The plasma membrane of focal adhesions has a high content of cholesterol and phosphatidylcholine with saturated acyl chains.

Cellular functions, such as differentiation and migration, are regulated by the extracellular microenvironment, including the extracellular matrix (ECM). Cells adhere to ECM through focal adhesions (FAs) and sense the surrounding microenvironments. Although FA proteins have been actively investigated, little is known about the lipids in the plasma membrane at FAs. In this study, we examine the lipid composition at FAs with imaging and biochemical approaches. Using the cholesterol-specific probe D4 with total internal reflection fluorescence microscopy and super-resolution microscopy, we show an enrichment of cholesterol at FAs simultaneously with FA assembly. Furthermore, we establish a method to isolate the lipid from FA-rich fractions, and biochemical quantification of the lipids reveals that there is a higher content of cholesterol and phosphatidylcholine with saturated fatty acid chains in the lipids of the FA-rich fraction than in either the plasma membrane fraction or the whole-cell membrane. These results demonstrate that plasma membrane at FAs has a locally distinct lipid composition compared to the bulk plasma membrane.

ABCA1 and cholesterol transfer protein Aster-A promote an asymmetric cholesterol distribution in the plasma membrane.

Cholesterol is a major and essential component of the mammalian cell plasma membrane (PM), and the loss of cholesterol homeostasis leads to various pathologies. Cellular cholesterol uptake and synthesis are regulated by a cholesterol sensor in the endoplasmic reticulum (ER). However, it remains unclear how changes in the cholesterol level of the PM are recognized. Here, we show that the sensing of cholesterol in the PM depends on ABCA1 and the cholesterol transfer protein Aster-A, which cooperatively maintain the asymmetric transbilayer cholesterol distribution in the PM. We demonstrate that ABCA1 translocates (flops) cholesterol from the inner leaflet of the PM to the outer leaflet of the PM to maintain a low inner leaflet cholesterol level. We also found that when inner cholesterol levels were increased, Aster-A was recruited to the PM-ER contact site to transfer cholesterol to the ER. These results suggest that ABCA1 could promote an asymmetric cholesterol distribution to suppress Aster-A recruitment to the PM-ER contact site to maintain intracellular cholesterol homeostasis.

ABCA13 dysfunction associated with psychiatric disorders causes impaired cholesterol trafficking.

ATP-binding cassette subfamily A member 13 (ABCA13) is predicted to be the largest ABC protein, consisting of 5058 amino acids and a long N-terminal region. Mutations in the ABCA13 gene were reported to increase the susceptibility to schizophrenia, bipolar disorder, and major depression. However, little is known about the molecular functions of ABCA13 or how they associate with psychiatric disorders. Here, we examined the biochemical activity of ABCA13 using HEK293 cells transfected with mouse ABCA13. The expression of ABCA13 induced the internalization of cholesterol and gangliosides from the plasma membrane to intracellular vesicles. Cholesterol internalization by ABCA13 required the long N-terminal region and ATP hydrolysis. To examine the physiological roles of ABCA13, we generated Abca13 KO mice using CRISPR/Cas and found that these mice exhibited deficits of prepulse inhibition. Vesicular cholesterol accumulation and synaptic vesicle endocytosis were impaired in primary cultures of Abca13 KO cortical neurons. Furthermore, mutations in ABCA13 gene associated with psychiatric disorders disrupted the protein's subcellular localization and impaired cholesterol trafficking. These findings suggest that ABCA13 accelerates cholesterol internalization by endocytic retrograde transport in neurons and that loss of this function is associated with the pathophysiology of psychiatric disorders.

Preoperative CA19-9 is a prognostic factor in pT3N0 gastric cancer patients undergoing curative resection.

BACKGROUND: The standard treatment for pT3N0 gastric cancer (GC) in Japanese guidelines is radical surgery without adjuvant chemotherapy. However, certain percentages of these patients develop recurrences; therefore, detecting the high-risk subgroup of recurrence may contribute to improve patient's outcome by adjuvant chemotherapy. In this study, we aimed to identify a predictive indicator of poor prognosis in pT3N0 GC. METHODS: Eighty-one patients who were diagnosed as pT3N0 GC after curative surgical resection and had not received adjuvant chemotherapy were included. The clinicopathological factors and laboratory parameters were evaluated by univariate and multivariate analyses to identify prognostic factors of tumor recurrence. Survival analysis was performed by Kaplan-Meier method. RESULTS: Male (P = 0.027), a high body mass index (BMI) (P = 0.031), a high CA19-9 value (P = 0.025), and a lower number of retrieved lymph nodes (P = 0.018) were found to be significantly associated with a shorter recurrence free survival (RFS). In a multivariate analysis, high CA19-9 value (> 37 U/ml) [(hazard ratio (HR): 3.326; 95% confidence interval (CI): 1.044 to 10.596; P = 0.042] was found to be an independent predictor of RFS. CONCLUSION: The preoperative high CA19-9 value is considered a useful prognostic marker for predicting cancer recurrence after curative surgery in pT3N0 GC patients. For those patients, adjuvant chemotherapy might be considered to improve the survival outcome.

In vivo FRET analyses reveal a role of ATP hydrolysis-associated conformational changes in human P-glycoprotein.

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

An amphipathic helix of vinexin α is necessary for a substrate stiffness-dependent conformational change in vinculin.

Extracellular matrix (ECM) stiffness regulates various cell behaviors, including cell differentiation, proliferation and migration. Vinculin and vinexin α (an isoform encoded by the SORBS3 gene), both of which localize to focal adhesions, cooperatively function as mechanosensors of ECM stiffness. On a rigid ECM, vinexin α interacts with vinculin and induces a conformational change in vinculin to give an 'open' form, which promotes nuclear localization of Yes-associated protein (YAP, also known as YAP1) and transcriptional coactivator with a PDZ-binding motif (TAZ, also known as WWTR1) (hereafter YAP/TAZ). However, the detailed mechanism by which vinexin α induces the conformational change in vinculin has not been revealed. Here, we identify an amphipathic helix named H2 as a novel vinculin-binding site in vinexin α. The H2 helix interacts with the vinculin D1b subdomain and promotes the formation of a talin-vinculin-vinexin α ternary complex. Mutations in the H2 region not only impair the ability of vinexin α to induce the ECM stiffness-dependent conformational change in vinculin but also to promote nuclear localization of YAP/TAZ on rigid ECM. Taken together, these results demonstrate that the H2 helix in vinexin α plays a critical role in ECM stiffness-dependent regulation of vinculin and cell behaviors.

Cholesterol asymmetry at the tip of filopodia during cell adhesion.

During adhesion, cells develop filopodia to facilitate the attachment to the extracellular matrix. The small guanosine triphosphate (GTP)-binding protein, Cdc42, plays a central role in the formation of filopodia. It has been reported that Cdc42 activity is regulated by cholesterol (Chol). We examined Chol distribution in filopodia using Chol-binding domain 4 (D4) fragment of bacterial toxin, perfringolysin O that senses high membrane concentration of Chol. Our results indicate that fluorescent D4 was enriched at the tip of the outer leaflet of filopodia in the initiation phase of cell adhesion. This enrichment was accompanied by a defect of D4 labeling in the inner leaflet. Steady phase adhered cell experiment indicated that both Cdc42 and ATP-binding cassette transporter, ABCA1, were involved in the binding of D4 to the cell surface. Depletion of Chol activated Cdc42. Our results suggest that asymmetric distribution of Chol at the tip of filopodia induces activation of Cdc42, and thus, facilitates filopodia formation.

Predictive Effectiveness of the Glasgow Prognostic Score for Gastrointestinal Stromal Tumors.

The aim of this study was to evaluate the significance of the Glasgow prognostic score (GPS) in patients with resected gastrointestinal stromal tumors (GISTs). Forty-six GIST patients who underwent radical resection between January 2004 and December 2011 were enrolled in this retrospective study. The clinicopathological parameters examined included predictors of recurrence-free survival (RFS). Univariate and multivariate analysis of prognostic factors related to RFS were calculated using Cox proportional hazards model. The GPS classification system revealed 37 (80.4%), 6 (13.1%), and 3 (6.5%) patients with a GPS of 0, 1, and 2, respectively. Patients with GPS 1/2 had a significantly shorter RFS compared to those with GPS 0 (P = 0.01). The 3- and 5-year RFS rates for patients with GPS 0 were 94.0% and 90.9%, respectively, compared to 66.7% and 53.3%, respectively, for patients with GPS 1/2. Univariate analyses indicated that tumor size (P < 0.01), mitotic rate (P < 0.01), higher GPS (P < 0.01), and platelet count (P = 0.04) were prognostic factors for RFS; tumor size (P = 0.01) and GPS (P = 0.04) were independent prognostic factors in multivariate analysis. Preoperative high GPS were predictors of long-term prognosis in patients with resected GISTs.

Stiffness of the extracellular matrix regulates differentiation into beige adipocytes.

Beige/brite adipocytes, which express high levels of uncoupling protein 1 (UCP1) to generate heat using stored triglycerides, are induced under specific stimuli such as cold exposure in inguinal white adipose tissue (iWAT). Although extracellular microenvironments such as extracellular matrix (ECM) stiffness are known to regulate cell behaviors, including cell differentiation into adipocytes, the effect on iWAT cells is unknown. In this study, we show that rigid ECM promotes the cell spreading of iWAT-derived preadipocytes. Furthermore, the expression of UCP1 and other thermogenic genes in iWAT cells is promoted when the cells are cultured on rigid ECM. The expression of mTOR, a kinase known to regulate the differentiation to beige adipocytes, is decreased on rigid substrates. These results suggest that ECM stiffness plays an important role in the differentiation to beige adipocytes.

CRISPR/Cas9 nickase-mediated efficient and seamless knock-in of lethal genes in the medaka fish Oryzias latipes.

The CRISPR/Cas system offers new opportunities for targeted gene modifications in a wide range of organisms. In medaka (Oryzias latipes), a vertebrate model organism, a wild-type Cas9-based approach is commonly used to establish desired strains, however, its use in lethal genes is still challenging due to excess gene disruptions triggered by DNA double strand breaks (DSBs). To overcome this problem, we aimed to develop a new knock-in system using Cas9 nickase (Cas9n) that can reduce DNA DSBs. We revealed that Cas9n allowed reduction of the DSB-induced unwanted mutagenesis via non-homologous end-joining at both on- and off- target sites. Further, with a new donor plasmid (p2BaitD) that provides a linear template through Cas9n-mediated nicks, we successfully integrated reporter cassettes via homology-directed repair (HDR) into all three loci tested, including a lethal gene. In the experiment targeting the lethal gene, the combination of p2BaitD and Cas9n achieved higher survival rates than the Cas9-based approach, which enabled the desired knock-in founders. Additionally, through a technical blend of our knock-in system with a recently developed One-step mating protocol, we successfully established a homozygous knock-in strain in one generation period. This study presents evidence of an effective method to generate an HDR-mediated gene knock-in in medaka and other organisms, which is useful for establishing screening platforms for genes or drugs toxicity or other applications.

C-terminal of ABCA1 separately regulates cholesterol floppase activity and cholesterol efflux activity.

ATP-Binding Cassette A1 (ABCA1) is a key lipid transporter for cholesterol homeostasis. We recently reported that ABCA1 not only exports excess cholesterol in an apoA-I dependent manner, but that it also flops cholesterol from the inner to the outer leaflet of the plasma membrane. However, the relationship between these two activities of ABCA1 is still unclear. In this study, we analyzed the subcellular localization of ABCA1 by using a newly generated monoclonal antibody against its extracellular domain and the functions of eleven chimera proteins, in which the C-terminal domain of ABCA1 was replaced with those of the other ABCA subfamily members. We identified two motifs important for the functions of ABCA1. Three periodically repeated leucine residues were necessary for the cholesterol floppase activity but not the cholesterol efflux activity, while a VFVNFA motif was essential for both activities of ABCA1. These results suggest that the C-terminal of ABCA1 separately regulates the cholesterol floppase activity and the cholesterol efflux activity.

Vinculin promotes nuclear localization of TAZ to inhibit ECM stiffness-dependent differentiation into adipocytes.

Extracellular matrix (ECM) stiffness regulates the lineage commitment of mesenchymal stem cells (MSCs). Although cells sense ECM stiffness through focal adhesions, how cells sense ECM stiffness and regulate ECM stiffness-dependent differentiation remains largely unclear. In this study, we show that the cytoskeletal focal adhesion protein vinculin plays a critical role in the ECM stiffness-dependent adipocyte differentiation of MSCs. ST2 mouse MSCs differentiate into adipocytes and osteoblasts in an ECM stiffness-dependent manner. We find that a rigid ECM increases the amount of cytoskeleton-associated vinculin and promotes the nuclear localization and activity of the transcriptional coactivator paralogs Yes-associated protein (YAP, also known as YAP1) and transcriptional coactivator with a PDZ-binding motif (TAZ, also known as WWTR1) (hereafter YAP/TAZ). Vinculin is necessary for enhanced nuclear localization and activity of YAP/TAZ on the rigid ECM but it does not affect the phosphorylation of the YAP/TAZ kinase LATS1. Furthermore, vinculin depletion promotes differentiation into adipocytes on rigid ECM, while it inhibits differentiation into osteoblasts. Finally, TAZ knockdown was less effective at promoting adipocyte differentiation in vinculin-depleted cells than in control cells. These results suggest that vinculin promotes the nuclear localization of transcription factor TAZ to inhibit the adipocyte differentiation on rigid ECM.

Vinexin family (SORBS) proteins play different roles in stiffness-sensing and contractile force generation.

Vinexin, c-Cbl associated protein (CAP) and Arg-binding protein 2 (ArgBP2) constitute an adaptor protein family called the vinexin (SORBS) family that is targeted to focal adhesions (FAs). Although numerous studies have focused on each of the SORBS proteins and partially elucidated their involvement in mechanotransduction, a comparative analysis of their function has not been well addressed. Here, we established mouse embryonic fibroblasts that individually expressed SORBS proteins and analysed their functions in an identical cell context. Both vinexin-α and CAP co-localized with vinculin at FAs and promoted the appearance of vinculin-rich FAs, whereas ArgBP2 co-localized with α-actinin at the proximal end of FAs and punctate structures on actin stress fibers (SFs), and induced paxillin-rich FAs. Furthermore, both vinexin-α and CAP contributed to extracellular matrix stiffness-dependent vinculin behaviors, while ArgBP2 stabilized α-actinin on SFs and enhanced intracellular contractile forces. These results demonstrate the differential roles of SORBS proteins in mechanotransduction.

Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol.

Controlled distribution of lipids across various cell membranes is crucial for cell homeostasis and regulation. We developed an imaging method that allows simultaneous in situ quantification of cholesterol in two leaflets of the plasma membrane (PM) using tunable orthogonal cholesterol sensors. Our imaging revealed marked transbilayer asymmetry of PM cholesterol (TAPMC) in various mammalian cells, with the concentration in the inner leaflet (IPM) being ∼12-fold lower than that in the outer leaflet (OPM). The asymmetry was maintained by active transport of cholesterol from IPM to OPM and its chemical retention at OPM. Furthermore, the increase in the IPM cholesterol level was triggered in a stimulus-specific manner, allowing cholesterol to serve as a signaling lipid. We found excellent correlation between the IPM cholesterol level and cellular Wnt signaling activity, suggesting that TAPMC and stimulus-induced PM cholesterol redistribution are crucial for tight regulation of cellular processes under physiological conditions.

Cell migration is negatively modulated by ABCA1.

Temporal and spatial changes of membrane lipid distribution in the plasma membrane are thought to be important for various cellular functions. ATP-Binding Cassette A1 (ABCA1) is a key lipid transporter for the generation of high density lipoprotein. Recently, we reported that ABCA1 maintains an asymmetric distribution of cholesterol in the plasma membrane. Here we report that ABCA1 suppresses cell migration by modulating signal pathways. ABCA1 knockdown in mouse embryonic fibroblasts accelerated cell migration and increased activation of Rac1 and its localization to detergent-resistant membranes. Phosphorylation of MEK and ERK also increased. Inhibition of Rac1 or MEK-ERK signals suppressed cell migration in ABCA1 knockdown cells. Because our experimental conditions for cell migration did not contain cholesterol or lipid acceptors for ABCA1, cellular cholesterol content was not changed. These data suggest that ABCA1 modulates cell migration via Rac1 and MEK-ERK signaling by altering lipid distribution in the plasma membrane.

Apolipoprotein A-I directly interacts with extracellular domain 1 of human ABCA1.

ATP-binding cassette transporter A1 (ABCA1) is critical for the generation of nascent high-density lipoprotein (HDL) and plays important roles in cholesterol homeostasis. ABCA1 has two large extracellular domains (ECDs), which may interact directly with apolipoprotein A-I (apoA-I). However, the molecular mechanisms underlying HDL formation and the importance of ABCA1-apoA-I interactions in HDL formation remain unclear. We investigated the ABCA1-apoA-I interaction in photo-activated crosslinking experiments using sulfo-SBED-labeled apoA-I. ApoA-I bound to cells expressing ABCA1, but not to untransfected cells or cells expressing non-functional ABCA1. Binding was inhibited by sulfo-SBED-labeled apoA-I, and crosslinking of sulfo-SBED-labeled apoA-I with ABCA1 was inhibited by non-labeled apoA-I, suggesting that sulfo-SBED-labeled apoA-I specifically binds and crosslinks with functional ABCA1. Proteolytic digestion of crosslinked ABCA1 revealed that apoA-I bound the N-terminal half of ABCA1, and that the first ECD of ABCA1 is an apoA-I binding site. Abbreviations: ABC: ATP-binding cassette; apoA-I: apolipoprotein A-I; ATP: adenosine triphosphate; CHAPS: 3-(3-cholamidepropyl)dimethylammonio-1- propanesulphonate; DTT: dithiothreitol; ECD: extra cellular domain; EDTA: ethylenediaminetetraacetic acid; GFP: green fluorescent protein; HA: hemagglutinin; HDL: high density lipoprotein; HEK: human embryonic kidney; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; sulfo-SBED: (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)hexanoamido] ethyl-1,3'-dithiopropionate; NHS-ester, N-hydroxysuccinimide-ester.

Leishmania LABCG2 transporter is involved in ATP-dependent transport of thiols.

The Leishmania LABCG2 transporter has a key role in the redox metabolism of these protozoan parasites. Recently, the involvement of LABCG2 in virulence, autophagy and oxidative stress has been described. Null mutant parasites for LABCG2 present an increase in the intracellular levels of glutathione (GSH) and trypanothione [T(SH)2]. On the other hand, parasites overexpressing LABCG2 transporter export non-protein thiols to the extracellular medium. To explore if LABCG2 may mediate an active transport of non-protein thiols, the effect of these molecules on ATPase activity of LABCG2 as well as the ability of LABCG2 to transport them was determined using a baculovirus-Sf9 insect cell system. Our results indicate that all thiols tested [GSH, T(SH)2] as well as their oxidized forms GSSG and TS2 (trypanothione disulfide) stimulate LABCG2-ATPase basal activity. We have measured the transport of [3H]-GSH in inside-out Sf9 cell membrane vesicles expressing LABCG2-GFP (green fluorescence protein), finding that LABCG2 was able to mediate a rapid and concentration-dependent uptake of [3H]-GSH in the presence of ATP. Finally, we have analyzed the ability of different thiol species to compete for this uptake, T(SH)2 and TS2 being the best competitors. The IC50 value for [3H]-GSH uptake in the presence of increasing concentrations of T(SH)2 was less than 100 µM, highlighting the affinity of this thiol for LABCG2. These results provide the first direct evidence that LABCG2 is an ABC transporter of reduced and oxidized non-protein thiols in Leishmania, suggesting that this transporter can play a role in the redox metabolism and related processes in this protozoan parasite.

Lysophosphatidylcholine export by human ABCA7.

The ATP-binding cassette transporter A7 (ABCA7), which is highly expressed in the brain, is associated with the pathogenesis of Alzheimer's disease (AD). However, the physiological function of ABCA7 and its transport substrates remain unclear. Immunohistochemical analyses of human brain sections from AD and non-AD subjects revealed that ABCA7 is expressed in neuron and microglia cells in the cerebral cortex. The transport substrates and acceptors were identified in BHK/ABCA7 cells and compared with those of ABCA1. Like ABCA1, ABCA7 exported choline phospholipids in the presence of apoA-I and apoE; however, unlike ABCA1, cholesterol efflux was marginal. Lipid efflux by ABCA7 was saturated by 5µg/ml apoA-I and was not dependent on apoE isoforms, whereas efflux by ABCA1 was dependent on apoA-I up to 20µg/ml and apoE isoforms. Liquid chromatography-tandem mass spectrometry analyses revealed that the two proteins had different preferences for phospholipid export: ABCA7 preferred phosphatidylcholine (PC)≥lysoPC>sphingomyelin (SM)=phosphatidylethanolamine (PE), whereas ABCA1 preferred PC>>SM>PE=lysoPC. The major difference in the pattern of lipid peaks between ABCA7 and ABCA1 was the high lysoPC/PC ratio of ABCA7. These results suggest that lysoPC is one of the major transport substrates for ABCA7 and that lysoPC export may be a physiologically important function of ABCA7 in the brain.

The distribution of vinculin to lipid rafts plays an important role in sensing stiffness of extracellular matrix.

Extracellular matrix (ECM) stiffness regulates cell differentiation, survival, and migration. Our previous study has shown that the interaction of the focal adhesion protein vinculin with vinexin α plays a critical role in sensing ECM stiffness and regulating stiffness-dependent cell migration. However, the mechanism how vinculin-vinexin α interaction affects stiffness-dependent cell migration is unclear. Lipid rafts are membrane microdomains that are known to affect ECM-induced signals and cell behaviors. Here, we show that vinculin and vinexin α can localize to lipid rafts. Cell-ECM adhesion, intracellular tension, and a rigid ECM promote vinculin distribution to lipid rafts. The disruption of lipid rafts with Methyl-ß-cyclodextrin impaired the ECM stiffness-mediated regulation of vinculin behavior and rapid cell migration on rigid ECM. These results indicate that lipid rafts play an important role in ECM-stiffness regulation of cell migration via vinculin.

Structural basis for gating mechanisms of a eukaryotic P-glycoprotein homolog.

P-glycoprotein is an ATP-binding cassette multidrug transporter that actively transports chemically diverse substrates across the lipid bilayer. The precise molecular mechanism underlying transport is not fully understood. Here, we present crystal structures of a eukaryotic P-glycoprotein homolog, CmABCB1 from Cyanidioschyzon merolae, in two forms: unbound at 2.6-Å resolution and bound to a unique allosteric inhibitor at 2.4-Å resolution. The inhibitor clamps the transmembrane helices from the outside, fixing the CmABCB1 structure in an inward-open conformation similar to the unbound structure, confirming that an outward-opening motion is required for ATP hydrolysis cycle. These structures, along with site-directed mutagenesis and transporter activity measurements, reveal the detailed architecture of the transporter, including a gate that opens to extracellular side and two gates that open to intramembranous region and the cytosolic side. We propose that the motion of the nucleotide-binding domain drives those gating apparatuses via two short intracellular helices, IH1 and IH2, and two transmembrane helices, TM2 and TM5.