Biology subjects

Furukawa, K.

Publications and source records attributed to Furukawa, K..

13 recordsLinked to original sources

bioRxiv2025

Aβ-HMGB1 complex is a pathogenic molecule at the advanced stage of Alzheimer's disease

Multiple molecules including A{beta}, tau and other inflammatory molecules mediate Alzheimers disease (AD) pathology. High mobility group box 1 (HMGB1), which is released from necrotic cells and binds to Toll-like receptors (TLRs) of surrounding neurons and microglia, also mediates AD pathology from the early stage. Paradoxically, HMGB1 concentration in cerebrospinal fluid (CSF) at the advanced stage of AD is not higher than that at the early stage. Here we show that A{beta}-HMGB1 complexes are generated in neurons undergoing secondary necrosis around A{beta} plaques in the AD brain at the advanced stage. A{beta}-HMGB1 complex triggers neurite degeneration and necrosis of human normal iPSC-derived neurons via binding to TLR4. Further, two anti-HMGB1 antibodies that inhibit its interaction with TLR4 successfully suppress the toxicity of A{beta}-HMGB1 complex to human iPSC-derived neurons, and recover cognitive impairment and A{beta}-HMGB1 complex-related brain pathology in AD model mice, while such therapeutic effects were not obvious with an anti-A{beta} antibody (lecanemab) approved for human AD patients. Enzyme-Linked Immuno Sorbent Assay (ELISA) revealed plasma level of A{beta}-HMGB1 complex was increased in a part of AD patients at the advanced stage. These findings for the molecular basis of toxicity to neurons suggest the significance of A{beta}-HMGB1 complex at the advanced stage of AD pathology, and might explain the discrepancy between A{beta} burdens and clinical symptoms of human AD patients treated with anti-A{beta} antibody.

neuroscience↗

bioRxiv2025

Mitochondrial fission during mitophagy requires both inner and outer mitofissins

Mitophagy maintains mitochondrial homeostasis through selective degradation of damaged or excess mitochondria. Recently, we identified mitofissin/Atg44, a mitochondrial intermembrane space-resident fission factor, which directly acts on lipid membranes and drives mitochondrial fission required for mitophagy in yeast. However, it remains unclear whether mitofissin is sufficient for mitophagy-associated mitochondrial fission and whether other factors act from outside the mitochondria. Here, we identify a mitochondrial outer membrane-resident mitofissin-like microprotein required for mitophagy, and we name it mitofissin 2/Mfi2 based on the following results. Overexpression of a C-terminally truncated form of Mfi2 induces mitochondrial fragmentation and partially restores mitophagy in atg44{Delta} cells. Mfi2 binds to lipid membranes and mediates membrane fission in vitro, demonstrating its intrinsic mitofissin activity. Genetic analyses reveal that Mfi2 and the dynamin-related protein Dnm1 independently facilitate mitochondrial fission during mitophagy. Thus, Atg44 and Mfi2, two mitofissins with distinct localizations, are required for mitophagy-associated mitochondrial fission.

cell biology↗

bioRxiv2024

Extracellular vesicles adhere to cells predominantly through the interaction of CD151-associated integrin heterodimers and GM1 with laminin

Tumor-derived extracellular vesicles (EVs) have attracted significant attention, yet the molecular mechanisms that govern their specific binding to recipient cells remain elusive. Our in vitro study utilizing single-particle tracking demonstrated that integrin heterodimers comprising 6{beta}4 and 6{beta}1 and ganglioside, GM1 are responsible for the binding of small-EV (sEV) subtypes to laminin. EVs derived from four distinct tumor cell lines, regardless of size, exhibited high binding affinities for laminin but not for fibronectin, although fibronectin receptors are abundant in EVs and have functional roles in EV-secreting cells. Our findings revealed that integrins in EVs bind to laminin via the conventional molecular interface, facilitated by CD151 rather than by inside-out signaling of talin-1 and kindlin-2. Super-resolution movie observation revealed that sEV integrins bind only to laminin on living recipient cells. Furthermore, sEVs bound to HUVEC and induced cell branching morphogenesis in a laminin-dependent manner. Thus, we demonstrated that EVs predominantly bind to laminin on recipient cells, which is indispensable for cell responses. SummaryQuantitative assessments using single-molecule imaging and super-resolution microscopy revealed that all extracellular vesicle subtypes derived from four distinct tumor cell lines, regardless of size, bind to laminin predominantly via CD151-facilitated integrin heterodimers and GM1, leading to response of recipient cells.

cell biology↗

bioRxiv2024

Discovery of anti-SARS-CoV-2 S2 protein antibody CV804 with broad-spectrum reactivity with various beta coronaviruses and analysis of its pharmacological properties in vitro and in vivo

SARS-CoV-2 pandemic alerts us that spillovers of various animal coronaviruses to human in the future may bring us enormous damages. Thus, there is a significant need of antibody-based drugs to treat patients infected with previously unseen coronaviruses.CV804 against the S2 domain of the spike protein, which is less prone to mutations. CV804 shows not only broad cross-reactivities with representative 20 animal-origin coronaviruses but also with diseases-associated human beta coronaviruses including SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-HKU1 and mutant strains of SARS-CoV-2. Other than that, the main characteristics of CV804 are that it has strong antibody-dependent cellular cytotoxicity (ADCC) activity to SARS-CoV2 spike protein-expressed cells in vitro and completely lacks virus-neutralization activity. Comprehensively in animal models, CV804 suppressed disease progression by SARS-CoV-2 infection. Structural studies using HDX-MS and point mutations of recombinant spike proteins revealed that CV804 binds to a unique epitope within the highly conserved S2 domain of the spike proteins of various coronaviruses. Based on the overall data, we suggest that the non-neutralizing CV804 antibody recognizes the conformational structure of the spike protein expressed on the surface of the infected cells and weakens the viral virulence by supporting host immune cells attack through ADCC activity in vivo. CV804 epitope identified in this study is not only useful for the design of pan-corona antibody therapeutics but also to design next-generation coronavirus vaccines and antiviral drugs.

microbiology↗

bioRxiv2023

Ras Activation by Hydrostatic Pressure is Enhanced by GAP and GEF in vitro

Hydrostatic pressure (HP) is a necessary stimulus for cell differentiation and growth in cultured chondrocytes. Assuming that Ras activation is involved in HP-induced reactions, if cellular Ras activity is increased by HP, Ras itself, Raf, Ras regulators including GTPase activating proteins (GAP) and guanine nucleotide exchange factors (GEF), then the upstream receptor and/or interactions between them should have HP sensitivity. Based on this hypothesis, we first attempted to examine whether Ras is activated by HP, and in the affirmative, identify which factors displayed HP sensitivity using an in vitro system to measure Ras activity. This in vitro system included mRaichu, a FRET-based Ras activity probe, to which two-point mutations were introduced to reduce Ras-independent signals. This improved Raichu was used to investigate the HP sensitivity of two components, the GAP domain (GAPd) derived from p120GAP and the GEF domain (GEFd) derived from hSOS-1. It was found that HP weakly activated Ras activity in the absence of GAPd and GEFd, presumably by facilitating GDP dissociation from Ras. A low concentration of GAPd enhanced HP-induced Ras activation by 16.3% whereas high concentrations of GAPd removed HP sensitivity, suggesting that HP partially dissociates GAPd from the GAPd-Ras-GDP complex and reduces the fraction of inactive Ras. Moreover, a broad concentration range of GEFd also enhanced HP-induced Ras activation. Given that HP also increased Ras activity under a condition mimicking cellular Ras activity, we propose that Ras activation is involved in the differentiation and growth stimulation of chondrocytes by HP. Statement of SignificanceThe Ras-cycle has been implicated in the regulation of growth and differentiation of eukaryotic cells. Here, we investigated the relationship between hydrostatic pressure (HP) and components of the Ras-cycle: Ras-Raf, the GAP domain, and the GEF domain. Generally, HP tends to weaken protein-protein and protein-ligand interactions, but in this study, a seemingly positive response was observed: HP-induced Ras activation. Additionally, this response was enhanced by the GEF domain and the GAP domain. Thus, given the multiple cellular functions of Ras and the responses of the Ras-cycle to HP, this study will help clarify the molecular mechanism by which HP modulates cellular functions, particularly in chondrocytes, which are subjected to repetitive HP stimuli in vivo.

biophysics↗

bioRxiv2023

The lipid Gb3 promotes germinal center B cell responses and anti-viral immunity

Influenza viruses escape immunity due to rapid antigenic evolution, which requires vaccination strategies that allow for broadly protective antibody responses. Here, we demonstrate that the lipid globotriaosylceramide (Gb3) expressed on germinal center (GC) B cells is essential for the production of high-affinity antibodies. Mechanistically, Gb3 binds and disengages CD19 from its chaperone CD81 for subsequent translocation to the B cell receptor (BCR) complex to trigger signaling. Abundance of Gb3 amplifies the PI3-kinase/Akt/Foxo1 pathway to drive affinity maturation. Moreover, this lipid regulates MHC-II expression to increase diversity of T follicular helper (Tfh) and GC B cells reactive with subdominant epitopes. In influenza infection, Gb3 promotes broadly reactive antibody responses and cross-protection. Thus, we show that Gb3 determines affinity as well as breadth in B cell immunity and propose this lipid as novel vaccine adjuvant against viral infection. One Sentence SummaryGb3 abundance on GC B cells selects antibodies with high affinity and broad epitope reactivities, which are cross-protective against heterologous influenza infection.

immunology↗

bioRxiv2023

Timing Control of Purkinje Cell Outputs by Dual cAMP Actions on Axonal Action Potential and Transmitter Release

All-or-none digital signaling based on high-fidelity action potentials (APs) in neuronal axons is pivotal for the temporally precise sending of identical outputs rapidly to widespread multiple target cells. However, technical limitation to directly measure the signaling in small size of intact axonal structures has hindered the evaluation of high-fidelity signal propagation. Here, using direct recordings from axonal trunks and/or terminals of cerebellar Purkinje cells in culture and slice, we demonstrate that the timing of axonal output is delayed by the second messenger cAMP without clear changes of transmission efficacy. Slowed axonal signaling upon cAMP increase was ascribed to negative control of axonal Na+ channels, leading to smaller and hence slower conduction of APs specifically at an axon. On the other hand, a facilitatory effect of cAMP on presynaptic transmitter release, which generally operates at various CNS synapses, was also evident as augmented release probability in Purkinje cell axon terminals, compensating for weakening of release by the reduction of Ca2+ influx upon smaller AP. Taken these results together, our tour-de-force functional dissection of inhibitory axonal signaling unveiled a dynamic control of synaptic output timing by cAMP keeping output strength constant.

neuroscience↗

bioRxiv2023

AP2A1 is upregulated upon replicative senescence of human fibroblasts to strengthen focal adhesions via integrin β1 translocation along stress fibers

Aging proceeds with accumulation of senescent cells in multiple organs. Senescent cells become large in size compared to young cells, which promotes further senescence and age-related diseases. Currently, the molecular mechanism behind the maintenance of such huge cell architecture undergoing senescence remains poorly understood. Here we focus on reorganization of actin stress fibers induced upon replicative senescence of human fibroblasts, typically used as a senescent cell model. We identified, together with our previous proteomic study, that AP2A1 (alpha 1 adaptin subunit of the adaptor protein 2) is upregulated in senescent cells along the length of stress fibers, which are enlarged following the increase in the whole cell size. We then revealed that knockdown of AP2A1 in senescent cells suppresses key senescence-associated phenotypes, which include decreased cell area and lowered expression of major senescence markers. Meanwhile, AP2A1 overexpression in young cells induced the opposite effects that rather advance senescence, suggesting that AP2A1 may be used as a senescence marker. We found that AP2A1 is colocalized with integrin {beta}1, and both of them move linearly along stress fibers. We further observed that focal adhesions are enlarged in senescent cells to reinforce cell adhesions to the substrate. These results suggest that senescent cells maintain their large size by strengthening the anchorage to the substrate by supplying integrin {beta}1 via translocation along stress fibers. This mechanism may work efficiently in senescent cells, compared with a case relying on random diffusion of integrin {beta}1, given the enlarged cell size and resulting increase in travel time and distance for endocytosed vesicle transportation.

cell biology↗

bioRxiv2023

A stress sensor IRE1α is required for bacterial exotoxin-induced inflammasome activation in tissue-resident macrophages

Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1{beta} (IL-1{beta}), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarified the roles of IRE1, an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1{beta} production from RPMs. In RPMs, CTB is incorporated into ER and induced ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1-deficient RPMs showed a significant impairment of CT- or CTB-induced IL-1{beta} production, indicating that IRE1 was required for CT- or CTB-induced IL-1{beta} production from RPMs. This study first demonstrates the critical roles of IRE1 in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages. One sentence summaryIRE1 is required for NLRP3 and pyrin-mediated IL-1{beta} production

immunology↗

bioRxiv2022

A model explaining environmental stiffness-dependent migration of fibroblasts with a focus on maturation of intracellular structures

Cell migration is fundamental to many biological processes, while it remains elusive how cells modulate their migration upon different environmental stiffness. In this work, we focus on the structural maturity of actin stress fibers to explain the substrate stiffness-dependent emergence of different cell migration velocity. We demonstrate that fibroblasts migrate longer distances on softer elastic substrates, and the distance is increased by lowering the myosin-driven contractile force. Stress fibers, the major intracellular structure to generate and sustain contractile forces, were found to be less mature in structure on soft substrate than on stiff substrate. Based on these experimental results, we present a minimal mathematical model to capture the salient features of how the substrate stiffness alters the migration velocity. Specifically, the ability of cells to generate large contractile forces is limited on soft substrate according to the Hookes law. The inverse relationship between the cellular force and migration velocity is described by the Hills muscle equation. These mathematical descriptions suggest that the migration velocity is raised on softer substrate where cells exert a lower magnitude of contractile forces. Cells undergoing faster movement make stress fibers less mature in structure as mathematically described by the maturation model, thereby limiting the ability to sustain the force and in turn allowing for consistent increase in cell migration velocity on soft substrate again according to the Hookes law and Hills muscle equation, respectively. Thus, our model, reproducing the basic trend of the experimental results, provides insights into the mechanisms of environmental cue-dependent migratory behavior of cells.

biophysics↗

bioRxiv2022

Novel monoclonal antibodies showing broad neutralizing activity for SARS-CoV-2 variants including Omicrons BA.5 and BA.2.75

We identified novel neutralizing monoclonal antibodies against SARS-CoV-2 variants (including Omicron) from individuals received two doses of mRNA vaccination after they had been infected with wildtype. We named them MO1, MO2 and MO3. MO1 shows high neutralizing activity against authentic variants: D614G, Delta, BA.1, BA.1.1, BA.2, and BA.2.75 and BA.5. Our findings confirm that the wildtype-derived vaccination can induce neutralizing antibodies that recognize the epitopes conserved among the SARS-CoV-2 variants (including BA.5 and BA.2.75). The monoclonal antibodies obtained herein could serve as novel prophylaxis and therapeutics against not only current SARS-CoV-2 viruses but also future variants that may arise.

microbiology↗

bioRxiv2022

Nardilysin-Regulated Scission Mechanism Activates Polo-like Kinase 3 to Suppress the Development of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) develops through step-wise genetic and molecular alterations including Kras mutation and inactivation of apoptotic pathways. Here, we find that development of anoikis resistance and metastasis of KrasG12D-driven PDAC in mice is accelerated by deleting Plk3, explaining the often reduced Plk3 expression in human PDAC. Importantly, a 41 kDa Plk3 (p41Plk3) that contained the entire kinase domain at the N-terminus (1-353 aa) is activated by scission of the precursor p72Plk3 at Arg354 by metalloendopeptidase Nardilysin (NRDC), and the resulting p32Plk3 C-terminal Polo-box domain (PBD) was quickly removed by proteasome degradation preventing the p41Plk3 inhibition by PBD. We found that p41Plk3 is the activated form of Plk3 that regulates a feedforward mechanism to promote anoikis and suppress PDAC and metastasis. p41Plk3 phosphorylates c-Fos on Thr164, which in turn, induces expression of Plk3 and pro-apoptotic genes. These findings uncovered an NRDC-regulated post-translational mechanism (PTM) that activates Plk3, establishing a prototypic regulation by scission mechanism.

cancer biology↗

bioRxiv2020

Phosphorylation and dephosphorylation of Ser852 and Ser889 control clustering, localization, and function of PAR-3

Cell polarity is essential for various asymmetric cellular events, where the partitioning defective (PAR) protein, PAR3, plays a unique role as a cellular landmark to establish polarity. In epithelial cells, PAR3 localizes at the subapical border such as the tight junction in vertebrates and functions as an apical determinant. Although there is much information about the regulators of PAR3 localization, the mechanism involved in PAR3 concentration and localization to the specific membrane domain remains an important question to be clarified. In this study, we demonstrate that ASPP2, a stimulator of PAR3 localization, can link PAR3 and protein phosphatase 1 (PP1). The ASPP2-PP1 complex dephosphorylates a novel phosphorylation site, Ser852, of PAR3. Furthermore, Ser852- or Ser889-unphosphorylatable PAR3 mutants form protein clusters and ectopically localize to the lateral membrane. Concomitance of clustering and ectopic localization suggests that PAR3 localization is a consequence of local clustering. We also demonstrate that unphosphorylatable forms of PAR3 are static in molecular turnover and fail to coordinate rapid reconstruction of the tight junction, supporting that both phosphorylated and dephosphorylated states are essential for the functional integrity of PAR3. Summary statementWe show that phosphorylation and dephosphorylation regulate clustering of PAR-3, a cell polarity-regulating factor, and how the clustering regulation affects localization of PAR-3 and cell-cell junction formation.

cell biology↗