variants (either ∆SKY or ∆SKYD49G) elongated mitochondrial networks with reduced mitochondrial recruitment of DRP1, despite DRP1 coimmunoprecipitates being highly enriched with ΔSKY variants. Ectopic wildtype FIS1 drove co-expressed YFP-TBC1D15 entirely from the cytoplasm to mitochondria as punctate structures concomitant with enhanced mitochondrial DRP1 recruitment. YFP-TBC1D15 co-expressed with the AAA variant further enhanced mitochondrial DRP1 recruitment, indicating a gain of function. In contrast, YFP-TBC1D15 co-expressed with deletion variants impaired mitochondrial DRP1 and YFP-TBC1D15 recruitment; however, mitochondrial fragmentation was restored. These phenotypes were not due to misfolding or poor expression of FIS1 variants, although ∆SKYD49G induced conformational heterogeneity that is lost upon deletion of the regulatory Fis1 arm, indicating SKY-arm interactions. Collectively, these results support a unifying model whereby FIS1 activity is effectively governed by intramolecular interactions between its regulatory arm and a noncanonical TPR insert that is conserved across eukaryotes.
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