The ATPase activity of ABCA1 is increased by cholesterol in the presence of anionic lipids.

High-density lipoprotein (HDL) transports excess cholesterol from peripheral tissues back to the liver, and plasma HDL levels are inversely related to cardiovascular disease incidence. ATP-binding cassette A1 (ABCA1) is a member of the ABC protein superfamily, and generates nascent HDL, which consists of several hundreds of phospholipids and cholesterol wrapped by apolipoprotein A-I (apoA-I). However, it remains unclear whether cholesterol is a transport substrate of ABCA1.

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.

Cholesterol accumulation in ovarian follicles causes ovulation defects in Japanese medaka ().

ATP-binding cassette A1 (ABCA1) is a membrane protein, which exports excess cellular cholesterol to generate HDL to reduce the risk of the onset of cardiovascular diseases (CVD). In addition, ABCA1 exerts pleiotropic effects on such as inflammation, tissue repair, and cell proliferation and migration. In this study, we explored the novel physiological roles of ABCA1 using Japanese medaka ( a small teleost fish.

Live Cell FRET Analysis of the Conformational Changes of Human P-glycoprotein.

The molecular mechanisms of P-glycoprotein (P-gp; also known as MDR1 or ABCB1) have been mainly investigated using artificial membranes such as lipid-detergent mixed micelles, artificial lipid bilayers, and membrane vesicles derived from cultured cells. Although these experiments help illustrate details about the molecular mechanisms of P-gp, they do not reflect physiological membrane environments in terms of lateral pressure, curvature, constituent lipid species, The protocol presented here includes a detailed guide for analyzing the conformational change of human P-gp in living HEK293 cells by using intramolecular fluorescence resonance energy transfer (FRET), in which excitation of the donor fluorophore is transferred to the acceptor without emission of a photon when two fluorescent proteins are in close proximity. Combining FRET analysis with membrane permeabilization, the contribution of small molecules such as nucleotides to the conformational change can be evaluated in living cells.

Characterization of novel SUMO family genes in the rice genome.

Small ubiquitin-related modifier (SUMO) is a post-translational modification factor composed of about 100 amino acid residues. Most plant species express a family of SUMO isoforms. We found three novel homologs of rice (Oryza sativa L.

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.

ABCB1/MDR1/P-gp employs an ATP-dependent twist-and-squeeze mechanism to export hydrophobic drugs.

ABCB1, also called MDR1 or P-glycoprotein, exports various hydrophobic compounds and plays an essential role as a protective physiological barrier in several organs, including the brain, testis, and placenta. However, little is known about the structural mechanisms that allow ABCB1 to recognize hydrophobic compounds of diverse structures or the coupling of ATP hydrolysis to uphill substrate export. High-resolution X-ray crystal structures of the pre- and post-transport states and FRET analyses in living cells have revealed that an aromatic hydrophobic network at the top of the inner cavity is key for the conformational change in ABCB1 that is triggered by a hydrophobic substrate.

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.

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.

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.

Roles of the Mesenchymal Stromal/Stem Cell Marker Meflin in Cardiac Tissue Repair and the Development of Diastolic Dysfunction.

Rationale: Myofibroblasts have roles in tissue repair following damage associated with ischemia, aging, and inflammation and also promote fibrosis and tissue stiffening, causing organ dysfunction. One source of myofibroblasts is mesenchymal stromal/stem cells that exist as resident fibroblasts in multiple tissues. We previously identified meflin (mesenchymal stromal cell- and fibroblast-expressing Linx paralogue), a glycosylphosphatidylinositol-anchored membrane protein, as a specific marker of mesenchymal stromal/stem cells and a regulator of their undifferentiated state.

Phosphorylation by protein kinase C stabilizes ABCG1 and increases cholesterol efflux.

ATP-binding cassette protein G1 (ABCG1) plays an important role in eliminating excess cholesterol from macrophages and in the formation of high-density lipoprotein (HDL), which contributes to the prevention and regression of atherosclerosis. The post-translational regulation of ABCG1 remains elusive, although phosphorylation by protein kinase A destabilizes ABCG1 proteins. We examined the phosphorylation of ABCG1 using HEK293 and Raw264.

Changes in the asymmetric distribution of cholesterol in the plasma membrane influence streptolysin O pore formation.

ATP-binding cassette A1 (ABCA1) plays a key role in generating high-density lipoprotein (HDL) and preventing atherosclerosis. ABCA1 exports cholesterol and phospholipid to apolipoprotein A-I (apoA-I) in serum to generate HDL. We found that streptolysin O (SLO), a cholesterol-dependent pore-forming toxin, barely formed pores in ABCA1-expressing cells, even in the absence of apoA-I.

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 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).

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.

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.

Vinexin family (SORBS) proteins regulate mechanotransduction in mesenchymal stem cells.

The stiffness of extracellular matrix (ECM) directs the differentiation of mesenchymal stem cells (MSCs) through the transcriptional co-activators Yes-associated protein (YAP) and transcriptional coactivator with a PDZ-binding motif (TAZ). Although a recent study revealed the involvement of vinexin α and CAP (c-Cbl-associated proteins), two of vinexin (SORBS) family proteins that bind to vinculin, in mechanosensing, it is still unclear whether these proteins regulate mechanotransduction and differentiation of MSCs. In the present study, we show that both vinexin α and CAP are necessary for the association of vinculin with the cytoskeleton and the promotion of YAP/TAZ nuclear localization in MSCs grown on rigid substrates.

Temporary sequestration of cholesterol and phosphatidylcholine within extracellular domains of ABCA1 during nascent HDL generation.

The quality and quantity of high-density lipoprotein (HDL) in blood plasma are important for preventing coronary artery disease. ATP-binding cassette protein A1 (ABCA1) and apolipoprotein A-I (apoA-I) play essential roles in nascent HDL formation, but controversy persists regarding the mechanism by which nascent HDL is generated. In the "direct loading model", apoA-I acquires lipids directly from ABCA1 while it is bound to the transporter.

Fluid shear stress applied by orbital shaking induces MG-63 osteosarcoma cells to activate ERK in two phases through distinct signaling pathways.

Fluid shear stress (FSS) induces a series of biochemical responses in osteoblasts, and this "mechanoresponse" regulates their survival, proliferation and differentiation. However, the events in cells immediately after FSS application are unclear, and how biochemical signals from soluble factors modify the mechanoresponses is largely unknown. We used the orbital shaking method, instead of the frequently used parallel plate method, to examine activation of ERK and AKT by FSS for detailed tracking of its temporal transition.

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.

Cellular force assay detects altered contractility caused by a nephritis-associated mutation in nonmuscle myosin IIA.

Recent progress in understanding the essential roles of mechanical forces in regulating various cellular processes expands the field of biology to one where interdisciplinary approaches with engineering techniques become indispensable. Contractile forces or contractility-inherently present in proliferative cells due to the activity of ubiquitous nonmuscle myosin II (NMII)-are one of such mechano-regulators, but because NMII works downstream of diverse signaling pathways, it is often difficult to predict how the inherent cellular forces change upon perturbations to particular molecules. Here, we determine whether the contractility of individual cells is upregulated or downregulated based on an assay analyzing specific deformations of silicone gel substrates.

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.

Vinculin association with actin cytoskeleton is necessary for stiffness-dependent regulation of vinculin behavior.

The extracellular matrix (ECM) is a major regulator of cell behavior. Recent studies have indicated the importance of the physical properties of the ECM, including its stiffness, for cell migration and differentiation. Using actomyosin-generated forces, cells pull the ECM and sense stiffness via cell-ECM adhesion structures called focal adhesions (FAs).

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.

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.