Restraining the Divider: A Drp1-Phospholipid Interaction Inhibits Drp1 Activity and Shifts the Balance from Mitochondrial Fission to Fusion.

In this issue of Molecular Cell, Adachi et al. (2016) describe a novel interaction between the mitochondrial fission GTPase Drp1 and phosphatidic acid that restrains Drp1 activity and shifts the balance toward mitochondrial fusion, adding another layer of complexity to the regulation of mitochondrial dynamics.

Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy.

Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5' AMP-activated protein kinase (AMPKα1/α2/ß2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment.

Erk2 phosphorylation of Drp1 promotes mitochondrial fission and MAPK-driven tumor growth.

Ras is mutated in up to 30% of cancers, including 90% of pancreatic ductal adenocarcinomas, causing it to be constitutively GTP-bound, and leading to activation of downstream effectors that promote a tumorigenic phenotype. As targeting Ras directly is difficult, there is a significant effort to understand the downstream biological processes that underlie its protumorigenic activity. Here, we show that expression of oncogenic Ras or direct activation of the MAPK pathway leads to increased mitochondrial fragmentation and that blocking this phenotype, through knockdown of the mitochondrial fission-mediating GTPase Drp1, inhibits tumor growth. This fission is driven by Erk2-mediated phosphorylation of Drp1 on Serine 616, and both this phosphorylation and mitochondrial fragmentation are increased in human pancreatic cancer. Finally, this phosphorylation is required for Ras-associated mitochondrial fission, and its inhibition is sufficient to block xenograft growth. Collectively, these data suggest mitochondrial fission may be a target for treating MAPK-driven malignancies.

Mitochondrial dynamics in cancer stem cells.

Many tumors are now understood to be heterogenous cell populations arising from a minority of epithelial-like cancer stem cells (CSCs). CSCs demonstrate distinctive metabolic signatures from the more differentiated surrounding tumor bulk that confer resistance to traditional chemotherapeutic regimens and potential for tumor relapse. Many CSC phenotypes including metabolism, epithelial-to-mesenchymal transition, cellular signaling pathway activity, and others, arise from altered mitochondrial function and turnover, which are regulated by constant cycles of mitochondrial fusion and fission. Further, recycling of mitochondria through mitophagy in CSCs is associated with maintenance of reactive oxygen species levels that dictate gene expression. The protein machinery that drives mitochondrial dynamics is surprisingly simple and may represent attractive new therapeutic avenues to target CSC metabolism and selectively eradicate tumor-generating cells to reduce the risks of metastasis and relapse for a variety of tumor types.

miR-206 family is important for mitochondrial and muscle function, but not essential for myogenesis in vitro.

miR-206, miR-1a-1, and miR-1a-2 are induced during differentiation of skeletal myoblasts and promote myogenesis in vitro. miR-206 is required for skeletal muscle regeneration in vivo. Although this miRNA family is hypothesized to play an essential role in differentiation, a triple knock-out (tKO) of the three genes has not been done to test this hypothesis. We report that tKO C2C12 myoblasts generated using CRISPR/Cas9 method differentiate despite the expected derepression of the miRNA targets. Surprisingly, their mitochondrial function is diminished. tKO mice demonstrate partial embryonic lethality, most likely due to the role of miR-1a in cardiac muscle differentiation. Two tKO mice survive and grow normally to adulthood with smaller myofiber diameter, diminished physical performance, and an increase in PAX7 positive satellite cells. Thus, unlike other miRNAs important in other differentiation pathways, the miR-206 family is not absolutely essential for myogenesis and is instead a modulator of optimal differentiation of skeletal myoblasts.

Dynamin-Related Protein 1 Deficiency Promotes Recovery from AKI.

The proximal tubule epithelium relies on mitochondrial function for energy, rendering the kidney highly susceptible to ischemic AKI. Dynamin-related protein 1 (DRP1), a mediator of mitochondrial fission, regulates mitochondrial function; however, the cell-specific and temporal role of DRP1 in AKI in vivo is unknown. Using genetic murine models, we found that proximal tubule-specific deletion of Drp1 prevented the renal ischemia-reperfusion-induced kidney injury, inflammation, and programmed cell death observed in wild-type mice and promoted epithelial recovery, which associated with activation of the renoprotective ß-hydroxybutyrate signaling pathway. Loss of DRP1 preserved mitochondrial structure and reduced oxidative stress in injured kidneys. Lastly, proximal tubule deletion of DRP1 after ischemia-reperfusion injury attenuated progressive kidney injury and fibrosis. These results implicate DRP1 and mitochondrial dynamics as an important mediator of AKI and progression to fibrosis and suggest that DRP1 may serve as a therapeutic target for AKI.

The regulation of tumor cell physiology by mitochondrial dynamics.

Mitochondrial dynamics are increasingly recognized to play an important role in regulating mitochondrial function in response to diverse stimuli. Given the overlap in the physiological processes influenced by mitochondria and the physiological processes disrupted in tumor cells, we speculate that tumor cells alter mitochondrial shape to promote the tumorigenic phenotype. Here, we briefly review the evidence linking changes in mitochondrial fusion and fission to a number of key tumorigenic processes, including metabolic rewiring, inhibition of cell death, cell migration, cell proliferation and self-renewal capacity. The role of mitochondrial dynamics in tumor growth is an important emerging area of research, a better understanding of which may lead to promising new therapeutic options for the treatment of cancer.

Label-Free Quantification of Intracellular Mitochondrial Dynamics Using Dielectrophoresis.

Mitochondrial dynamics play an important role within several pathological conditions, including cancer and neurological diseases. For the purpose of identifying therapies that target aberrant regulation of the mitochondrial dynamics machinery and characterizing the regulating signaling pathways, there is a need for label-free means to detect the dynamic alterations in mitochondrial morphology. We present the use of dielectrophoresis for label-free quantification of intracellular mitochondrial modifications that alter cytoplasmic conductivity, and these changes are benchmarked against label-based image analysis of the mitochondrial network. This is validated by quantifying the mitochondrial alterations that are carried out by entirely independent means on two different cell lines: human embryonic kidney cells and mouse embryonic fibroblasts. In both cell lines, the inhibition of mitochondrial fission that leads to a mitochondrial structure of higher connectivity is shown to substantially enhance conductivity of the cell interior, as apparent from the significantly higher positive dielectrophoresis levels in the 0.5-15 MHz range. Using single-cell velocity tracking, we show ∼10-fold higher positive dielectrophoresis levels at 0.5 MHz for cells with a highly connected versus those with a highly fragmented mitochondrial structure, suggesting the feasibility for frequency-selective dielectrophoretic isolation of cells to aid the discovery process for development of therapeutics targeting the mitochondrial machinery.

Ral GTPases in tumorigenesis: emerging from the shadows.

Oncogenic Ras proteins rely on a series of key effector pathways to drive the physiological changes that lead to tumorigenic growth. Of these effector pathways, the RalGEF pathway, which activates the two Ras-related GTPases RalA and RalB, remains the most poorly understood. This review will focus on key developments in our understanding of Ral biology, and will speculate on how aberrant activation of the multiple diverse Ral effector proteins might collectively contribute to oncogenic transformation and other aspects of tumor progression.

Tumour maintenance is mediated by eNOS.

Tumour cells become addicted to the expression of initiating oncogenes like Ras, such that loss of oncogene expression in established tumours leads to tumour regression. HRas, NRas or KRas are mutated to remain in the active GTP-bound oncogenic state in many cancers. Although Ras activates several proteins to initiate human tumour growth, only PI3K, through activation of protein kinase B (PKB; also known as AKT), must remain activated by oncogenic Ras to maintain this growth. Here we show that blocking phosphorylation of the AKT substrate, endothelial nitric oxide synthase (eNOS or NOS3), inhibits tumour initiation and maintenance. Moreover, eNOS enhances the nitrosylation and activation of endogenous wild-type Ras proteins, which are required throughout tumorigenesis. We suggest that activation of the PI3K-AKT-eNOS-(wild-type) Ras pathway by oncogenic Ras in cancer cells is required to initiate and maintain tumour growth.

PIM1 drives lipid droplet accumulation to promote proliferation and survival in prostate cancer.

Lipid droplets (LDs) are dynamic organelles with a neutral lipid core surrounded by a phospholipid monolayer. Solid tumors exhibit LD accumulation, and it is believed that LDs promote cell survival by providing an energy source during energy deprivation. However, the precise mechanisms controlling LD accumulation and utilization in prostate cancer are not well known. Here, we show peroxisome proliferator-activated receptor α (PPARα) acts downstream of PIM1 kinase to accelerate LD accumulation and promote cell proliferation in prostate cancer. Mechanistically, PIM1 inactivates glycogen synthase kinase 3 beta (GSK3ß) via serine 9 phosphorylation. GSK3ß inhibition stabilizes PPARα and enhances the transcription of genes linked to peroxisomal biogenesis (PEX3 and PEX5) and LD growth (Tip47). The effects of PIM1 on LD accumulation are abrogated with GW6471, a specific inhibitor for PPARα. Notably, LD accumulation downstream of PIM1 provides a significant survival advantage for prostate cancer cells during nutrient stress, such as glucose depletion. Inhibiting PIM reduces LD accumulation in vivo alongside slow tumor growth and proliferation. Furthermore, TKO mice, lacking PIM isoforms, exhibit suppression in circulating triglycerides. Overall, our findings establish PIM1 as an important regulator of LD accumulation through GSK3ß-PPARα signaling axis to promote cell proliferation and survival during nutrient stress.

HSP70-mediated mitochondrial dynamics and autophagy represent a novel vulnerability in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is one of the deadliest forms of cancer with limited therapy options. Overexpression of the heat shock protein 70 (HSP70) is a hallmark of cancer that is strongly associated with aggressive disease and worse clinical outcomes. However, the underlying mechanisms by which HSP70 allows tumor cells to thrive under conditions of continuous stress have not been fully described. Here, we report that PDAC has the highest expression of HSP70 relative to normal tissue across all cancers analyzed. Furthermore, HSP70 expression is associated with tumor grade and is further enhanced in metastatic PDAC. We show that genetic or therapeutic ablation of HSP70 alters mitochondrial subcellular localization, impairs mitochondrial dynamics, and promotes mitochondrial swelling to induce apoptosis. Mechanistically, we find that targeting HSP70 suppresses the PTEN-induced kinase 1 (PINK1) mediated phosphorylation of dynamin-related protein 1 (DRP1). Treatment with the HSP70 inhibitor AP-4-139B was efficacious as a single agent in primary and metastatic mouse models of PDAC. In addition, we demonstrate that HSP70 inhibition promotes the AMP-activated protein kinase (AMPK) mediated phosphorylation of Beclin-1, a key regulator of autophagic flux. Accordingly, we find that the autophagy inhibitor hydroxychloroquine (HCQ) enhances the ability of AP-4-139B to mediate anti-tumor activity in vivo. Collectively, our results suggest that HSP70 is a multi-functional driver of tumorigenesis that orchestrates mitochondrial dynamics and autophagy. Moreover, these findings support the rationale for concurrent inhibition of HSP70 and autophagy as a novel therapeutic approach for HSP70-driven PDAC.

Opa1 and Drp1 reciprocally regulate cristae morphology, ETC function, and NAD+ regeneration in KRas-mutant lung adenocarcinoma.

Oncogenic KRas activates mitochondrial fission through Erk-mediated phosphorylation of the mitochondrial fission GTPase Drp1. Drp1 deletion inhibits tumorigenesis of KRas-driven pancreatic cancer, but the role of mitochondrial dynamics in other Ras-driven malignancies is poorly defined. Here we show that in vitro and in vivo growth of KRas-driven lung adenocarcinoma is unaffected by deletion of Drp1 but is inhibited by deletion of Opa1, the GTPase that regulates inner membrane fusion and proper cristae morphology. Mechanistically, Opa1 knockout disrupts cristae morphology and inhibits electron transport chain (ETC) assembly and activity, which inhibits tumor cell proliferation through loss of NAD+ regeneration. Simultaneous inactivation of Drp1 and Opa1 restores cristae morphology, ETC activity, and cell proliferation indicating that mitochondrial fission activity drives ETC dysfunction induced by Opa1 knockout. Our results support a model in which mitochondrial fission events disrupt cristae structure, and tumor cells with hyperactive fission activity require Opa1 activity to maintain ETC function.

Regulation of mitochondrial fission by GIPC-mediated Drp1 retrograde transport.

Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission, a large cytoplasmic GTPase recruited to the mitochondrial surface via transmembrane adaptors to initiate scission. While Brownian motion likely accounts for the local interactions between Drp1 and the mitochondrial adaptors, how this essential enzyme is targeted from more distal regions like the cell periphery remains unknown. Based on proteomic interactome screening and cell-based studies, we report that GAIP/RGS19-interacting protein (GIPC) mediates the actin-based retrograde transport of Drp1 toward the perinuclear mitochondria to enhance fission. Drp1 interacts with GIPC through its atypical C-terminal PDZ-binding motif. Loss of this interaction abrogates Drp1 retrograde transport resulting in cytoplasmic mislocalization and reduced fission despite retaining normal intrinsic GTPase activity. Functionally, we demonstrate that GIPC potentiates the Drp1-driven proliferative and migratory capacity in cancer cells. Together, these findings establish a direct molecular link between altered GIPC expression and Drp1 function in cancer progression and metabolic disorders.

ISL2 is a putative tumor suppressor whose epigenetic silencing reprograms the metabolism of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) cells reprogram their transcriptional and metabolic programs to survive the nutrient-poor tumor microenvironment. Through in vivo CRISPR screening, we discovered islet-2 (ISL2) as a candidate tumor suppressor that modulates aggressive PDA growth. Notably, ISL2, a nuclear and chromatin-associated transcription factor, is epigenetically silenced in PDA tumors and high promoter DNA methylation or its reduced expression correlates with poor patient survival. The exogenous ISL2 expression or CRISPR-mediated upregulation of the endogenous loci reduces cell proliferation. Mechanistically, ISL2 regulates the expression of metabolic genes, and its depletion increases oxidative phosphorylation (OXPHOS). As such, ISL2-depleted human PDA cells are sensitive to the inhibitors of mitochondrial complex I in vitro and in vivo. Spatial transcriptomic analysis shows heterogeneous intratumoral ISL2 expression, which correlates with the expression of critical metabolic genes. These findings nominate ISL2 as a putative tumor suppressor whose inactivation leads to increased mitochondrial metabolism that may be exploitable therapeutically.

cPLA2 regulates the expression of type I interferons and intracellular immunity to Chlamydia trachomatis.

Infection with the obligate bacterial intracellular pathogen Chlamydia trachomatis leads to the sustained activation of the small GTPase RAS and many of its downstream signaling components. In particular, the mitogen-activated protein kinase ERK and the calcium-dependent phospholipase cPLA(2) are activated and are important for the onset of inflammatory responses. In this study we tested if activation of ERK and cPLA(2) occurred as a result of RAS signaling during infection and determined the relative contribution of these signaling components to chlamydial replication and survival. We provide genetic and pharmacological evidence that during infection RAS, ERK, and, to a lesser extent, cPLA(2) activation are uncoupled, suggesting that Chlamydia activates individual components of this signaling pathway in a non-canonical manner. In human cell lines, inhibition of ERK or cPLA(2) signaling did not adversely impact C. trachomatis replication. In contrast, in murine cells, inhibition of ERK and cPLA(2) played a significant protective role against C. trachomatis. We determined that cPLA(2)-deficient murine cells are permissive for C. trachomatis replication because of their impaired expression of beta interferon and the induction of immunity-related GTPases (IRG) important for the containment of intracellular pathogens. Furthermore, the MAPK p38 was primarily responsible for cPLA(2) activation in Chlamydia-infected cells and IRG expression. Overall, these findings define a previously unrecognized role for cPLA(2) in the induction of cell autonomous cellular immunity to Chlamydia and highlight the many non-canonical signaling pathways engaged during infection.

RalA and PLD1 promote lipid droplet growth in response to nutrient withdrawal.

Lipid droplets (LDs) are dynamic organelles that undergo dynamic changes in response to changing cellular conditions. During nutrient depletion, LD numbers increase to protect cells against toxic fatty acids generated through autophagy and provide fuel for beta-oxidation. However, the precise mechanisms through which these changes are regulated have remained unclear. Here, we show that the small GTPase RalA acts downstream of autophagy to directly facilitate LD growth during nutrient depletion. Mechanistically, RalA performs this function through phospholipase D1 (PLD1), an enzyme that converts phosphatidylcholine (PC) to phosphatidic acid (PA) and that is recruited to lysosomes during nutrient stress in a RalA-dependent fashion. RalA inhibition prevents recruitment of the LD-associated protein perilipin 3, which is required for LD growth. Our data support a model in which RalA recruits PLD1 to lysosomes during nutrient deprivation to promote the localized production of PA and the recruitment of perilipin 3 to expanding LDs.

Mito Hacker: a set of tools to enable high-throughput analysis of mitochondrial network morphology.

Mitochondria are highly dynamic organelles that can exhibit a wide range of morphologies. Mitochondrial morphology can differ significantly across cell types, reflecting different physiological needs, but can also change rapidly in response to stress or the activation of signaling pathways. Understanding both the cause and consequences of these morphological changes is critical to fully understanding how mitochondrial function contributes to both normal and pathological physiology. However, while robust and quantitative analysis of mitochondrial morphology has become increasingly accessible, there is a need for new tools to generate and analyze large data sets of mitochondrial images in high throughput. The generation of such datasets is critical to fully benefit from rapidly evolving methods in data science, such as neural networks, that have shown tremendous value in extracting novel biological insights and generating new hypotheses. Here we describe a set of three computational tools, Cell Catcher, Mito Catcher and MiA, that we have developed to extract extensive mitochondrial network data on a single-cell level from multi-cell fluorescence images. Cell Catcher automatically separates and isolates individual cells from multi-cell images; Mito Catcher uses the statistical distribution of pixel intensities across the mitochondrial network to detect and remove background noise from the cell and segment the mitochondrial network; MiA uses the binarized mitochondrial network to perform more than 100 mitochondria-level and cell-level morphometric measurements. To validate the utility of this set of tools, we generated a database of morphological features for 630 individual cells that encode 0, 1 or 2 alleles of the mitochondrial fission GTPase Drp1 and demonstrate that these mitochondrial data could be used to predict Drp1 genotype with 87% accuracy. Together, this suite of tools enables the high-throughput and automated collection of detailed and quantitative mitochondrial structural information at a single-cell level. Furthermore, the data generated with these tools, when combined with advanced data science approaches, can be used to generate novel biological insights.

PIM kinases alter mitochondrial dynamics and chemosensitivity in lung cancer.

Resistance to chemotherapy represents a major obstacle to the successful treatment of non-small-cell lung cancer (NSCLC). The goal of this study was to determine how PIM kinases impact mitochondrial dynamics, ROS production, and response to chemotherapy in lung cancer. Live-cell imaging and microscopy were used to determine the effect of PIM loss or inhibition on mitochondrial phenotype and ROS. Inhibition of PIM kinases caused excessive mitochondrial fission and significant upregulation of mitochondrial superoxide, increasing intracellular ROS. Mechanistically, we define a signaling axis linking PIM1 to Drp1 and mitochondrial fission in lung cancer. PIM inhibition significantly increased the protein levels and mitochondrial localization of Drp1, causing marked fragmentation of mitochondria. An inverse correlation between PIM1 and Drp1 was confirmed in NSCLC patient samples. Inhibition of PIM sensitized NSCLC cells to chemotherapy and produced a synergistic antitumor response in vitro and in vivo. Immunohistochemistry and transmission electron microscopy verified that PIM inhibitors promote mitochondrial fission and apoptosis in vivo. These data improve our knowledge about how PIM1 regulates mitochondria and provide justification for combining PIM inhibition with chemotherapy in NSCLC.

RalA and RalB relocalization to depolarized mitochondria depends on clathrin-mediated endocytosis and facilitates TBK1 activation.

Healthy mitochondria use an electrochemical gradient across the inner mitochondrial membrane (IMM) to generate energy in the form of ATP. A variety of endogenous and exogenous factors can lead to transient or sustained depolarization of the IMM, including mitochondrial fission events, expression of uncoupling proteins, electron transport chain (ETC) inhibitors, or chemical uncouplers. This depolarization in turn leads to a variety of physiological responses, ranging from selective mitochondrial clearance (mitophagy) to cell death. How cells recognize and ultimately respond to depolarized mitochondria remains incompletely understood. Here we show that the small GTPases RalA and RalB both relocalize to mitochondria following depolarization in a process dependent on clathrin-mediated endocytosis (CME). Furthermore, both genetic and pharmacologic inhibition of RalA and RalB leads to an increase in the activity of the atypical IκB kinase TBK1 both basally and in response to mitochondrial depolarization. This phenotype was also observed following inhibition of Ral relocalization. Collectively, these data suggest a model in which RalA and RalB inhibit TBK1 and that relocalization of Ral to depolarized mitochondria facilitates TBK1 activation through release of this inhibition.