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. Since ATP hydrolysis of ABC proteins is typically increased by their transport substrates, we characterized the effects of cholesterol on the ATPase activity of purified ABCA1 using liposomes of various lipid compositions. ABCA1 showed substantial ATPase activity (20-30 nmol$\cdot$min-1$\cdot$mg-1) only in liposomes containing anionic lipids, including phosphatidylserine. Cholesterol increased the ATPase activity by 1.6- to 3-fold in the presence of anionic lipids. Moreover, phosphatidylserine addition to BHK/ABCA1 cells increased phosphatidylcholine and cholesterol efflux to apoA-I. Next, we investigated the sterol specificity of ABCA1. The ATPase activity of ABCA1 was strongly enhanced by desmosterol and zymosterol, similar to cholesterol. In contrast, 7-dehydrocholesterol and lathosterol weakly increased the ATPase activity, and no increase was observed with stigmasterol or brassicasterol. These findings suggest that ABCA1 transports cholesterol and prefers cholesterol over plant sterols as a transport substrate.

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.

Cholesterol accumulation in ovarian follicles causes ovulation defects in Abca1a -/- Japanese medaka (Oryzias latipes).

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 (Oryzias latipes), a small teleost fish. Three Abca1 genes were found in the medaka genome. ABCA1A and ABCA1C exported cholesterol to generate nascent HDL as human ABCA1 when expressed in HEK293 cells. To investigate their physiological roles, each Abca1-deficient fish was generated using the CRISPR-Cas9 system. Abca1a -/- female medaka was found to be infertile, while Abca1b -/- and Abca1c -/- female medaka were fertile. In vitro ovarian follicle culture suggested that Abca1a deficiency causes ovulation defects. In the ovary, ABCA1A was expressed in theca cells, an outermost layer of the ovarian follicle. Total cholesterol content of Abca1a -/- ovary was significantly higher than that of the wild-type, while estrogen and progestin contents were compatible with those of the wild-type. Furthermore, cholesterol loading to the wild-type follicles caused ovulation defects. These results suggest that ABCA1A in theca cells regulates cholesterol content in the ovarian follicles and its deficiency inhibits successful ovulation through cholesterol accumulation in the ovarian follicle.

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 in vitro 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, etc. 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.

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.

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. ATP binding, but not hydrolysis, induces a progressive network that results in a twisting motion of the whole protein, squeezing out the substrate directly to the extracellular space. This twist-and-squeeze mechanism by which ABCB1 exports hydrophobic substrates is distinct from those of other transporters.

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.

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.

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.

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. The roles of meflin in the development of heart disease, however, have not been investigated. OBJECTIVE: We examined the expression of meflin in the heart and its involvement in cardiac repair after ischemia, fibrosis, and the development of heart failure. METHODS AND RESULTS: We found that meflin has an inhibitory role in myofibroblast differentiation of cultured mesenchymal stromal/stem cells. Meflin expression was downregulated by stimulation with TGF (transforming growth factor)-ß, substrate stiffness, hypoxia, and aging. Histological analysis revealed that meflin-positive fibroblastic cells and their lineage cells proliferated in the hearts after acute myocardial infarction and pressure-overload heart failure mouse models. Analysis of meflin knockout mice revealed that meflin is essential for the increase in the number of cells that highly express type I collagen in the heart walls after myocardial infarction induction. When subjected to pressure overload by transverse aortic constriction, meflin knockout mice developed marked cardiac interstitial fibrosis with defective compensation mechanisms. Analysis with atomic force microscopy and hemodynamic catheterization revealed that meflin knockout mice developed stiff failing hearts with diastolic dysfunction. Mechanistically, we found that meflin interacts with bone morphogenetic protein 7, an antifibrotic cytokine that counteracts the action of TGF-ß and augments its intracellular signaling. CONCLUSIONS: These data suggested that meflin is involved in cardiac tissue repair after injury and has an inhibitory role in myofibroblast differentiation of cardiac fibroblastic cells and the development of cardiac fibrosis.

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 Raw264.7 cells. ABCG1 phosphorylation was enhanced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator. PKC activation by TPA increased ABCG1 protein levels and promoted ABCG1-dependent cholesterol efflux to HDL. This activity was suppressed by Go6976, a PKCα/ßI inhibitor, suggesting that PKC activation stabilizes ABCG1. To confirm this, the degradation rate of ABCG1 was analysed; ABCG1 degradation was suppressed upon PKC activation, suggesting that PKC phosphorylation regulates ABCG1 levels. To confirm this involvement, we co-expressed ABCG1 and a constitutively active form of PKCα in HEK cells. ABCG1 was increased upon co-expression. These results suggest that PKC-mediated phosphorylation, probably PKCα, stabilizes ABCG1, consequently increasing ABCG1-mediated cholesterol efflux, by suppressing ABCG1 degradation. PKC activation could thus be a therapeutic target to suppress the development of atherosclerosis.

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. Neither cholesterol content in cell membranes nor the amount of SLO bound to cells was affected by ABCA1. On the other hand, binding of the D4 domain of perfringolysin O (PFO) to ABCA1-expressing cells increased, suggesting that the amount of cholesterol in the outer leaflet of the plasma membrane (PM) increased and that the cholesterol dependences of these two toxins differ. Addition of cholesterol to the PM by the MßCD-cholesterol complex dramatically restored SLO pore formation in ABCA1-expressing cells. Therefore, exogenous expression of ABCA1 causes reduction in the cholesterol level in the inner leaflet, thereby suppressing SLO pore formation.

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.

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.

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. Furthermore, CAP is involved in the MSC differentiation in a stiffness-dependent manner, whereas vinexin depletion suppresses adipocyte differentiation independently of YAP/TAZ. These observations reveal a critical role of vinexin α and CAP in mechanotransduction and MSC differentiation.

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. By contrast, in the "indirect model", apoA-I acquires lipids from the specific membrane domains created by ABCA1. In this study, we found that trypsin treatment causes rapid release of phosphatidylcholine (PC) and cholesterol from BHK/ABCA1 cells, and that the time course of lipid release coincides with those of trypsin digestion of extracellular domains (ECDs) of surface ABCA1 and of release of ECD fragments into the medium. This trypsin-dependent lipid release was dependent on ABCA1 ATPase activity, and did not occur in cells that express ABCG1, which exports lipids like ABCA1 but does not have large ECDs. These results suggest that the trypsin-sensitive sites on the cell surface are the large ECDs of ABCA1, and that lipids transported by ABCA1 are temporarily sequestered within the ECDs during nascent HDL formation.

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.

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. We found that ERK activation by orbital shaking was biphasic. The early phase was independent of Ca2+, PI3-kinase, and Rho kinase but required RAF activity. The late phase was dependent on Ca2+ but not RAF. These results suggest that the superior time-resolving capability of the orbital shaking method to separate the previously unrecognized Ca2+-independent early phase of ERK activation from the late phase. We also found that a certain combination of serum starvation and medium renewal affected ERK activation by FSS, suggesting that a soluble factor(s) may be secreted during serum starvation, which modified the phosphorylation level of ERK. These findings revealed novel aspects of the osteoblastic mechanoresponses and indicated that the orbital shaking method would be a useful, complementary alternative to the parallel plate method for certain types of study on cellular mechanoresponses.

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. We focus on the effect of mutations in the human MYH9 gene that encodes NMIIA, which have been implicated in the pathogenesis of various diseases including nephritis. Our assay equipped with a high-throughput data analysis capability reveals that a point mutation of E1841K but not I1816V significantly reduces the magnitude of the endogenous forces of human embryonic kidney (HEK293) cells. Given the increasingly recognized roles of the endogenous forces as a critical mechano-regulator as well as that no apparent morphological changes were induced to cells even by introducing the mutations, our findings suggest a possibility that the detected reduction in the force magnitude at the individual cellular level may underlie the pathogenesis of the kidney disease.