PPARdelta activation induces metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes.

Pluripotent stem-cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease, but they are functionally and structurally immature. Here, we induce efficient human PSC-CM (hPSC-CM) maturation through metabolic-pathway modulations. Specifically, we find that peroxisome-proliferator-associated receptor (PPAR) signaling regulates glycolysis and fatty acid oxidation (FAO) in an isoform-specific manner. While PPARalpha (PPARa) is the most active isoform in hPSC-CMs, PPARdelta (PPARd) activation efficiently upregulates the gene regulatory networks underlying FAO, increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation, and augments FAO flux. PPARd activation further increases binucleation, enhances myofibril organization, and improves contractility. Transient lactate exposure, which is frequently used for hPSC-CM purification, induces an independent cardiac maturation program but, when combined with PPARd activation, still enhances oxidative metabolism. In summary, we investigate multiple metabolic modifications in hPSC-CMs and identify a role for PPARd signaling in inducing the metabolic switch from glycolysis to FAO in hPSC-CMs.

Repeated hypoglycemia remodels neural inputs and disrupts mitochondrial function to blunt glucose-inhibited GHRH neuron responsiveness.

Hypoglycemia is a frequent complication of diabetes, limiting therapy and increasing morbidity and mortality. With recurrent hypoglycemia, the counterregulatory response (CRR) to decreased blood glucose is blunted, resulting in hypoglycemia-associated autonomic failure (HAAF). The mechanisms leading to these blunted effects are only poorly understood. Here, we report, with ISH, IHC, and the tissue-clearing capability of iDISCO+, that growth hormone releasing hormone (GHRH) neurons represent a unique population of arcuate nucleus neurons activated by glucose deprivation in vivo. Repeated glucose deprivation reduces GHRH neuron activation and remodels excitatory and inhibitory inputs to GHRH neurons. We show that low glucose sensing is coupled to GHRH neuron depolarization, decreased ATP production, and mitochondrial fusion. Repeated hypoglycemia attenuates these responses during low glucose. By maintaining mitochondrial length with the small molecule mitochondrial division inhibitor-1, we preserved hypoglycemia sensitivity in vitro and in vivo. Our findings present possible mechanisms for the blunting of the CRR, significantly broaden our understanding of the structure of GHRH neurons, and reveal that mitochondrial dynamics play an important role in HAAF. We conclude that interventions targeting mitochondrial fission in GHRH neurons may offer a new pathway to prevent HAAF in patients with diabetes.

Cell death through the ages: The ICDS 25th Anniversary Meeting.

In June of 2019, the International Cell Death Society (ICDS) held its 25th anniversary meeting in New York City at the Icahn School of Medicine at Mount Sinai organized by Drs. Richard A. Lockshin (St. John's University, USA), Zahra Zakeri (Queens College, USA), and Jerry Edward Chipuk (Icahn School of Medicine at Mount Sinai, USA). The three-day event, entitled 'Cell death through the ages: The ICDS 25th anniversary meeting', hosted ninety-one delegates including thirty-four speakers and twenty-two poster presentations. Additionally, the organizers gave special recognition to the twenty-one previous ICDS Lifetime Achievement awardees-those who have significantly contributed to the field of cell death and the growth of the organization. Here, we provide a summary of the meeting and highlight trending research in the fields of cell death, autophagy, immunology, and their impact on health and disease.

MDM2 Integrates Cellular Respiration and Apoptotic Signaling through NDUFS1 and the Mitochondrial Network.

Signaling diversity and subsequent complexity in higher eukaryotes is partially explained by one gene encoding a polypeptide with multiple biochemical functions in different cellular contexts. For example, mouse double minute 2 (MDM2) is functionally characterized as both an oncogene and a tumor suppressor, yet this dual classification confounds the cell biology and clinical literatures. Identified via complementary biochemical, organellar, and cellular approaches, we report that MDM2 negatively regulates NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1), leading to decreased mitochondrial respiration, marked oxidative stress, and commitment to the mitochondrial pathway of apoptosis. MDM2 directly binds and sequesters NDUFS1, preventing its mitochondrial localization and ultimately causing complex I and supercomplex destabilization and inefficiency of oxidative phosphorylation. The MDM2 amino-terminal region is sufficient to bind NDUFS1, alter supercomplex assembly, and induce apoptosis. Finally, this pathway is independent of p53, and several mitochondrial phenotypes are observed in Drosophila and murine models expressing transgenic Mdm2.

Late-onset megaconial myopathy in mice lacking group I Paks.

BACKGROUND: Group I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42, and they regulate cytoskeletal dynamics, cell polarity, and transcription. We previously demonstrated that Pak1 and Pak2 function redundantly to promote skeletal myoblast differentiation during postnatal development and regeneration in mice. However, the roles of Pak1 and Pak2 in adult muscle homeostasis are unknown. Choline kinase ß (Chk ß) is important for adult muscle homeostasis, as autosomal recessive mutations in CHKß are associated with two human muscle diseases, megaconial congenital muscular dystrophy and proximal myopathy with focal depletion of mitochondria. METHODS: We analyzed mice conditionally lacking Pak1 and Pak2 in the skeletal muscle lineage (double knockout (dKO) mice) over 1 year of age. Muscle integrity in dKO mice was assessed with histological stains, immunofluorescence, electron microscopy, and western blotting. Assays for mitochondrial respiratory complex function were performed, as was mass spectrometric quantification of products of choline kinase. Mice and cultured myoblasts deficient for choline kinase ß (Chk ß) were analyzed for Pak1/2 phosphorylation. RESULTS: dKO mice developed an age-related myopathy. By 10 months of age, dKO mouse muscles displayed centrally-nucleated myofibers, fibrosis, and signs of degeneration. Disease severity occurred in a rostrocaudal gradient, hindlimbs more strongly affected than forelimbs. A distinctive feature of this myopathy was elongated and branched intermyofibrillar (megaconial) mitochondria, accompanied by focal mitochondrial depletion in the central region of the fiber. dKO muscles showed reduced mitochondrial respiratory complex I and II activity. These phenotypes resemble those of rmd mice, which lack Chkß and are a model for human diseases associated with CHKß deficiency. Pak1/2 and Chkß activities were not interdependent in mouse skeletal muscle, suggesting a more complex relationship in regulation of mitochondria and muscle homeostasis. CONCLUSIONS: Conditional loss of Pak1 and Pak2 in mice resulted in an age-dependent myopathy with similarity to mice and humans with CHKß deficiency. Protein kinases are major regulators of most biological processes but few have been implicated in muscle maintenance or disease. Pak1/Pak2 dKO mice offer new insights into these processes.

FBXW7 regulates a mitochondrial transcription program by modulating MITF.

FBXW7 is well characterized as a tumor suppressor in many human cancers including melanoma; however, the mechanisms of tumor-suppressive function have not been fully elucidated. We leveraged two distinct RNA sequencing datasets: human melanoma cell lines (n = 10) with control versus silenced FBXW7 and a cohort of human melanoma tumor samples (n = 51) to define the transcriptomic fingerprint regulated by FBXW7. Here, we report that loss of FBXW7 enhances a mitochondrial gene transcriptional program that is dependent on MITF in human melanoma and confers poor patient outcomes. MITF is a lineage-specific master regulator of melanocytes and together with PGC-1alpha is a marker for melanoma subtypes with dependence for mitochondrial oxidative metabolism. We found that inactivation of FBXW7 elevates MITF protein levels in melanoma cells. In vitro studies examining loss of FBXW7 and MITF alone or in combination showed that FBXW7 is an upstream regulator for the MITF/PGC-1 signaling.

Dual suppression of inner and outer mitochondrial membrane functions augments apoptotic responses to oncogenic MAPK inhibition.

Mitogen-activated protein kinase (MAPK) pathway inhibitors show promise in treating melanoma, but are unsuccessful in achieving long-term remission. Concordant with clinical data, BRAFV600E melanoma cells eliminate glycolysis upon inhibition of BRAFV600E or MEK with the targeted therapies Vemurafenib or Trametinib, respectively. Consequently, exposure to these therapies reprograms cellular metabolism to increase mitochondrial respiration and restrain cell death commitment. As the inner mitochondrial membrane (IMM) is sub-organellar site of oxidative phosphorylation (OXPHOS), and the outer mitochondrial membrane (OMM) is the major site of anti-apoptotic BCL-2 protein function, we hypothesized that suppressing these critical mitochondrial membrane functions would be a rational approach to maximize the pro-apoptotic effect of MAPK inhibition. Here, we demonstrate that disruption of OXPHOS with the mitochondria-specific protonophore BAM15 promotes the mitochondrial pathway of apoptosis only when oncogenic MAPK signaling is inhibited. Based on RNA-sequencing analyses of nevi and primary melanoma samples, increased pro-apoptotic BCL-2 family expression positively correlates with high-risk disease suggesting a highly active anti-apoptotic BCL-2 protein repertoire likely contributes to worse outcome. Indeed, combined inhibition of the anti-apoptotic BCL-2 repertoire with BH3-mimetics, OXPHOS, and oncogenic MAPK signaling induces fulminant apoptosis and eliminates clonogenic survival. Altogether, these data suggest that dual suppression of IMM and OMM functions may unleash the normally inadequate pro-apoptotic effects of oncogenic MAPK inhibition to eradicate cancer cells, thus preventing the development of resistant disease, and ultimately, supporting long-term remission.

RAF/MEK/extracellular signal-related kinase pathway suppresses dendritic cell migration and traps dendritic cells in Langerhans cell histiocytosis lesions.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by granulomatous lesions containing pathological CD207+ dendritic cells (DCs) with constitutively activated mitogen-activated protein kinase (MAPK) pathway signaling. Approximately 60% of LCH patients harbor somatic BRAFV600E mutations localizing to CD207+ DCs within lesions. However, the mechanisms driving BRAFV600E+ LCH cell accumulation in lesions remain unknown. Here we show that sustained extracellular signal-related kinase activity induced by BRAFV600E inhibits C-C motif chemokine receptor 7 (CCR7)-mediated DC migration, trapping DCs in tissue lesions. Additionally, BRAFV600E increases expression of BCL2-like protein 1 (BCL2L1) in DCs, resulting in resistance to apoptosis. Pharmacological MAPK inhibition restores migration and apoptosis potential in a mouse LCH model, as well as in primary human LCH cells. We also demonstrate that MEK inhibitor-loaded nanoparticles have the capacity to concentrate drug delivery to phagocytic cells, significantly reducing off-target toxicity. Collectively, our results indicate that MAPK tightly suppresses DC migration and augments DC survival, rendering DCs in LCH lesions trapped and resistant to cell death.

Lymphatic endothelial S1P promotes mitochondrial function and survival in naive T cells.

Effective adaptive immune responses require a large repertoire of naive T cells that migrate throughout the body, rapidly identifying almost any foreign peptide. Because the production of T cells declines with age, naive T cells must be long-lived. However, it remains unclear how naive T cells survive for years while constantly travelling. The chemoattractant sphingosine 1-phosphate (S1P) guides T cell circulation among secondary lymphoid organs, including spleen, lymph nodes and Peyer's patches, where T cells search for antigens. The concentration of S1P is higher in circulatory fluids than in lymphoid organs, and the S1P1 receptor (S1P1R) directs the exit of T cells from the spleen into blood, and from lymph nodes and Peyer's patches into lymph. Here we show that S1P is essential not only for the circulation of naive T cells, but also for their survival. Using transgenic mouse models, we demonstrate that lymphatic endothelial cells support the survival of T cells by secreting S1P via the transporter SPNS2, that this S1P signals through S1P1R on T cells, and that the requirement for S1P1R is independent of the established role of the receptor in guiding exit from lymph nodes. S1P signalling maintains the mitochondrial content of naive T cells, providing cells with the energy to continue their constant migration. The S1P signalling pathway is being targeted therapeutically to inhibit autoreactive T cell trafficking, and these findings suggest that it may be possible simultaneously to target autoreactive or malignant cell survival.

Disruption of mitochondrial electron transport chain function potentiates the pro-apoptotic effects of MAPK inhibition.

The mitochondrial network is a major site of ATP production through the coupled integration of the electron transport chain (ETC) with oxidative phosphorylation. In melanoma arising from the V600E mutation in the kinase v-RAF murine sarcoma viral oncogene homolog B (BRAFV600E), oncogenic signaling enhances glucose-dependent metabolism while reducing mitochondrial ATP production. Likewise, when BRAFV600E is pharmacologically inhibited by targeted therapies (e.g. PLX-4032/vemurafenib), glucose metabolism is reduced, and cells increase mitochondrial ATP production to sustain survival. Therefore, collateral inhibition of oncogenic signaling and mitochondrial respiration may help enhance the therapeutic benefit of targeted therapies. Honokiol (HKL) is a well tolerated small molecule that disrupts mitochondrial function; however, its underlying mechanisms and potential utility with targeted anticancer therapies remain unknown. Using wild-type BRAF and BRAFV600E melanoma model systems, we demonstrate here that HKL administration rapidly reduces mitochondrial respiration by broadly inhibiting ETC complexes I, II, and V, resulting in decreased ATP levels. The subsequent energetic crisis induced two cellular responses involving cyclin-dependent kinases (CDKs). First, loss of CDK1-mediated phosphorylation of the mitochondrial division GTPase dynamin-related protein 1 promoted mitochondrial fusion, thus coupling mitochondrial energetic status and morphology. Second, HKL decreased CDK2 activity, leading to G1 cell cycle arrest. Importantly, although pharmacological inhibition of oncogenic MAPK signaling increased ETC activity, co-treatment with HKL ablated this response and vastly enhanced the rate of apoptosis. Collectively, these findings integrate HKL action with mitochondrial respiration and shape and substantiate a pro-survival role of mitochondrial function in melanoma cells after oncogenic MAPK inhibition.

Mitochondrial Fission in Human Diseases.

Mitochondria are an essential component of multicellular life - from primitive organisms, to highly complex entities like mammals. The importance of mitochondria is underlined by their plethora of well-characterized essential functions such as energy production through oxidative phosphorylation (OX-PHOS), calcium and reactive oxygen species (ROS) signaling, and regulation of apoptosis. In addition, novel roles and attributes of mitochondria are coming into focus through the recent years of mitochondrial research. In particular, over the past decade the study of mitochondrial shape and dynamics has achieved special significance, as they are found to impact mitochondrial function. Recent advances indicate that mitochondrial function and dynamics are inter-connected, and maintain the balance between health and disease at a cellular and an organismal level. For example, excessive mitochondrial division (fission) is associated with functional defects, and is implicated in multiple human diseases from neurodegenerative diseases to cancer. In this chapter we examine the recent literature on the mitochondrial dynamics-function relationship, and explore how it impacts on the development and progression of human diseases. We will also highlight the implications of therapeutic manipulation of mitochondrial dynamics in treating various human pathologies.

Mitochondrial metabolism in hematopoietic stem cells requires functional FOXO3.

Hematopoietic stem cells (HSC) are primarily dormant but have the potential to become highly active on demand to reconstitute blood. This requires a swift metabolic switch from glycolysis to mitochondrial oxidative phosphorylation. Maintenance of low levels of reactive oxygen species (ROS), a by-product of mitochondrial metabolism, is also necessary for sustaining HSC dormancy. Little is known about mechanisms that integrate energy metabolism with hematopoietic stem cell homeostasis. Here, we identify the transcription factor FOXO3 as a new regulator of metabolic adaptation of HSC. ROS are elevated in Foxo3(-/-) HSC that are defective in their activity. We show that Foxo3(-/-) HSC are impaired in mitochondrial metabolism independent of ROS levels. These defects are associated with altered expression of mitochondrial/metabolic genes in Foxo3(-/-) hematopoietic stem and progenitor cells (HSPC). We further show that defects of Foxo3(-/-) HSC long-term repopulation activity are independent of ROS or mTOR signaling. Our results point to FOXO3 as a potential node that couples mitochondrial metabolism with HSC homeostasis. These findings have critical implications for mechanisms that promote malignant transformation and aging of blood stem and progenitor cells.

Mitochondrial division is requisite to RAS-induced transformation and targeted by oncogenic MAPK pathway inhibitors.

Mitochondrial division is essential for mitosis and metazoan development, but a mechanistic role in cancer biology remains unknown. Here, we examine the direct effects of oncogenic RAS(G12V)-mediated cellular transformation on the mitochondrial dynamics machinery and observe a positive selection for dynamin-related protein 1 (DRP1), a protein required for mitochondrial network division. Loss of DRP1 prevents RAS(G12V)-induced mitochondrial dysfunction and renders cells resistant to transformation. Conversely, in human tumor cell lines with activating MAPK mutations, inhibition of these signals leads to robust mitochondrial network reprogramming initiated by DRP1 loss resulting in mitochondrial hyper-fusion and increased mitochondrial metabolism. These phenotypes are mechanistically linked by ERK1/2 phosphorylation of DRP1 serine 616; DRP1(S616) phosphorylation is sufficient to phenocopy transformation-induced mitochondrial dysfunction, and DRP1(S616) phosphorylation status dichotomizes BRAF(WT) from BRAF(V600E)-positive lesions. These findings implicate mitochondrial division and DRP1 as crucial regulators of transformation with leverage in chemotherapeutic success.

Mitochondrial shape governs BAX-induced membrane permeabilization and apoptosis.

Proapoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define proapoptotic BCL-2 protein composition after stress and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for proapoptotic BCL-2 family function. Cells with hyperfragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAXα9·membrane interactions. This work identifies a mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis.

Putting the pieces together: How is the mitochondrial pathway of apoptosis regulated in cancer and chemotherapy?

In order to solve a jigsaw puzzle, one must first have the complete picture to logically connect the pieces. However, in cancer biology, we are still gaining an understanding of all the signaling pathways that promote tumorigenesis and how these pathways can be pharmacologically manipulated by conventional and targeted therapies. Despite not having complete knowledge of the mechanisms that cause cancer, the signaling networks responsible for cancer are becoming clearer, and this information is serving as a solid foundation for the development of rationally designed therapies. One goal of chemotherapy is to induce cancer cell death through the mitochondrial pathway of apoptosis. Within this review, we present the pathways that govern the cellular decision to undergo apoptosis as three distinct, yet connected puzzle pieces: (1) How do oncogene and tumor suppressor pathways regulate apoptosis upstream of mitochondria? (2) How does the B-cell lymphoma 2 (BCL-2) family influence tumorigenesis and chemotherapeutic responses? (3) How is post-mitochondrial outer membrane permeabilization (MOMP) regulation of cell death relevant in cancer? When these pieces are united, it is possible to appreciate how cancer signaling directly impacts upon the fundamental cellular mechanisms of apoptosis and potentially reveals novel pharmacological targets within these pathways that may enhance chemotherapeutic success.

Functional expression of frog and rainbow trout melanocortin 2 receptors using heterologous MRAP1s.

Analysis of the functional expression of the melanocortin 2 receptor (MC2R) from a rather broad spectrum of vertebrates indicates that MC2R is exclusively selective for the ligand, ACTH, and the melanocortin receptor accessory protein 1 (MRAP1) is required for high affinity ACTH binding and activation of MC2R. A phylogenetic analysis of MRAP1 suggested that tetrapod sequences and bony fish sequences may represent two distinct trends in the evolution of the mrap1 gene. To test this hypothesis, a frog (Xenopus tropicalis) MC2R was expressed in CHO cells either in the presence of a tetrapod (mouse) MRAP1 or a bony fish (zebrafish) MRAP1. The response of frog MC2R to different concentrations of human ACTH(1-24) was more robust in the presence of mouse MRAP1 than in the presence of zebrafish MRAP1. Conversely, the cAMP response mediated by the rainbow trout (Oncorhynchus mykiss) MC2R was almost twofold higher and occurred at 1000-fold lower ACTH concentration in the presence of zebrafish MRAP1 than in the presence of mouse MRAP1. Collectively, these experiments raise the possibility that at least two distinct trends have emerged in the co-evolution of MC2R/MRAP1 interactions during the radiation of the vertebrates.

Use of chimeric melanocortin-2 and -4 receptors to identify regions responsible for ligand specificity and dependence on melanocortin 2 receptor accessory protein.

The melanocortin 2 (MC(2)) receptor differs from other melanocortin family members in its pharmacological profile and reliance on an accessory protein, MC(2) receptor accessory protein (MRAP), for surface expression and signal transduction. To identify features of the MC(2) receptor responsible for these characteristics, we created chimeras between MC(2) and MC(4) receptors and expressed these in CHO cells, where MRAP is essential for trafficking and signaling by MC(2) but not MC(4) receptors. Replacing the first transmembrane segment of the MC(2) receptor with the corresponding region from the MC(4) receptor allowed some surface expression in the absence of an accessory protein, while ACTH-induced cAMP production remained entirely MRAP-dependent. On the other hand, replacing the last two transmembrane domains, third extracellular loop and C-terminal tail of the MC(4) receptor with the corresponding regions from the MC(2) receptor resulted in MRAP-dependent signaling. Surprisingly, replacing the second and third transmembrane domains and the intervening first extracellular loop of MC(2) receptors with MC(4) sequences generated a chimera (2C2) that responded to both adrenocorticotropic hormone (ACTH) and to the potent MSH analog 4-norleucine-7-d-phenylalanine-α-melanocyte stimulating hormone (NDP-α-MSH), which does not activate native MC(2) receptors. The 2C2 chimeric receptor was able to respond to NDP-α-MSH without MRAP, but MRAP shifted the EC50 value for NDP-α-MSH to the left and caused constitutive activity. These results identify the first transmembrane domain as important for surface expression and regions from the second to third transmembrane segments of the MC(2) receptor as important for MRAP dependent-signal transduction and ligand specificity.

Identification and characterization of unique proline-rich peptides binding to the mitochondrial fission protein hFis1.

Mammalian mitochondrial fission requires at least two proteins, hFis1 and the dynamin-like GTPase DLP1/Drp1. The mitochondrial protein hFis1 is anchored at the outer membrane by a C-terminal transmembrane domain. The cytosolic domain of hFis1 contains six alpha helices [alpha1-alpha6] out of which [alpha2-alpha5] form tetratricopeptide repeat (TPR)-like motifs. DLP1 and possibly other proteins are thought to interact with the hFis1 TPR region during the fission process. It has also been suggested that the alpha1-helix regulates protein-protein interactions at the TPR. We performed random peptide phage display screening using the hFis1[alpha2-alpha6] as the target and identified ten different peptide sequences. Phage ELISA using mutant hFis1 indicates that the peptide binding requires the alpha2 and alpha3 helices and the intact TPR structure. Competition experiments and surface plasmon resonance analyses confirmed that a subset of free peptides enriched with proline residues directly bind to the target. Two of these peptides bind to the alpha1-containing intact cytosolic domain of hFis1 with decreased affinity. Peptide microinjection into cells abolished the mitochondrial swelling induced by overexpression of alpha1-deleted hFis1, and significantly decreased cytochrome c release from mitochondria upon apoptotic induction. Our data demonstrate that hFis1 can bind to multiple amino acid sequences selectively, and that the TPR constitutes the main binding region of hFis1, providing a first insight into the hFis1 TPR as a potential therapeutic target.

The mitochondrial outer membrane protein hFis1 regulates mitochondrial morphology and fission through self-interaction.

Mitochondrial fission in mammals is mediated by at least two proteins, DLP1/Drp1 and hFis1. DLP1 mediates the scission of mitochondrial membranes through GTP hydrolysis, and hFis1 is a putative DLP1 receptor anchored at the mitochondrial outer membrane by a C-terminal single transmembrane domain. The cytosolic domain of hFis1 contains six alpha-helices (alpha1-alpha6) out of which alpha2-alpha5 form two tetratricopeptide repeat (TPR) folds. In this study, by using chimeric constructs, we demonstrated that the cytosolic domain contains the necessary information for hFis1 function during mitochondrial fission. By using transient expression of different mutant forms of the hFis1 protein, we found that hFis1 self-interaction plays an important role in mitochondrial fission. Our results show that deletion of the alpha1 helix greatly increased the formation of dimeric and oligomeric forms of hFis1, indicating that alpha1 helix functions as a negative regulator of the hFis1 self-interaction. Further mutational approaches revealed that a tyrosine residue in the alpha5 helix and the linker between alpha3 and alpha4 helices participate in hFis1 oligomerization. Mutations causing oligomerization defect greatly reduced the ability to induce not only mitochondrial fragmentation by full-length hFis1 but also the formation of swollen ball-shaped mitochondria caused by alpha1-deleted hFis1. Our data suggest that oligomerization of hFis1 in the mitochondrial outer membrane plays a role in mitochondrial fission, potentially through participating in fission factor recruitment.