The mechanosensitive ion channel PIEZO1 promotes satellite cell function in muscle regeneration.

Muscle satellite cells (MuSCs), myogenic stem cells in skeletal muscles, play an essential role in muscle regeneration. After skeletal muscle injury, quiescent MuSCs are activated to enter the cell cycle and proliferate, thereby initiating regeneration; however, the mechanisms that ensure successful MuSC division, including chromosome segregation, remain unclear. Here, we show that PIEZO1, a calcium ion (Ca2+)-permeable cation channel activated by membrane tension, mediates spontaneous Ca2+ influx to control the regenerative function of MuSCs. Our genetic engineering approach in mice revealed that PIEZO1 is functionally expressed in MuSCs and that Piezo1 deletion in these cells delays myofibre regeneration after injury. These results are, at least in part, due to a mitotic defect in MuSCs. Mechanistically, this phenotype is caused by impaired PIEZO1-Rho signalling during myogenesis. Thus, we provide the first concrete evidence that PIEZO1, a bona fide mechanosensitive ion channel, promotes proliferation and regenerative functions of MuSCs through precise control of cell division.

Cellular function of (a)symmetric biological membranes.

In mammalian cells, phospholipids are asymmetrically distributed between the outer and inner leaflets of the plasma membrane. The maintenance of asymmetric phospholipid distribution has been demonstrated to be required for a wide range of cellular functions including cell division, cell migration, and signal transduction. However, we recently reported that asymmetric phospholipid distribution is disrupted in Drosophila cell membranes, and this unique phospholipid distribution leads to the formation of highly deformable cell membranes. In addition, it has become clear that asymmetry in the trans-bilayer distribution of phospholipids is disturbed even in living mammalian cells under certain circumstances. In this article, we introduce our recent studies while focusing on the trans-bilayer distribution of phospholipids, and discuss the cellular functions of (a)symmetric biological membranes.

Regulation of thermoregulatory behavior by commensal bacteria in Drosophila.

Commensal bacteria affect many aspects of host physiology. In this study, we focused on the role of commensal bacteria in the thermoregulatory behavior of Drosophila melanogaster. We demonstrated that the elimination of commensal bacteria caused an increase in the preferred temperature of Drosophila third-instar larvae without affecting the activity of transient receptor potential ankyrin 1 (TRPA1)-expressing thermosensitive neurons. We isolated eight bacterial strains from the gut and culture medium of conventionally reared larvae and found that the preferred temperature of the larvae was decreased by mono-association with Lactobacillus plantarum or Corynebacterium nuruki. Mono-association with these bacteria did not affect the indices of energy metabolism such as ATP and glucose levels of larvae, which are closely linked to thermoregulation in animals. Thus, we show a novel role for commensal bacteria in host thermoregulation and identify two bacterial species that affect thermoregulatory behavior in Drosophila.

Cell-autonomous control of intracellular temperature by unsaturation of phospholipid acyl chains.

Intracellular temperature affects a wide range of cellular functions in living organisms. However, it remains unclear whether temperature in individual animal cells is controlled autonomously as a response to fluctuations in environmental temperature. Using two distinct intracellular thermometers, we find that the intracellular temperature of steady-state Drosophila S2 cells is maintained in a manner dependent on Δ9-fatty acid desaturase DESAT1, which introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA. The DESAT1-mediated increase of intracellular temperature is caused by the enhancement of F1Fo-ATPase-dependent mitochondrial respiration, which is coupled with thermogenesis. We also reveal that F1Fo-ATPase-dependent mitochondrial respiration is potentiated by cold exposure through the remodeling of mitochondrial cristae structures via DESAT1-dependent unsaturation of mitochondrial phospholipid acyl chains. Based on these findings, we propose a cell-autonomous mechanism for intracellular temperature control during environmental temperature changes.

Temperature sensitivity of Notch signaling underlies species-specific developmental plasticity and robustness in amniote brains.

Ambient temperature significantly affects developmental timing in animals. The temperature sensitivity of embryogenesis is generally believed to be a consequence of the thermal dependency of cellular metabolism. However, the adaptive molecular mechanisms that respond to variations in temperature remain unclear. Here, we report species-specific thermal sensitivity of Notch signaling in the developing amniote brain. Transient hypothermic conditions increase canonical Notch activity and reduce neurogenesis in chick neural progenitors. Increased biosynthesis of phosphatidylethanolamine, a major glycerophospholipid components of the plasma membrane, mediates hypothermia-induced Notch activation. Furthermore, the species-specific thermal dependency of Notch signaling is associated with developmental robustness to altered Notch signaling. Our results reveal unique regulatory mechanisms for temperature-dependent neurogenic potentials that underlie developmental and evolutionary adaptations to a range of ambient temperatures in amniotes.

Extreme deformability of insect cell membranes is governed by phospholipid scrambling.

Organization of dynamic cellular structure is crucial for a variety of cellular functions. In this study, we report that Drosophila and Aedes have highly elastic cell membranes with extremely low membrane tension and high resistance to mechanical stress. In contrast to other eukaryotic cells, phospholipids are symmetrically distributed between the bilayer leaflets of the insect plasma membrane, where phospholipid scramblase (XKR) that disrupts the lipid asymmetry is constitutively active. We also demonstrate that XKR-facilitated phospholipid scrambling promotes the deformability of cell membranes by regulating both actin cortex dynamics and mechanical properties of the phospholipid bilayer. Moreover, XKR-mediated construction of elastic cell membranes is essential for hemocyte circulation in the Drosophila cardiovascular system. Deformation of mammalian cells is also enhanced by the expression of Aedes XKR, and thus phospholipid scrambling may contribute to formation of highly deformable cell membranes in a variety of living eukaryotic cells.

Organelle membrane-specific chemical labeling and dynamic imaging in living cells.

Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking.

Functional expression of Δ12 fatty acid desaturase modulates thermoregulatory behaviour in Drosophila.

Polyunsaturated fatty acids (PUFAs) play crucial roles in adaptation to cold environments in a wide variety of animals and plants. However, the mechanisms by which PUFAs affect thermoregulatory behaviour remain elusive. Thus, we investigated the roles of PUFAs in thermoregulatory behaviour of Drosophila melanogaster. To this end, we generated transgenic flies expressing Caenorhabditis elegans Δ12 fatty acid desaturase (FAT-2), which converts mono-unsaturated fatty acids to PUFAs such as linoleic acid [C18:2 (n-6)] and linolenic acid [C18:3 (n-3)]. Neuron-specific expression of FAT-2 using the GAL4/UAS expression system led to increased contents of C18:2 (n-6)-containing phospholipids in central nerve system (CNS) and caused significant decreases in preferred temperature of third instar larvae. In genetic screening and calcium imaging analyses of thermoreceptor-expressing neurons, we demonstrated that ectopic expression of FAT-2 in TRPA1-expressing neurons led to decreases in preferred temperature by modulating neuronal activity. We conclude that functional expression of FAT-2 in a subset of neurons changes the thermoregulatory behaviour of D. melanogaster, likely by modulating quantities of PUFA-containing phospholipids in neuronal cell membranes.

Changes in the physicochemical properties of fish cell membranes during cellular senescence.

Fish cell lines are widely used for the studies of developmental biology, virology, biology of aging, and nutrition physiology. However, little is known about their physicochemical properties. Here, we report the phospholipid compositions and mechanical properties of cell membranes derived from freshwater, anadromous and marine fish species. Biophysical analyses revealed that fish cell lines have highly deformable cell membranes with significantly low membrane tensions and Young's moduli compared with those of mammalian cell lines. The induction of cellular senescence by DNA demethylation using 5-Aza-2'-deoxycytidine significantly reduced the deformability of fish cell membrane, but hydrogen peroxide-induced oxidative stress did not affect the deformability. Mass spectrometry analysis of phospholipids revealed that the level of phosphatidylethanolamine molecules containing polyunsaturated fatty acids significantly increased during the 5-Aza-2'-deoxycytidine-induced cellular senescence. Fish cell lines provide a useful model system for studying the changes in the physicochemical properties of cell membranes during cellular senescence.Abbreviations: 2D-TLC: two-dimensional thin layer chromatography; 5-Aza-dC: 5-Aza-2'-deoxycytidine; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; FBS: fetal bovine serum; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; PS: phosphatidylserine; PUFA: polyunsaturated fatty acid; SA-ß-gal: senescence-associated beta-galactosidase; SM: sphingomyelin.

Different mechanisms for selective transport of fatty acids using a single class of lipoprotein in Drosophila.

In mammals, lipids are selectively transported to specific sites using multiple classes of lipoproteins. However, in Drosophila, a single class of lipoproteins, lipophorin, carries more than 95% of the lipids in the hemolymph. Although a unique ability of the insect lipoprotein system for cargo transport has been demonstrated, it remains unclear how this single class of lipoproteins selectively transports lipids. In this study, we carried out a comparative analysis of the fatty-acid composition among lipophorin, the CNS, and CNS-derived cell lines and investigated the transport mechanism of fatty acids, particularly focusing on the transport of PUFAs in Drosophila We showed that PUFAs are selectively incorporated into the acyl chains of lipophorin phospholipids and effectively transported to CNS through lipophorin receptor-mediated endocytosis of lipophorin. In addition, we demonstrated that C14 fatty acids are selectively incorporated into the diacylglycerols (DAGs) of lipophorin and that C14 fatty-acid-containing DAGs are spontaneously transferred from lipophorin to the phospholipid bilayer. These results suggest that PUFA-containing phospholipids and C14 fatty-acid-containing DAGs in lipophorin could be transferred to different sites by different mechanisms to selectively transport fatty acids using a single class of lipoproteins.

Structure and Function of Δ9-Fatty Acid Desaturase.

Δ9-Fatty acid desaturase (Δ9-desaturase) is a rate-limiting enzyme of unsaturated fatty acid biosynthesis in animal cells and specifically introduces a cis-double bond at the Δ9-position of acyl-CoA. Since the chemical structure of fatty acids determines the physicochemical properties of cellular membrane and modulates a broad range of cellular functions, double bond introduction into a fatty acid by Δ9-desaturase should be specifically carried out. Reported crystal structures of stearoyl-CoA desaturase (SCD)1, one of the most studied Δ9-desaturases, have revealed the mechanism underlying the determination of substrate preference, as well as the position (Δ9) and conformation (cis) of double bond introduction. The crystal structures of SCD1 have also provided insights into the function of other Δ9-desaturases, including Drosophila homologs. Moreover, the amino-terminal sequences of Δ9-desaturases are shown to have unique roles in protein degradation. In this review, we introduce recent advances in the understanding of the function and regulation of Δ9-desaturase from the standpoint of protein structure.

Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation.

Myotube formation by fusion of myoblasts and subsequent elongation of the syncytia is essential for skeletal muscle formation. However, molecules that regulate myotube formation remain elusive. Here we identify PIEZO1, a mechanosensitive Ca2+ channel, as a key regulator of myotube formation. During myotube formation, phosphatidylserine, a phospholipid that resides in the inner leaflet of the plasma membrane, is transiently exposed to cell surface and promotes myoblast fusion. We show that cell surface phosphatidylserine inhibits PIEZO1 and that the inward translocation of phosphatidylserine, which is driven by the phospholipid flippase complex of ATP11A and CDC50A, is required for PIEZO1 activation. PIEZO1-mediated Ca2+ influx promotes RhoA/ROCK-mediated actomyosin assemblies at the lateral cortex of myotubes, thus preventing uncontrolled fusion of myotubes and leading to polarized elongation during myotube formation. These results suggest that cell surface flip-flop of phosphatidylserine acts as a molecular switch for PIEZO1 activation that governs proper morphogenesis during myotube formation.

Hijacking of multiple phospholipid biosynthetic pathways and induction of membrane biogenesis by a picornaviral 3CD protein.

RNA viruses induce specialized membranous structures for use in genome replication. These structures are often referred to as replication organelles (ROs). ROs exhibit distinct lipid composition relative to other cellular membranes. In many picornaviruses, phosphatidylinositol-4-phosphate (PI4P) is a marker of the RO. Studies to date indicate that the viral 3A protein hijacks a PI4 kinase to induce PI4P by a mechanism unrelated to the cellular pathway, which requires Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1, GBF1, and ADP ribosylation factor 1, Arf1. Here we show that a picornaviral 3CD protein is sufficient to induce synthesis of not only PI4P but also phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylcholine (PC). Synthesis of PI4P requires GBF1 and Arf1. We identified 3CD derivatives: 3CDm and 3CmD, that we used to show that distinct domains of 3CD function upstream of GBF1 and downstream of Arf1 activation. These same 3CD derivatives still supported induction of PIP2 and PC, suggesting that pathways and corresponding mechanisms used to induce these phospholipids are distinct. Phospholipid induction by 3CD is localized to the perinuclear region of the cell, the outcome of which is the proliferation of membranes in this area of the cell. We conclude that a single viral protein can serve as a master regulator of cellular phospholipid and membrane biogenesis, likely by commandeering normal cellular pathways.

Synthesis of omega-3 long-chain polyunsaturated fatty acid-rich triacylglycerols in an endemic goby, Gymnogobius isaza, from Lake Biwa, Japan.

It is commonly observed that freshwater fish contain lower amounts of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), such as eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3), than marine fish species. In this study, we performed a detailed comparative analysis of phospholipids (PLs) and triacylglycerols (TAGs) from Gymnogobius isaza, a freshwater goby endemic to Lake Biwa inhabiting the lake bottom, and Gymnogobius urotaenia, a related goby that inhabits the shore of Lake Biwa. We found that tissues from G. isaza contain remarkably high amounts of omega-3 LC-PUFAs in both PLs and TAGs. Mass spectrometry analysis of TAGs demonstrated that the most abundant TAG molecular species were TAG (16:0/18:1/20:5), followed by TAG (14:0/18:1/20:5), in which EPA is incorporated into TAG at either the sn-1 or sn-3 positions. We isolated cDNAs encoding acyl-CoA: diacylglycerol acyltransferase designated as GiDGAT1 and GiDGAT2, from G. isaza. Expression studies using a neutral lipid-deficient Saccharomyces cerevisiae mutant strain demonstrated that both GiDGAT1 and GiDGAT2 possessed diacylglycerol acyltransferase activity, and preferential incorporation of LC-PUFA into TAG was observed in the presence of GiDGAT1. This study revealed the novel lipid profiles of G. isaza and identified the enzymes that were involved in the production of PUFA-containing TAGs.

Pleckstrin homology domain of p210 BCR-ABL interacts with cardiolipin to regulate its mitochondrial translocation and subsequent mitophagy.

Chronic myeloid leukemia (CML) is caused by the chimeric protein p210 BCR-ABL encoded by a gene on the Philadelphia chromosome. Although the kinase domain of p210 BCR-ABL is an active driver of CML, the pathological role of its pleckstrin homology (PH) domain remains unclear. Here, we carried out phospholipid vesicle-binding assays to show that cardiolipin (CL), a characteristic mitochondrial phospholipid, is a unique ligand of the PH domain. Arg726, a basic amino acid in the ligand-binding region, was crucial for ligand recognition. A subset of wild-type p210 BCR-ABL that was transiently expressed in HEK293 cells was dramatically translocated from the cytosol to mitochondria in response to carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment, which induces mitochondrial depolarization and subsequent externalization of CL to the organelle's outer membrane, whereas an R726A mutant of the protein was not translocated. Furthermore, only wild-type p210 BCR-ABL, but not the R726A mutant, suppressed CCCP-induced mitophagy and subsequently enhanced reactive oxygen species production. Thus, p210 BCR-ABL can change its intracellular localization via interactions between the PH domain and CL to cope with mitochondrial damage. This suggests that p210 BCR-ABL could have beneficial effects for cancer proliferation, providing new insight into the PH domain's contribution to CML pathogenesis.

An N-terminal di-proline motif is essential for fatty acid-dependent degradation of Δ9-desaturase in Drosophila.

The Δ9-fatty acid desaturase introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA and regulates the cellular levels of unsaturated fatty acids. However, it is unclear how Δ9-desaturase expression is regulated in response to changes in the levels of fatty acid desaturation. In this study, we found that the degradation of DESAT1, the sole Δ9-desaturase in the Drosophila cell line S2, was significantly enhanced when the amounts of unsaturated acyl chains of membrane phospholipids were increased by supplementation with unsaturated fatty acids, such as oleic and linoleic acids. In contrast, inhibition of DESAT1 activity remarkably suppressed its degradation. Of note, removal of the DESAT1 N-terminal domain abolished the responsiveness of DESAT1 degradation to the level of fatty acid unsaturation. Further truncation and amino acid replacement analyses revealed that two sequential prolines, the second and third residues of DESAT1, were responsible for the unsaturated fatty acid-dependent degradation. Although degradation of mouse stearoyl-CoA desaturase 1 (SCD1) was unaffected by changes in fatty acid unsaturation, introduction of the N-terminal sequential proline residues into SCD1 conferred responsiveness to unsaturated fatty acid-dependent degradation. Furthermore, we also found that the Ca2+-dependent cysteine protease calpain is involved in the sequential proline-dependent degradation of DESAT1. In light of these findings, we designated the sequential prolines at the second and third positions of DESAT1 as a "di-proline motif," which plays a crucial role in the regulation of Δ9-desaturase expression in response to changes in the level of cellular unsaturated fatty acids.

Development of a Novel Tetravalent Synthetic Peptide That Binds to Phosphatidic Acid.

We employed a multivalent peptide-library screening technique to identify a peptide motif that binds to phosphatidic acid (PA), but not to other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). A tetravalent peptide with the sequence motif of MARWHRHHH, designated as PAB-TP (phosphatidic acid-binding tetravalent peptide), was shown to bind as low as 1 mol% of PA in the bilayer membrane composed of PC and cholesterol. Kinetic analysis of the interaction between PAB-TP and the membranes containing 10 mol% of PA showed that PAB-TP associated with PA with a low dissociation constant of KD = 38 ± 5 nM. Coexistence of cholesterol or PE with PA in the membrane enhanced the PAB-TP binding to PA by increasing the ionization of the phosphomonoester head group as well as by changing the microenvironment of PA molecules in the membrane. Amino acid replacement analysis demonstrated that the tryptophan residue at position 4 of PAB-TP was involved in the interaction with PA. Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA. Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane. The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates.

Ceramide phosphoethanolamine (CPE), a sphingomyelin analog, is a major sphingolipid in invertebrates and parasites, whereas only trace amounts are present in mammalian cells. In this study, mushroom-derived proteins of the aegerolysin family­pleurotolysin A2 (PlyA2; K(D) = 12 nM), ostreolysin (Oly; K(D) = 1.3 nM), and erylysin A (EryA; K(D) = 1.3 nM)­strongly associated with CPE/cholesterol (Chol)-containing membranes, whereas their low affinity to sphingomyelin/Chol precluded establishment of the binding kinetics. Binding specificity was determined by multilamellar liposome binding assays, supported bilayer assays, and solid-phase studies against a series of neutral and negatively charged lipid classes mixed 1:1 with Chol or phosphatidylcholine. No cross-reactivity was detected with phosphatidylethanolamine. Only PlyA2 also associated with CPE, independent of Chol content (K(D) = 41 µM), rendering it a suitable tool for visualizing CPE in lipid-blotting experiments and biologic samples from sterol auxotrophic organisms. Visualization of CPE enrichment in the CNS of Drosophila larvae (by PlyA2) and in the bloodstream form of the parasite Trypanosoma brucei (by EryA) by fluorescence imaging demonstrated the versatility of aegerolysin family proteins as efficient tools for detecting and visualizing CPE.

Targeting cholesterol in a liquid-disordered environment by theonellamides modulates cell membrane order and cell shape.

Roles of lipids in the cell membrane are poorly understood. This is partially due to the lack of methodologies, for example, tool chemicals that bind to specific membrane lipids and modulate membrane function. Theonellamides (TNMs), marine sponge-derived peptides, recognize 3ß-hydroxysterols in lipid membranes and induce major morphological changes in cultured mammalian cells through as yet unknown mechanisms. Here, we show that TNMs recognize cholesterol-containing liquid-disordered domains and induce phase separation in model lipid membranes. Modulation of membrane order was also observed in living cells following treatment with TNM-A, in which cells shrank considerably in a cholesterol-, cytoskeleton-, and energy-dependent manner. These findings present a previously unrecognized mode of action of membrane-targeting natural products. Meanwhile, we demonstrated the importance of membrane order, which is maintained by cholesterol, for proper cell morphogenesis.

Transbilayer distribution of lipids at nano scale.

There is a limited number of methods to examine transbilayer lipid distribution in biomembranes. We employed freeze-fracture replica-labelling immunoelectron microscopy in combination with lipid-binding proteins and a peptide to examine both transbilayer distribution and lateral distribution of various phospholipids in mammalian cells. Our results indicate that phospholipids are exclusively distributed either in the outer or inner leaflet of human red blood cell (RBC) membranes. In contrast, in nucleated cells, such as human skin fibroblasts and neutrophils, sphingomyelin was distributed in both leaflets while exhibiting characteristic lipid domains in the inner leaflet. Similar to RBCs, lipid asymmetry was maintained both in resting and thrombin-activated platelets. However, the microparticles released from thrombin-activated platelets lost membrane asymmetry. Our results suggest that the microparticles were shed from platelet plasma membrane domains enriched with phosphatidylserine and/or phosphatidylinositol at the outer leaflet. These findings underscore the strict regulation and cell-type specificity of lipid asymmetry in the plasma membrane.