Drp1 Recruitment Research Articles

COX assembly factor ccdc56 regulates mitochondrial morphology by affecting mitochondrial recruitment of Drp1

Mitochondria are dynamic organelles that alter their morphology in response to cellular signaling and differentiation through balanced fusion and fission. In this study, we found that the mitochondrial inner membrane ATPase ATAD3A interacted with ccdc56/MITRAC12/COA3, a subunit of the cytochrome oxidase (COX)-assembly complex. Overproduction of ccdc56 in HeLa cells resulted in fragmented mitochondrial morphology, while mitochondria were highly elongated in ccdc56-repressed cells by the defective recruitment of the fission factor Drp1. We also found that mild and chronic inhibition of COX led to mitochondrial elongation, as seen in ccdc56-repressed cells. These results indicate that ccdc56 positively regulates mitochondrial fission via regulation of COX activity and the mitochondrial recruitment of Drp1, and thus, suggest a novel relationship between COX assembly and mitochondrial morphology.

Crystal structure and functional analysis of MiD49, a receptor for the mitochondrial fission protein Drp1.

Mitochondrial fission requires recruitment of dynamin-related protein 1 (Drp1) to the mitochondrial surface, where assembly leads to activation of its GTP-dependent scission function. MiD49 and MiD51 are two receptors on the mitochondrial outer membrane that can recruit Drp1 to facilitate mitochondrial fission. Structural studies indicated that MiD51 has a variant nucleotidyl transferase fold that binds an ADP co-factor essential for activation of Drp1 function. MiD49 shares sequence homology with MiD51 and regulates Drp1 function. However, it is unknown if MiD49 binds an analogous co-factor. Because MiD49 does not readily crystallize, we used structural predictions and biochemical screening to identify a surface entropy reduction mutant that facilitated crystallization. Using molecular replacement, we determined the atomic structure of MiD49 to 2.4 Å. Like MiD51, MiD49 contains a nucleotidyl transferase domain; however, the electron density provides no evidence for a small-molecule ligand. Structural changes in the putative nucleotide-binding pocket make MiD49 incompatible with an extended ligand like ADP, and critical nucleotide-binding residues found in MiD51 are not conserved. MiD49 contains a surface loop that physically interacts with Drp1 and is necessary for Drp1 recruitment to the mitochondrial surface. Our results suggest a structural basis for the differential regulation of MiD51- versus MiD49-mediated fission.

Mfn2 downregulation in excitotoxicity causes mitochondrial dysfunction and delayed neuronal death

Mitochondrial fusion and fission is a dynamic process critical for the maintenance of mitochondrial function and cell viability. During excitotoxicity neuronal mitochondria are fragmented, but the mechanism underlying this process is poorly understood. Here, we show that Mfn2 is the only member of the mitochondrial fusion/fission machinery whose expression is reduced in in vitro and in vivo models of excitotoxicity. Whereas in cortical primary cultures, Drp1 recruitment to mitochondria plays a primordial role in mitochondrial fragmentation in an early phase that can be reversed once the insult has ceased, Mfn2 downregulation intervenes in a delayed mitochondrial fragmentation phase that progresses even when the insult has ceased. Downregulation of Mfn2 causes mitochondrial dysfunction, altered calcium homeostasis, and enhanced Bax translocation to mitochondria, resulting in delayed neuronal death. We found that transcription factor MEF2 regulates basal Mfn2 expression in neurons and that excitotoxicity-dependent degradation of MEF2 causes Mfn2 downregulation. Thus, Mfn2 reduction is a late event in excitotoxicity and its targeting may help to reduce excitotoxic damage and increase the currently short therapeutic window in stroke.

Specific interaction with cardiolipin triggers functional activation of Dynamin-Related Protein 1.

Dynamin-Related Protein 1 (Drp1), a large GTPase of the dynamin superfamily, is required for mitochondrial fission in healthy and apoptotic cells. Drp1 activation is a complex process that involves translocation from the cytosol to the mitochondrial outer membrane (MOM) and assembly into rings/spirals at the MOM, leading to membrane constriction/division. Similar to dynamins, Drp1 contains GTPase (G), bundle signaling element (BSE) and stalk domains. However, instead of the lipid–interacting Pleckstrin Homology (PH) domain present in the dynamins, Drp1 contains the so-called B insert or variable domain that has been suggested to play an important role in Drp1 regulation. Different proteins have been implicated in Drp1 recruitment to the MOM, although how MOM-localized Drp1 acquires its fully functional status remains poorly understood. We found that Drp1 can interact with pure lipid bilayers enriched in the mitochondrion-specific phospholipid cardiolipin (CL). Building on our previous study, we now explore the specificity and functional consequences of this interaction. We show that a four lysine module located within the B insert of Drp1 interacts preferentially with CL over other anionic lipids. This interaction dramatically enhances Drp1 oligomerization and assembly-stimulated GTP hydrolysis. Our results add significantly to a growing body of evidence indicating that CL is an important regulator of many essential mitochondrial functions.

Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance

Autosomal recessive early-onset Parkinson's disease is most often caused by mutations in the genes encoding the cytosolic E3 ubiquitin ligase Parkin and the mitochondrial serine/threonine kinase PINK1. Studies in Drosophila models and mammalian cells have demonstrated that these proteins regulate various aspects of mitochondrial physiology, including organelle transport, dynamics and turnover. How PINK1 and Parkin orchestrate these processes, and whether they always do so within a common pathway remain to be clarified.We have revisited the role of PINK1 and Parkin in mitochondrial dynamics, and explored its relation to the mitochondrial clearance program controlled by these proteins. We show that PINK1 and Parkin promote Drp1-dependent mitochondrial fission by mechanisms that are at least in part independent. Parkin-mediated mitochondrial fragmentation was abolished by treatments interfering with the calcium/calmodulin/calcineurin signaling pathway, suggesting that it requires dephosphorylation of serine 637 of Drp1. Parkinson's disease-causing mutations with differential impact on mitochondrial morphology and organelle degradation demonstrated that the pro-fission effect of Parkin is not required for efficient mitochondrial clearance. In contrast, the use of Förster energy transfer imaging microscopy revealed that Drp1 and Parkin are co-recruited to mitochondria in proximity of PINK1 following mitochondrial depolarization, indicating spatial coordination between these events in mitochondrial degradation. Our results also hint at a major role of the outer mitochondrial adaptor MiD51 in Drp1 recruitment and Parkin-dependent mitophagy. Altogether, our observations provide new insight into the mechanisms underlying the regulation of mitochondrial dynamics by Parkin and its relation to the mitochondrial clearance program mediated by the PINK1/Parkin pathway.

Regulation of mitochondrial fission by MiD51 (757.1)

Regulation of mitochondrial fission by MiD51 Oliver C. Losόn1, Michael E. Rome1, Jens T. Kaiser2, Shu‐ou Shan2 and David C. Chan1,3 1Division of Biology and Biological Engineering, 2Division of Chemistry and Chemical Engineering and 3Howard Hughes Medical Institute, California Institute of Technology, Pasadena CA, 91125, USA Mitochondrial fission requires the recruitment of dynamin‐related protein 1 (Drp1) to mitochondria and activation of its GTP‐dependent scission function. In mammals, the recruitment of Drp1 is primarily regulated by the outer membrane receptors Mff, MiD49 and MiD51, while Fis1 plays a minor role. We recently demonstrated that these receptors can function independently of one another to recruit Drp1 and mediate mitochondrial division. Intriguingly, exogenous expression of MiD49 and MiD51 causes constitutive recruitment of Drp1 but inhibits its scission activity. With mitochondrial stress, however, these receptors can rapidly activate pre‐recruited Drp1 and cause rapid mitochondrial fragmentation independently of Fis1 and Mff. This behavior suggests an ability to sense mitochondrial function, but the molecular mechanism is unknown. To better understand the role of the MiDs in regulating mitochondrial fission, we produced recombinant protein pertaining to the cytosolic domain of each MiD, and performed crystallographic screens. We obtained crystals for MiD51 that diffracted to high resolution, and we were able to determine its structure. MiD51 possesses a nucleotidyl transferase fold and uses a variant set of residues to tightly bind a ligand. We have performed a structure‐function analysis of MiD51 by mutating structurally implicated residues and studying their importance in vivo and in vitro to activate Drp1. Our analysis reveals how MiD51 activates mitochondrial fission during mitochondrial stress.

Structural and functional analysis of MiD51, a dynamin receptor required for mitochondrial fission

Mitochondrial fission is important for organelle transport, inheritance, and turnover, and alterations in fission are seen in neurological disease. In mammals, mitochondrial fission is executed by dynamin-related protein 1 (Drp1), a cytosolic guanosine triphosphatase that polymerizes and constricts the organelle. Recruitment of Drp1 to mitochondria involves receptors including Mff, MiD49, and MiD51. MiD49/51 form foci at mitochondrial constriction sites and coassemble with Drp1 to drive fission. Here, we solved the crystal structure of the cytosolic domain of human MiD51, which adopts a nucleotidyltransferase fold. Although MiD51 lacks catalytic residues for transferase activity, it specifically binds guanosine diphosphate and adenosine diphosphate. MiD51 mutants unable to bind nucleotides were still able to recruit Drp1. Disruption of an additional region in MiD51 that is not part of the nucleotidyltransferase fold blocked Drp1 recruitment and assembly of MiD51 into foci. MiD51 foci are also dependent on the presence of Drp1, and after scission they are distributed to daughter organelles, supporting the involvement of MiD51 in the fission apparatus.

Novel roles for actin in mitochondrial fission.

Mitochondrial dynamics, including fusion, fission and translocation, are crucial to cellular homeostasis, with roles in cellular polarity, stress response and apoptosis. Mitochondrial fission has received particular attention, owing to links with several neurodegenerative diseases. A central player in fission is the cytoplasmic dynamin-related GTPase Drp1, which oligomerizes at the fission site and hydrolyzes GTP to drive membrane ingression. Drp1 recruitment to the outer mitochondrial membrane (OMM) is a key regulatory event, which appears to require a pre-constriction step in which the endoplasmic reticulum (ER) and mitochondrion interact extensively, a process termed ERMD (ER-associated mitochondrial division). It is unclear how ER-mitochondrial contact generates the force required for pre-constriction or why pre-constriction leads to Drp1 recruitment. Recent results, however, show that ERMD might be an actin-based process in mammals that requires the ER-associated formin INF2 upstream of Drp1, and that myosin II and other actin-binding proteins might be involved. In this Commentary, we present a mechanistic model for mitochondrial fission in which actin and myosin contribute in two ways; firstly, by supplying the force for pre-constriction and secondly, by serving as a coincidence detector for Drp1 binding. In addition, we discuss the possibility that multiple fission mechanisms exist in mammals.

Impaired OMA1 dependent OPA1 cleavage and reduced DRP1 fission activity combine to prevent mitophagy in OXPHOS dependent cells

ABSTRACTMitochondrial dynamics play crucial roles in mitophagy-based mitochondrial quality control, but how these pathways are regulated to meet cellular energy demands remains obscure. Using non-transformed human RPE1 cells, we report that upregulation of mitochondrial oxidative phosphorylation alters mitochondrial dynamics to inhibit Parkin-mediated mitophagy. Despite the basal mitophagy rates remaining stable upon the switch to dependence on oxidative phosphorylation, mitochondria resist fragmentation when RPE1 cells are treated with the protonophore carbonyl cyanide m-chlorophenyl hydrazone. Mechanistically, we show that this is because cleavage of the inner membrane fusion factor L-OPA1 is prevented due to the failure to activate the inner membrane protease OMA1 in mitochondria that have a collapsed membrane potential. In parallel, mitochondria that use oxidative phosphorylation are protected from damage-induced fission through the impaired recruitment and activation of mitochondrial DRP1. Using OMA1-deficient MEF cells, we show that the preservation of a stable pool of L-OPA1 at the inner mitochondrial membrane is sufficient to delay mitophagy, even in the presence of Parkin. The capacity of cells that are dependent on oxidative phosphorylation to maintain substantial mitochondrial content in the face of acute damage has important implications for mitochondrial quality control in vivo.

Adaptor Proteins MiD49 and MiD51 Can Act Independently of Mff and Fis1 in Drp1 Recruitment and Are Specific for Mitochondrial Fission

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.

Mitochondria-targeted heme oxygenase-1 induces oxidative stress and mitochondrial dysfunction in macrophages, kidney fibroblasts and in chronic alcohol hepatotoxicity

The inducible form of Heme Oxygenase-1 (HO-1), a major endoplasmic reticulum (ER) associated heme protein, is known to play important roles in protection against oxidative and chemical stress by degrading free heme released from degradation of heme proteins. In this study we show that induced expression of HO-1 by subjecting macrophage RAW-264.7 cells to chemical or physiological hypoxia resulted in significant translocation of HO-1 protein to mitochondria. Transient transfection of COS-7 cells with cloned cDNA also resulted in mitochondrial translocation of HO-1. Deletion of N-terminal ER targeting domain increased mitochondrial translocation under the transient transfection conditions. Mitochondrial localization of both intact HO-1 and N-terminal truncated HO-1 caused loss of heme aa-3 and cytochrome c oxidase (CcO) activity in COS-7 cells. The truncated protein, which localizes to mitochondria at higher levels, induced substantially steeper loss of CcO activity and reduced heme aa3 content. Furthermore, cells expressing mitochondria targeted HO-1 also induced higher ROS production. Consistent with dysfunctional state of mitochondria induced by HO-1, the mitochondrial recruitment of autophagy markers LC-3 and Drp-1 was also increased in these cells. Chronic ethanol feeding in rats also caused an increase in mitochondrial HO-1 and decrease in CcO activity. These results show that as opposed to the protective effect of the ER associated HO-1, mitochondria targeted HO-1 under normoxic conditions induces mitochondrial dysfunction.

A Bcl-xL–Drp1 complex regulates synaptic vesicle membrane dynamics during endocytosis

Following exocytosis, the rate of recovery of neurotransmitter release is determined by vesicle retrieval from the plasma membrane and by recruitment of vesicles from reserve pools within the synapse, the latter of which is dependent on mitochondrial ATP. The Bcl-2 family protein Bcl-xL, in addition to its role in cell death, regulates neurotransmitter release and recovery in part by increasing ATP availability from mitochondria. We now find, however, that, Bcl-xL directly regulates endocytotic vesicle retrieval in hippocampal neurons through protein/protein interaction with components of the clathrin complex. Our evidence suggests that, during synaptic stimulation, Bcl-xL translocates to clathrin-coated pits in a calmodulin-dependent manner and forms a complex of proteins with the GTPase Drp1, Mff and clathrin. Depletion of Drp1 produces misformed endocytotic vesicles. Mutagenesis studies suggest that formation of the Bcl-xL-Drp1 complex is necessary for the enhanced rate of vesicle endocytosis produced by Bcl-xL, thus providing a mechanism for presynaptic plasticity.

Fis1, Mff, MiD49 and MiD51 facilitate Drp1 recruitment for mitochondrial fission

Several mitochondrial outer membrane proteins—Fis1, Mff, MiD49, and MiD51—have been proposed to promote mitochondrial fission by recruiting the cytosolic GTPase 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, Mffnull, and Fis1/Mff‐null cells, we show that both Fis1 and Mff have roles in mitochondrial fission. We employ several independent, quantitative techniques to show that these cell lines have significantly different mitochondrial elongation phenotypes. Using a specialized image analysis technique, we find that Fis1 and Mff are important for regulating the number and size of Drp1 fluorescent 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. We also demonstrate that a significant enhancement of Drp1 phosphorylation at S637 is causal for the elongation phenotype during MiD overexpression. These results demonstrate that multiple receptors can recruit Drp1 to mediate mitochondrial fission, and suggest that these proteins may function in parallel pathways. This work was supported by the National Institutes of Health (GM062967). OCL was supported by a R. L. Kirschstein National Research Service Award (5F31GM089327).

Cadmium induced Drp1-dependent mitochondrial fragmentation by disturbing calcium homeostasis in its hepatotoxicity

Mitochondria are critical targets in the hepatotoxicity of cadmium (Cd). Abnormal mitochondrial dynamics have been increasingly implicated in mitochondrial dysfunction in pathophysiological conditions. Therefore, our study aimed to investigate the effects and underlying mechanism of Cd on mitochondrial dynamics during hepatotoxicity. In the L02 liver cell lines, 12 μM cadmium chloride (CdCl2) exposure induced excessive mitochondrial fragmentation as early as 3 h post-treatment with Cd, which preceded the mitochondrial dysfunction such as reactive oxygen species (ROS) overproduction, mitochondrial membrane potential (ΔΨm) loss and ATP reduction. Concurrent to mitochondrial fragmentation, CdCl2 treatment increased the protein levels of dynamin-related protein (Drp1) and promoted the recruitment of Drp1 into mitochondria. Strikingly, mitochondrial fragmentation also occurred in the liver tissue of rats exposed to CdCl2, accompanied by enhanced recruitment of Drp1 into mitochondria. Moreover, in L02 cells, Drp1 silencing could effectively reverse Cd-induced mitochondrial fragmentation and mitochondrial dysfunction. Furthermore, the increased expression and mitochondrial recruitment of Drp1 were tightly related to the disturbance of calcium homeostasis, which could be prevented by both chelating [Ca2+]i and inhibiting [Ca2+]m uptake. Overall, our study indicated that Cd induced Drp1-dependent mitochondrial fragmentation by disturbing calcium homeostasis to promote hepatotoxicity. Manipulation of Drp1 may be the potential avenue for developing novel strategies to protect against cadmium-induced hepatotoxicity.

Fis1, Mff, MiD49, and MiD51 mediate Drp1 recruitment in mitochondrial fission

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.

Regulation of mitochondrial dynamics: convergences and divergences between yeast and vertebrates

In eukaryotic cells, the shape of mitochondria can be tuned to various physiological conditions by a balance of fusion and fission processes termed mitochondrial dynamics. Mitochondrial dynamics controls not only the morphology but also the function of mitochondria, and therefore is crucial in many aspects of a cell’s life. Consequently, dysfunction of mitochondrial dynamics has been implicated in a variety of human diseases including cancer. Several proteins important for mitochondrial fusion and fission have been discovered over the past decade. However, there is emerging evidence that there are as yet unidentified proteins important for these processes and that the fusion/fission machinery is not completely conserved between yeast and vertebrates. The recent characterization of several mammalian proteins important for the process that were not conserved in yeast, may indicate that the molecular mechanisms regulating and controlling the morphology and function of mitochondria are more elaborate and complex in vertebrates. This difference could possibly be a consequence of different needs in the different cell types of multicellular organisms. Here, we review recent advances in the field of mitochondrial dynamics. We highlight and discuss the mechanisms regulating recruitment of cytosolic Drp1 to the mitochondrial outer membrane by Fis1, Mff, and MIEF1 in mammals and the divergences in regulation of mitochondrial dynamics between yeast and vertebrates.

Mitochondrial Fission Triggered by Hyperglycemia Is Mediated by ROCK1 Activation in Podocytes and Endothelial Cells

Several lines of evidence suggest that mitochondrial dysfunction plays a critical role in the pathogenesis of microvascular complications of diabetes, including diabetic nephropathy. However, the signaling pathways by which hyperglycemia leads to mitochondrial dysfunction are not fully understood. Here we examined the role of Rho-associated coiled coil-containing protein kinase 1 (ROCK1) on mitochondrial dynamics by generating two diabetic mouse models with targeted deletions of ROCK1 and an inducible podocyte-specific knockin mouse expressing a constitutively active (cA) mutant of ROCK1. Our findings suggest that ROCK1 mediates hyperglycemia-induced mitochondrial fission by promoting dynamin-related protein-1 (Drp1) recruitment to the mitochondria. Deletion of ROCK1 in diabetic mice prevented mitochondrial fission, whereas podocyte-specific cA-ROCK1 mice exhibited increased mitochondrial fission. Importantly, we found that ROCK1 triggers mitochondrial fission by phosphorylating Drp1 at serine 600 residue. These findings provide insights into the unexpected role of ROCK1 in a signaling cascade that regulates mitochondrial dynamics.

Fis1 acts as a mitochondrial recruitment factor for TBC1D15 that is involved in regulation of mitochondrial morphology

In yeast, C-tail-anchored mitochondrial outer membrane protein Fis1 recruits the mitochondrial-fission-regulating GTPase Dnm1 to mitochondrial fission sites. However, the function of its mammalian homologue remains enigmatic because it has been reported to be dispensable for the mitochondrial recruitment of Drp1, a mammalian homologue of Dnm1. We identified TBC1D15 as a Fis1-binding protein in HeLa cell extracts. Immunoprecipitation revealed that Fis1 efficiently interacts with TBC1D15 but not with Drp1. Bacterially expressed Fis1 and TBC1D15 formed a direct and stable complex. Exogenously expressed TBC1D15 localized mainly in cytoplasm in HeLa cells, but when coexpressed with Fis1 it localized to mitochondria. Knockdown of TBC1D15 induced highly developed mitochondrial network structures similar to the effect of Fis1 knockdown, suggesting that the TBC1D15 and Fis1 are associated with the regulation of mitochondrial morphology independently of Drp1. These data suggest that Fis1 acts as a mitochondrial receptor in the recruitment of mitochondrial morphology protein in mammalian cells.

MIEF1 as Fission Relief

MIEF1 promotes mitochondrial fusion despite recruiting the fission-promoting GTPase Drp1 to the mitochondrial membrane.

How to split up: lessons from mitochondria

How to split up: lessons from mitochondria