Search

Background: The number of mutations in nuclear encoded genes causing mitochondrial disease is ever increasing. Identification of these mutations is particularly important in the diagnosis of neuromuscular disorders as their presentation may mimic other acquired disorders.We present a novel heterozygous variant in mitochondrial fission factor (MFF) which mimics myasthenia gravis. Objective: To determine if the MFF c.937G>A, p.E313K variant causes a mild mitochondrial phenotype. Methods: We used whole exome sequencing (WES) to identify a novel heterozygous variant in MFF in a patient with ptosis, fatigue and muscle weakness. Using patient derived fibroblasts, we performed assays to evaluate mitochondrial and peroxisome dynamics. Results: We show that fibroblasts derived from this patient are defective in mitochondrial fission, despite normal recruitment of Drp1 to the mitochondria. Conclusions: The MFF c.937G>A, p.E313K variant leads to a mild mitochondrial phenotype and is associated with defective mitochondrial fission in patient-derived fibroblasts.

Many human diseases, including cancer and neurological abnormalities, are linked to deficiencies of phosphatase and tensin homolog deleted on chromosome ten (PTEN), a dual phosphatase that dephosphorylates both lipids and proteins. PTEN functions in multiple intracellular locations, including the plasma membrane and nucleus. Therefore, a critical challenge to understand the pathogenesis of PTEN-associated diseases is to determine the specific role of PTEN at different locations. Toward this goal, the current study generated a mouse line in which lysine 13, which is critical for the nuclear localization of PTEN, is changed to arginine in the lipid-binding domain using the CRISPR-Ca9 gene-editing system. We found that PTENK13R mice show a strong decrease in the localization of PTEN in the nucleus without affecting the protein stability, phosphatase activity, and phosphorylation in the C-terminal tail region. PTENK13R mice are viable but produce smaller neurons and develop microcephaly. These data demonstrate that PTENK13R mice provide a useful animal model to study the role of PTEN in the nucleus in vivo.

The mtHSP60/HSPD1-mtHSP10/HSPE1 system prevents protein misfolding and maintains proteostasis in the mitochondrial matrix. Altered activities of this chaperonin system have been implicated in human diseases, such as cancer and neurodegeneration. However, how defects in HSPD1 and HSPE1 affect mitochondrial structure and dynamics remains elusive. In the current study, we address this fundamental question in a human cell line, HEK293T. We found that the depletion of HSPD1 or HSPE1 results in fragmentation of mitochondria, suggesting a decrease in mitochondrial fusion. Supporting this notion, HSPE1 depletion led to proteolytic inactivation of OPA1, a dynamin-related GTPase that fuses the mitochondrial membrane. This OPA1 inactivation was mediated by a stress-activated metalloprotease, OMA1. In contrast, HSPD1 depletion did not induce OMA1 activation or OPA1 cleavage. These data suggest that HSPE1 controls mitochondrial morphology through a mechanism separate from its chaperonin activity.

Non-alcoholic steatohepatitis (NASH) is a most common chronic liver disease that is manifested by steatosis, inflammation, fibrosis, and tissue damage. Hepatocytes produce giant mitochondria termed megamitochondria in patients with NASH. It has been shown that gene knockout of OPA1, a mitochondrial dynamin-related GTPase that mediates mitochondrial fusion, prevents megamitochondria formation and liver damage in a NASH mouse model induced by a methionine-choline-deficient (MCD) diet. However, it is unknown whether blocking mitochondrial fusion mitigates NASH pathologies. Here, we acutely depleted OPA1 using antisense oligonucleotides in the NASH mouse model before or after megamitochondria formation. When OPA1 ASOs were applied at the disease onset, they effectively prevented megamitochondria formation and liver pathologies in the MCD model. Notably, even when applied after mice robustly developed NASH pathologies, OPA1 targeting effectively regressed megamitochondria and the disease phenotypes. Thus, our data show the efficacy of mitochondrial dynamics as a unique therapy for megamitochondria-associated liver disease.

The ubiquitination of mitochondrial proteins labels damaged mitochondria for degradation through mitophagy. We recently developed an in vivo system in which mitophagy is slowed by inhibiting mitochondrial division through knockout of Dnm1l/Drp1, a dynamin related GTPase that mediates mitochondrial division. Using this system, we revealed that the ubiquitination of mitochondrial proteins required SQSTM1/p62, but not the ubiquitin E3 ligase PRKN/parkin, during mitophagy. Here, we tested the role of PINK1, a mitochondrial protein kinase that activates mitophagy by phosphorylating ubiquitin, in mitochondrial ubiquitination by knocking out Pink1 in dnm1l-knockout liver. We found mitochondrial ubiquitination did not decrease in the absence of PINK1; instead, PINK1 was required for the degradation of MFN1 (mitofusin 1) and MFN2, two homologous outer membrane proteins that mediate mitochondrial fusion in dnm1l-knockout hepatocytes. These data suggest that mitochondrial ubiquitination is promoted by SQSTM1 independently of PINK1 and PRKN during mitophagy. PINK1 and PRKN appear to control the balance between mitochondrial division and fusion in vivo. Abbreviations: DNM1L/DRP1: dynamin 1-like; KEAP1: kelch-like ECH-associated protein 1; KO: knockout; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFN1/2: mitofusin 1/2; OPA1: OPA1, mitochondrial dynamin like GTPase; PDH: pyruvate dehydrogenase E1; PINK1: PTEN induced putative kinase 1; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase.

Defects in PTEN, a critical tumor suppressor, are associated with tumorigenesis and aberrant organ sizes. It has been shown that heterozygous PTEN loss increases brains and neuron size, while the specific loss of nuclear PTEN has the opposite effect. Here, we investigate the impact of a combination of heterozygous PTEN loss and nuclear PTEN loss on the size of various organs, including the brain, liver, thymus, spleen, and inguinal lymph node. We found that the effect of the combination varies among organs. Notably, the combination of heterozygous PTEN loss and nuclear PTEN loss restored the normal size of brains and neurons. In contrast, the liver's size was unaffected by either single PTEN defects or their combination. Strikingly, the size of the inguinal lymph node was greatly increased due to lymphoma by the combination of the two PTEN defects. These data suggest that nuclear PTEN and non-nuclear PTEN function in an antagonistic manner in the brain while acting synergistically in the inguinal lymph node.

PTEN and p53 are highly mutated in many cancers. These two tumor suppressors have critical functions in the nucleus, such as DNA repair, cell cycle progression, and genome maintenance. However, the in vivo functional relationship of nuclear PTEN and p53 is unknown. Here, we analyzed the liver of mice in which nuclear PTEN and p53 are individually or simultaneously depleted. We found that nuclear PTEN loss greatly upregulates p53 expression upon oxidative stress, while the loss of p53 potentiates stress-induced accumulation of PTEN in the nucleus. Next, we examined oxidative stress-induced DNA damage in hepatocytes, and found that nuclear PTEN loss aggravated the damage while p53 loss did not. Notably, mice lacking nuclear PTEN had increased hepatocellular carcinoma under oxidative stress, while mice lacking p53 in hepatocytes had accelerated hepatocellular carcinoma and intrahepatic cholangiocarcinoma. The formation of cholangiocarcinoma appears to involve the transformation of hepatocytes into cholangiocarcinoma. Simultaneous loss of nuclear PTEN and p53 exacerbated both types of liver cancers. These data suggest that nuclear PTEN and p53 suppress liver cancers through distinct mechanisms.

Background Cells show directed migration response to electric signals, namely electrotaxis or galvanotaxis. PI3K and PTEN jointly play counterbalancing roles in this event via a bilateral regulation of PIP3 signaling. PI3K has been proved essential in anterior signaling of electrotaxing cells, whilst the role of PTEN remains elusive. Methods Dictyostelium cells with different genetic backgrounds were treated with direct current electric signals to investigate the genetic regulation of electrotaxis. Results We demonstrated that electric signals promoted PTEN phosphatase activity and asymmetrical translocation to the posterior plasma membrane of the electrotaxing cells. Electric stimulation produced a similar but delayed rear redistribution of myosin II, immediately before electrotaxis started. Actin polymerization is required for the asymmetric membrane translocation of PTEN and myosin. PTEN signaling is also responsible for the asymmetric anterior redistribution of PIP3/F-actin, and a biased redistribution of pseudopod protrusion in the forwarding direction of electrotaxing cells. Conclusions PTEN controls electrotaxis by coordinately regulating asymmetric redistribution of myosin to the posterior, and PIP3/F-actin to the anterior region of the directed migration cells.

The connectivity of mitochondria is regulated by a balance between fusion and division. Many human diseases are associated with excessive mitochondrial connectivity due to impaired Drp1, a dynamin-related GTPase that mediates division. Here, we report a mitochondrial stress response, named mitochondrial safeguard, that adjusts the balance of fusion and division in response to increased mitochondrial connectivity. In cells lacking Drp1, mitochondria undergo hyperfusion. However, hyperfusion does not completely connect mitochondria because Opa1 and mitofusin 1, two other dynamin-related GTPases that mediate fusion, become proteolytically inactivated. Pharmacological and genetic experiments show that the activity of Oma1, a metalloprotease that cleaves Opa1, is regulated by short pulses of the membrane depolarization without affecting the overall membrane potential in Drp1-knockout cells. Re-activation of Opa1 and Mitofusin 1 in Drp1-knockout cells further connects mitochondria beyond hyperfusion, termed extreme fusion, leading to bioenergetic deficits. These findings reveal an unforeseen safeguard mechanism that prevents extreme fusion of mitochondria, thereby maintaining mitochondrial function when the balance is shifted to excessive connectivity.

This study experimentally analyzes differences in interpersonal cognition, focusing on face-to-face meetings and video conferences in the context of group work at a university. We attempt to quantify the situation in relation to communication by focusing on the elements that comprise the process of interpersonal cognition, in which the self acts toward others, others react, and the self observes this. We analyze differences in interpersonal cognition in the context of meetings/conferencing based on physical measurement of study participants’ vocal utterances, observation of the non-verbal expressions, and evaluation of participants’ overall impressions in order to ascertain psychological reactions. We seek to identify the communication problems associated with the use of video conferencing systems from the results of measurements relating to three types of meeting content.

Mitochondria are highly dynamic organelles that continuously grow, divide, and fuse. The division of mitochondria is crucial for human health. During mitochondrial division, the mechano-guanosine triphosphatase (GTPase) dynamin-related protein (Drp1) severs mitochondria at endoplasmic reticulum (ER)-mitochondria contact sites, where peripheral ER tubules interact with mitochondria. Here, we report that Drp1 directly shapes peripheral ER tubules in human and mouse cells. This ER-shaping activity is independent of GTP hydrolysis and located in a highly conserved peptide of 18 amino acids (termed D-octadecapeptide), which is predicted to form an amphipathic α helix. Synthetic D-octadecapeptide tubulates liposomes in vitro and the ER in cells. ER tubules formed by Drp1 promote mitochondrial division by facilitating ER-mitochondria interactions. Thus, Drp1 functions as a two-in-one protein during mitochondrial division, with ER tubulation and mechano-GTPase activities.

Purkinje neurons, one of the largest neurons in the brain, are critical for controlling body movements, and the dysfunction and degeneration of these cells cause ataxia. Purkinje neurons require a very efficient energy supply from mitochondria because of their large size and extensive dendritic arbors. We have previously shown that mitochondrial division mediated by dynamin-related protein 1 (Drp1) is critical for the development and survival of Purkinje neurons. Drp1 deficiency has been associated with one of the major types of ataxia: autosomal recessive spastic ataxia of Charlevoix Saguenay. Using post-mitotic Purkinje neuron-specific Drp1 knockout (KO) in mice, we investigated the molecular mechanisms that mediate the progressive degeneration of Drp1-KO Purkinje neurons in vivo. In these Purkinje neurons, p62/sequestosome-1, a multi-functional adaptor protein that balances apoptotic cell death and cell survival, was recruited to large mitochondria resulting from unopposed fusion in the absence of mitochondrial division. To test the role of p62 in Drp1-deficient neurodegeneration, we created mice lacking both Drp1 and p62 and found that the additional loss of p62 significantly extended the survival of Purkinje neurons lacking Drp1. These results provide insights into the neurodegenerative mechanisms of mitochondrial ataxia and a critical foundation for therapeutic interventions for this disease.

Membrane organelles comprise both proteins and lipids. Remodeling of these membrane structures is controlled by interactions between specific proteins and lipids. Mitochondrial structure and function depend on regulated fusion and the division of both the outer and inner membranes. Here we discuss recent advances in the regulation of mitochondrial dynamics by two critical phospholipids, phosphatidic acid (PA) and cardiolipin (CL). These two lipids interact with the core components of mitochondrial fusion and division (Opa1, mitofusin, and Drp1) to activate and inhibit these dynamin-related GTPases. Moreover, lipid-modifying enzymes such as phospholipases and lipid phosphatases may organize local lipid composition to spatially and temporarily coordinate a balance between fusion and division to establish mitochondrial morphology.

AKT is a serine/threonine kinase that plays an important role in metabolism, cell growth, and cytoskeletal dynamics. AKT is activated by two kinases, PDK1 and mTORC2. Although the regulation of PDK1 is well understood, the mechanism that controls mTORC2 is unknown. Here, by investigating insulin receptor signaling in human cells and biochemical reconstitution, we found that insulin induces the activation of mTORC2 toward AKT by assembling a supercomplex with KRAS4B and RHOA GTPases, termed KARATE (KRAS4B-RHOA-mTORC2 Ensemble). Insulin-induced KARATE assembly is controlled via phosphorylation of GTP-bound KRAS4B at S181 and GDP-bound RHOA at S188 by protein kinase A. By developing a KARATE inhibitor, we demonstrate that KRAS4B-RHOA interaction drives KARATE formation. In adipocytes, KARATE controls insulin-dependent translocation of the glucose transporter GLUT4 to the plasma membrane for glucose uptake. Thus, our work reveals a fundamental mechanism that activates mTORC2 toward AKT in insulin-regulated glucose homeostasis.

PTEN is a PIP3 phosphatase that antagonizes oncogenic PI3-kinase signalling. Due to its critical role in suppressing the potent signalling pathway, it is one of the most mutated tumour suppressors, especially in brain tumours. It is generally thought that PTEN deficiencies predominantly result from either loss of expression or enzymatic activity. By analysing PTEN in malignant glioblastoma primary cells derived from 16 of our patients, we report mutations that block localization of PTEN at the plasma membrane and nucleus without affecting lipid phosphatase activity. Cellular and biochemical analyses as well as structural modelling revealed that two mutations disrupt intramolecular interaction of PTEN and open its conformation, enhancing polyubiquitination of PTEN and decreasing protein stability. Moreover, promoting mono-ubiquitination increases protein stability and nuclear localization of mutant PTEN. Thus, our findings provide a molecular mechanism for cancer-associated PTEN defects and may lead to a brain cancer treatment that targets PTEN mono-ubiquitination.

The mitochondrion is an essential organelle for a wide range of cellular processes, including energy production, metabolism, signal transduction and cell death. To execute these functions, mitochondria regulate their size, number, morphology and distribution in cells via mitochondrial division and fusion. In addition, mitochondrial division and fusion control the autophagic degradation of dysfunctional mitochondria to maintain a healthy population. Defects in these dynamic membrane processes are linked to many human diseases that include metabolic syndrome, myopathy and neurodegenerative disorders. In the last several years, our fundamental understanding of mitochondrial fusion, division and degradation has been significantly advanced by high resolution structural analyses, protein-lipid biochemistry, super resolution microscopy and in vivo analyses using animal models. Here, we summarize and discuss this exciting recent progress in the mechanism and function of mitochondrial division and fusion.

We have been developing the innovative e-learning system using Manga for international students. We have blended a variety of learning events and have built up the original training method of the cross-cultural communication to cultivate mindset and skillset. We have solved the several problems of existing teaching materials to learn the cross cultural communication. And we have evaluated the effect of the learning system. After learning several courses on our e-learning system, each international student had a test to check the ability of the cross cultural communication. By analyzing a lot of feedback from our users who are international students and educational institutions, we were able to confirm that we had solved the several problems of existing teaching materials. And, we were able to confirm the learning effectiveness of our e-learning by comparing the international students who had learned with the ones who had not learned. We describe the result that we have investigated the effectiveness of the e-learning system for international students to learn cross-cultural understanding through Japanese domestic business manner.

Dynamin-related protein 1 (Drp1) divides mitochondria as a mechano-chemical GTPase. However, the function of Drp1 beyond mitochondrial division is largely unknown. Multiple Drp1 isoforms are produced through mRNA splicing. One such isoform, Drp1ABCD, contains all four alternative exons and is specifically expressed in the brain. Here, we studied the function of Drp1ABCD in mouse neurons in both culture and animal systems using isoform-specific knockdown by shRNA and isoform-specific knockout by CRISPR/Cas9. We found that the expression of Drp1ABCD is induced during postnatal brain development. Drp1ABCD is enriched in dendritic spines and regulates postsynaptic clathrin-mediated endocytosis by positioning the endocytic zone at the postsynaptic density, independently of mitochondrial division. Drp1ABCD loss promotes the formation of ectopic dendrites in neurons and enhanced sensorimotor gating behavior in mice. These data reveal that Drp1ABCD controls postsynaptic endocytosis, neuronal morphology and brain function.

Mitochondria divide to control their size, distribution, turnover, and function. Dynamin-related protein 1 (Drp1) is a critical mechanochemical GTPase that drives constriction during mitochondrial division. It is generally believed that mitochondrial division is regulated during recruitment of Drp1 to mitochondria and its oligomerization into a division apparatus. Here, we report an unforeseen mechanism that regulates mitochondrial division by coincident interactions of Drp1 with the head group and acyl chains of phospholipids. Drp1 recognizes the head group of phosphatidic acid (PA) and two saturated acyl chains of another phospholipid by penetrating into the hydrophobic core of the membrane. The dual phospholipid interactions restrain Drp1 via inhibition of oligomerization-stimulated GTP hydrolysis that promotes membrane constriction. Moreover, a PA-producing phospholipase, MitoPLD, binds Drp1, creating a PA-rich microenvironment in the vicinity of a division apparatus. Thus, PA controls the activation of Drp1 after the formation of the division apparatus.

The cycle of mitochondrial division and fusion disconnect and reconnect individual mitochondria in cells to remodel this energy-producing organelle. Although dynamin-related protein 1 (Drp1) plays a major role in mitochondrial division in cells, a reduced level of mitochondrial division still persists even in the absence of Drp1. It is unknown how much Drp1-mediated mitochondrial division accounts for the connectivity of mitochondria. The role of a Parkinson’s disease-associated protein—parkin, which biochemically and genetically interacts with Drp1—in mitochondrial connectivity also remains poorly understood. Here, we quantified the number and connectivity of mitochondria using mitochondria-targeted photoactivatable GFP in cells. We show that the loss of Drp1 increases the connectivity of mitochondria by 15-fold in mouse embryonic fibroblasts (MEFs). While a single loss of parkin does not affect the connectivity of mitochondria, the connectivity of mitochondria significantly decreased compared with a single loss of Drp1 when parkin was lost in the absence of Drp1. Furthermore, the loss of parkin decreased the frequency of depolarization of the mitochondrial inner membrane that is caused by increased mitochondrial connectivity in Drp1-knockout MEFs. Therefore, our data suggest that parkin negatively regulates Drp1-indendent mitochondrial division.

A novel protein, GflB, is found to control both Ras and Rho to optimize the reorganization of actin cytoskeletons for directed cell migration. GflB is subjected to feedback regulation from actin cytoskeletons, allowing cells to detect and control the size of actin-rich pseudopods and navigate their movements with extremely high precision., Directional sensing, a process in which cells convert an external chemical gradient into internal signaling events, is essential in chemotaxis. We previously showed that a Rho GTPase, RacE, regulates gradient sensing in Dictyostelium cells. Here, using affinity purification and mass spectrometry, we identify a novel RacE-binding protein, GflB, which contains a Ras GEF domain and a Rho GAP domain. Using biochemical and gene knockout approaches, we show that GflB balances the activation of Ras and Rho GTPases, which enables cells to precisely orient signaling events toward higher concentrations of chemoattractants. Furthermore, we find that GflB is located at the leading edge of migrating cells, and this localization is regulated by the actin cytoskeleton and phosphatidylserine. Our findings provide a new molecular mechanism that connects directional sensing and morphological polarization.

Summary The activation of G-protein-coupled receptors (GPCRs) leads to the activation of mTORC2 in cell migration and metabolism. However, the mechanism that links GPCRs to mTORC2 remains unknown. Here, using Dictyostelium cells, we show that GPCR-mediated chemotactic stimulation induces hetero-oligomerization of phosphorylated GDP-bound Rho GTPase and GTP-bound Ras GTPase in directed cell migration. The Rho-Ras hetero-oligomers directly and specifically stimulate mTORC2 activity toward AKT in cells and after biochemical reconstitution using purified proteins in vitro. The Rho-Ras hetero-oligomers do not activate ERK/MAPK, another kinase that functions downstream of GPCRs and Ras. Human KRas4B functionally replace Dictyostelium Ras in mTORC2 activation. In contrast to GDP-Rho, GTP-Rho antagonizes mTORC2-AKT signaling by inhibiting the oligomerization of GDP-Rho with GTP-Ras. These data reveal that GPCR-stimulated hetero-oligomerization of Rho and Ras provides a critical regulatory step that controls mTORC2-AKT signaling.

Summary The PTEN gene is highly mutated in many cancers, including hepatocellular carcinoma. The PTEN protein is located at different subcellular regions—PTEN at the plasma membrane suppresses PI3-kinase signaling in cell growth, whereas PTEN in the nucleus maintains genome integrity. Here, using nuclear PTEN-deficient mice, we analyzed the role of PTEN in the nucleus in hepatocellular carcinoma that is induced by carcinogen and oxidative stress-producing hepatotoxin. Upon oxidative stress, PTEN was accumulated in the nucleus of the liver, and this accumulation promoted repair of DNA damage in wild-type mice. In contrast, nuclear PTEN-deficient mice had increased DNA damage and accelerated hepatocellular carcinoma formation. Both basal and oxidative stress-induced localization of PTEN in the nucleus require ubiquitination of lysine 13 in PTEN. Taken together, these data suggest the critical role of nuclear PTEN in the protection from DNA damage and tumorigenesis in vivo., Graphical Abstract, Highlights • The nuclear localization of PTEN is regulated by ubiquitination of lysine 13 • Nuclear PTEN suppresses DNA damage in vivo • Nuclear PTEN loss causes tumorigenesis in a mouse model for hepatocellular carcinoma, Cell Biology; Cancer

mTORC2 plays critical roles in metabolism, cell survival and actin cytoskeletal dynamics through the phosphorylation of AKT. Despite its importance to biology and medicine, it is unclear how mTORC2-mediated AKT phosphorylation is controlled. Here, we identify an unforeseen principle by which a GDP-bound form of the conserved small G protein Rho GTPase directly activates mTORC2 in AKT phosphorylation in social amoebae (Dictyostelium discoideum) cells. Using biochemical reconstitution with purified proteins, we demonstrate that Rho-GDP promotes AKT phosphorylation by assembling a supercomplex with Ras-GTP and mTORC2. This supercomplex formation is controlled by the chemoattractant-induced phosphorylation of Rho-GDP at S192 by GSK-3. Furthermore, Rho-GDP rescues defects in both mTORC2-mediated AKT phosphorylation and directed cell migration in Rho-null cells in a manner dependent on phosphorylation of S192. Thus, in contrast to the prevailing view that the GDP-bound forms of G proteins are inactive, our study reveals that mTORC2-AKT signalling is activated by Rho-GDP.

Defects in phosphatase and tensin homolog (PTEN) are associated with neurological disorders and tumors. PTEN functions at two primary intracellular locations: the plasma membrane and the nucleus. At the membrane, PTEN functions as a phosphatidylinositol (3,4,5)-trisphosphate phosphatase and suppresses PI 3-kinase signaling that drives cell growth and tumorigenesis. However, the in vivo function of nuclear PTEN is unclear. Here, using CRISPR/Cas9, we generated a mouse model in which PTEN levels in the nucleus are decreased. Nuclear PTEN-deficient mice were born with microcephaly and maintained a small brain during adulthood. The size of neuronal soma was significantly smaller in the cerebellum, cerebral cortex, and hippocampus. Also, these mice were prone to seizure. No changes in PI 3-kinase signaling were observed. By contrast, the size of other organs was unaffected. Therefore, nuclear PTEN is essential for the health of the brain by promoting the growth of neuronal soma size during development.

Many tumors are associated with deficiency of the tumor suppressor, PTEN, a PIP3 phosphatase that turns off PIP3 signaling. The major site of PTEN action is the plasma membrane, where PIP3 is produced by PI3 kinases. However, the mechanism and functional importance of PTEN membrane recruitment are poorly defined. Using the heterologous expression system in which human PTEN is expressed in Dictyostelium discoideum, we defined the molecular mechanisms that regulate the membrane-binding site through inhibitory interactions with the phosphorylated C-terminal tail. In addition, we potentiated mechanisms that mediate PTEN membrane association and engineered an enhanced PTEN with increased tumor suppressor functions. Moreover, we identified a new class of cancer-associated PTEN mutations that are specifically defective in membrane association. In this review, we summarize recent advances in PTEN-membrane interactions and methods useful in addressing PTEN function.

Mitochondria govern many metabolic processes. In addition, mitochondria sense the status of metabolism and change their functions to regulate energy production, cell death, and thermogenesis. Recent studies have revealed that mitochondrial structural remodeling through division and fusion is critical to the organelle’s function. It has also become clear that abnormalities in mitochondrial division and fusion are linked to the pathophysiology of metabolic diseases such as diabetes and obesity. Here, we discuss the current understanding of the mechanisms of mitochondrial dynamics and their role in cellular and organismal metabolism.

Dynamin-related protein 1 (Drp1) constricts mitochondria as a mechanochemical GTPase during mitochondrial division. The Drp1 gene contains several alternative exons and produces multiple isoforms through RNA splicing. Here we performed a systematic analysis of Drp1 transcripts in different mouse tissues and identified a previously uncharacterized isoform that is highly enriched in the brain. This Drp1 isoform is termed Drp1(ABCD) because it contains four alterative exons: A, B, C, and D. Remarkably, Drp1(ABCD) is located at lysosomes, late endosomes, and the plasma membrane in addition to mitochondria. Furthermore, Drp1(ABCD) is concentrated at the interorganelle interface between mitochondria and lysosomes/late endosomes. The localizations of Drp1(ABCD) at lysosomes, late endosomes, and the plasma membrane require two exons, A and B, that are present in the GTPase domain. Drp1(ABCD) assembles onto these membranes in a manner that is regulated by its oligomerization and GTP hydrolysis. Experiments using lysosomal inhibitors show that the association of Drp1(ABCD) with lysosomes/late endosomes depends on lysosomal pH but not their protease activities. Thus, Drp1 may connect mitochondria to endosomal–lysosomal pathways in addition to mitochondrial division.

Sung and Weaver discuss new work from Kriebel et al. showing how extracellular vesicles generate and sustain chemotactic signals., Directional cell motility toward a chemical gradient, chemotaxis, is critical during inflammation, embryogenesis, and cancer metastasis. In this issue, Kriebel et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201710170) demonstrate that the key cAMP chemoattractant for Dictyostelium discoideum amoebas is synthesized within and released from extracellular vesicles to promote chemotaxis.

It is unknown what occurs if both mitochondrial division and fusion are completely blocked. Here, we introduced mitochondrial stasis by deleting two dynamin-related GTPases for division (Drp1) and fusion (Opa1) in livers. Mitochondrial stasis rescues liver damage and hypotrophy caused by the single knockout (KO). At the cellular level, mitochondrial stasis re-establishes mitochondrial size and rescues mitophagy defects caused by division deficiency. Using Drp1KO livers, we found that the autophagy adaptor protein p62/sequestosome-1-which is thought to function downstream of ubiquitination-promotes mitochondrial ubiquitination. p62 recruits two subunits of a cullin-RING ubiquitin E3 ligase complex, Keap1 and Rbx1, to mitochondria. Resembling Drp1KO, diet-induced nonalcoholic fatty livers enlarge mitochondria and accumulate mitophagy intermediates. Resembling Drp1Opa1KO, Opa1KO rescues liver damage in this disease model. Our data provide a new concept that mitochondrial stasis leads the spatial dimension of mitochondria to a stationary equilibrium and a new mechanism for mitochondrial ubiquitination in mitophagy.

Dynamin-related protein 1 (Drp1) is a dynamin superfamily GTPase, which drives membrane constriction during mitochondrial division. To mediate mitochondrial division, Drp1 is recruited to the mitochondrial outer membrane and is assembled into the division machinery. We previously showed that Drp1 interacts with phosphatidic acid (PA) and saturated phospholipids in the mitochondrial membrane, and this interaction restrains Drp1 in initiating the constriction of mitochondria. Here, we show that the role of saturated acyl chains of phospholipids is independent of their contribution to the membrane curvature or lipid packing suggesting their direct interaction with Drp1. We further show that an unstructured loop in the stalk domain of Drp1 is critical for interaction with unsaturated PA. Our data significantly advance our understanding of this unique protein-lipid interaction involved in mitochondrial division.

Mitochondrial dynamics and mitophagy have been linked to cardiovascular and neurodegenerative diseases. Here, we demonstrate that the mitochondrial division dynamin Drp1 and the Parkinson's disease-associated E3 ubiquitin ligase parkin synergistically maintain the integrity of mitochondrial structure and function in mouse heart and brain. Mice lacking cardiac Drp1 exhibited lethal heart defects. In Drp1KO cardiomyocytes, mitochondria increased their connectivity, accumulated ubiquitinated proteins, and decreased their respiration. In contrast to the current views of the role of parkin in ubiquitination of mitochondrial proteins, mitochondrial ubiquitination was independent of parkin in Drp1KO hearts, and simultaneous loss of Drp1 and parkin worsened cardiac defects. Drp1 and parkin also play synergistic roles in neuronal mitochondrial homeostasis and survival. Mitochondrial degradation was further decreased by combination of Drp1 and parkin deficiency, compared with their single loss. Thus, the physiological importance of parkin in mitochondrial homeostasis is revealed in the absence of mitochondrial division in mammals.

Mitochondria grow, divide, and fuse in cells. Mitochondrial division is critical for the maintenance of the structure and function of mitochondria. Alterations in this process have been linked to many human diseases, including peripheral neuropathies and aging-related neurological disorders. In this review, we discuss recent progress inmitochondrial division by focusing on molecular and invivo analyses of the evolutionarily conserved, centralcomponent of mitochondrial division, dynamin-related protein 1 (Drp1), in the yeast and mouse model organisms. This article is part of a Special Issue entitled: Misfolded Proteins, Mitochondrial Dysfunction, and Neurodegenerative Diseases.