Biology subjects

Fujimoto, T.

Publications and source records attributed to Fujimoto, T..

15 recordsLinked to original sources

bioRxiv2026

Prior Bombyx mori latent virus exposure enhances subsequent BmNPV infection and suppresses antiviral responses in the silkworm, Bombyx mori

Bombyx mori latent virus (BmLV) is a persistent RNA virus frequently detected in silkworm-derived cell lines and has been reported to interact with B. mori nucleopolyhedrovirus (BmNPV). However, whether previous BmLV exposure alters subsequent BmNPV infection in silkworm larvae remains unclear. Here, we examined how BmLV exposure history affects subsequent BmNPV infection in cultured cells and fifth-instar B. mori larvae. Persistent BmLV infection increased BmNPV-mediated reporter protein expression and BmNPV genome abundance in cultured cells, whereas short-term BmLV exposure or simultaneous BmLV and BmNPV coinfection did not reproduce the increases observed in persistently BmLV-positive cultures. In silkworm larvae (n22 strain), 24 h BmLV pre-exposure before BmNPV challenge increased BmNPV-mediated reporter protein expression and viral genome accumulation compared with BmNPV infection alone. BmLV antigenome RNA was detected in multiple larval tissues after BmLV exposure, indicating BmLV replication during the pre-exposure period. Notably, this prior BmLV exposure was associated with altered antiviral responses upon subsequent BmNPV infection. Specifically, BmLV-pre-exposed larvae showed reduced induction of Dicer-2, R2D2, and stimulator of interferon genes (STING) in the fat body. These findings indicate that prior BmLV exposure enhances subsequent BmNPV infection and is associated with attenuated induction of host antiviral genes. This study expands our understanding of how sequential viral infections shape host-virus interactions and suggests a novel approach for increasing recombinant protein production in baculovirus-based expression systems.

microbiology↗

bioRxiv2026

High-fat diet-induced obesity differentially alters circadian gene expression across peripheral tissues

Circadian rhythms regulate diverse physiological processes, including metabolism, and their disruption has been implicated in metabolic disorders such as obesity. However, the tissue-specific effects of obesity on peripheral circadian clocks remain incompletely understood. Here, we investigated the impact of high-fat diet (HFD)-induced obesity on circadian gene expression in skeletal muscle, liver, and white adipose tissue (WAT). Mice were fed either a regular diet (RD) or HFD for 6 weeks, followed by tissue collection at 4-hour intervals over a 24-hour period. Under RD conditions, key circadian regulators and their downstream targets exhibited robust 24-hour oscillations across all tissues. In contrast, HFD feeding induced distinct, tissue-specific alterations. In the liver, Per2, Dbp, and Rev-erb showed phase-advanced expression patterns, whereas in WAT, rhythmic expression was markedly attenuated. Notably, skeletal muscle largely preserved circadian gene expression patterns, indicating relative resistance to HFD-induced circadian disruption. In addition, HFD feeding altered metabolic gene expression in adipose tissue, characterized by reduced Pgc1 expression and increased Leptin expression. Together, these findings demonstrate that HFD-induced obesity differentially disrupts peripheral circadian clocks in a tissue-specific manner and highlight skeletal muscle as a relatively resilient tissue. These results provide insight into how circadian dysregulation contributes to metabolic abnormalities in obesity.

physiology↗

bioRxiv2026

ATG deficiency impairs stationary-phase microlipophagy through acetic acid-induced clustering of Niemann-Pick type C proteins

While the role of autophagy-related (ATG) proteins in microautophagy remains unclear, their absence in budding yeast has been reported to impair stationary-phase microlipophagy. Here, we show that this defect in ATG-deficient (atg{Delta}) cells arises not from a direct requirement of ATG proteins for the execution of microlipophagy but from accumulation of acetic acid (AA) in the medium. High concentrations of AA in the medium of atg{Delta} cells trigger the clustering of Niemann-Pick type C (NPC) proteins, causing impairment of raft-like vacuolar microdomain formation and suppression of microlipophagy. Lowering extracellular AA rapidly dissolves NPC protein clusters, restores vacuolar microdomains, and rescues microlipophagy in atg{Delta} cells. Conversely, elevating AA concentrations in the medium of wild-type cells induces NPC protein clusters and microlipophagy defects. These findings demonstrate that stationary-phase microlipophagy can proceed independently of ATG proteins and that the defect in atg{Delta} cells can be rescued by normalizing extracellular AA levels.

cell biology↗

bioRxiv2024

Phase separation promotes Atg8 lipidation for autophagy progression

Upon starvation, the autophagy-initiating Atg1 complex undergoes phase separation to organize the pre-autophagosomal structure (PAS) in yeast, from which autophagosome formation is considered to proceed. However, the physiological roles of the PAS as a liquid droplet remain unclear. Here we show that core Atg proteins are recruited into early PAS droplets that are formed by phase separation of the Atg1 complex with different efficiencies in vitro. The Atg12-Atg5-Atg16 E3 ligase complex for Atg8 lipidation is the most efficiently condensed in the droplets via specific Atg12-Atg17 interaction, which is also important for the PAS targeting of the E3 complex in vivo. In vitro reconstitution experiments reveal that E3-enriched early PAS droplets promote Atg8 lipidation and incorporate Atg8-coated vesicles to the interior, thereby protecting them from Atg4-mediated delipidation. These data suggest that the PAS utilizes its liquid-like property to function as an efficient production site for lipidated Atg8 and pool membrane seeds to drive autophagosome formation.

cell biology↗

bioRxiv2024

Oxidized low-density lipoprotein potentiates angiotensin II-induced Gq activation through the AT1-LOX1 receptor complex: Implications for renal dysfunction

Chronic kidney disease (CKD) and atherosclerotic heart disease, frequently associated with dyslipidemia and hypertension, represent significant health concerns. We investigated the interplay among these conditions, focusing on the role of oxidized low-density lipoprotein (oxLDL) and angiotensin II (Ang II) in renal injury via G protein q subunit (Gq) signaling. We hypothesized that oxLDL enhances Ang II-induced Gq signaling via the AT1 (Ang II type 1 receptor)-LOX1 (lectin-like oxLDL receptor) complex. Based on CHO and renal cell model experiments, oxLDL alone did not activate Gq signaling. However, when combined with Ang II, it significantly potentiated Gq-mediated inositol phosphate 1 production and calcium influx in cells expressing both LOX-1 and AT1 but not in AT1-expressing cells. This suggests a critical synergistic interaction between oxLDL and Ang II in the AT1-LOX1 complex. Conformational studies using AT1 biosensors have indicated a unique receptor conformational change due to the oxLDL-Ang II combination. In vivo, wild-type mice fed a high-fat diet with Ang II infusion presented exacerbated renal dysfunction, whereas LOX-1 knockout mice did not, underscoring the pathophysiological relevance of the AT1-LOX1 interaction in renal damage. These findings highlight a novel mechanism of renal dysfunction in CKD driven by dyslipidemia and hypertension and suggest the therapeutic potential of AT1-LOX1 receptor complex in patients with these comorbidities.

molecular biology↗

bioRxiv2024

Exercising with virtual reality is potentially better for the working memory and positive mood than cycling alone

Although virtual reality (VR) exercise has attracted attention as a factor in exercise habituation due to its mood-enhancing effects, its impact on brain function remains unclear. This study, involving 23 healthy university students, used functional magnetic resonance imaging (fMRI) to explore how VR exercise affects working memory, a key executive function, and its underlying neural mechanisms. Our findings indicate that a 10-min VR exercise session improved mood (arousal and vitality level) and working memory task performance (3-back task) more effectively than exercise or rest alone. Furthermore, the results confirmed that increased vitality from exercise and VR exercise interventions was associated with improved 3-back task performance. However, specific brain regions contributing to this enhancement remain unidentified. These results highlight VR exercise as the optimal exercise program for enhancing working memory function by increasing vitality level. These insights underscore VRs potential as a novel exercise modality with benefits extending beyond exercise adherence to potentially preventing dementia and depression.

neuroscience↗

bioRxiv2024

Fetal Kidney Transplantation for In Utero Fetuses

Potter sequence, characterized by bilateral renal agenesis, oligohydramnios, and consequent pulmonary hypoplasia, presents a significant challenge in the management of affected neonates. Due to their prematurity and associated abdominal complications, these infants often fail to reach a stage where dialysis can be safely initiated and sustained, leading to an exceedingly high mortality rate. Therefore, there is hopeful anticipation that interventions serving as a bridge to achieve a state where dialysis can be safely performed will markedly improve life expectancy. We have developed a unique approach of "transplantation of fetal kidneys from a different species during the fetal period" as a bridge therapy until stable dialysis therapy can be implemented. This is a new concept of fetal therapy, targeting the fetus in utero and utilizing fetal kidneys of an appropriate size for transplantation. In this study, we first validated the approach using allogeneic transplantation. Fetal kidneys with bladders from GFP-expressing rats (gestational age 14.0-16.5 days) were transplanted subcutaneously into allogeneic rat fetuses in utero (gestational age 18.0-18.5 days) using a special needle transuterinally, and live pups were successfully obtained. The transplanted fetal kidneys with bladders were confirmed to have urine production capability. By periodic aspiration of the subcutaneous urinary cyst after birth, urine produced by the transplanted fetal kidney was successfully drained outside the body for an extended period (up to 150 days). Biochemical tests confirmed the solute removal capacity of the transplanted fetal kidney. Furthermore, despite allogeneic transplantation, long-term urine production was sustained without the use of immunosuppressants, confirming that organ transplantation into fetuses is associated with lower rejection compared to adult transplantation. Next, xenotransplantation was performed. When GFP-expressing mouse fetal kidneys (gestational age 13.0-13.5 days) were transplanted into rat fetuses in utero, maturation of renal tissue structures was confirmed even in the interspecies setting.

bioengineering↗

bioRxiv2024

Transposon fusion gave birth to Fem, Bombyx mori female determining gene

Fem is a W-linked piRNA precursor, and Fem piRNA is a master gene of female determination in Bombyx mori. Since Fem has significantly low similarity to any known sequences and the origin of Fem remains unclear, two hypotheses have been proposed for the origin of Fem. The first one is that Fem is an allele of Masc, while the second one is that Fem arose by transposition of Masc. To draw a solid conclusion, we determined the W chromosome sequences of B. mori and a closely relative bombycid species of Trilocha varians with a Fem-independent sex determination system. Comprehensive genome comparison led us to the "third" hypothesis: Fem is a chimeric sequence of transposons. Although we still cannot completely exclude the first hypothesis, at least a large portion of the parts other than the 44-bp Masc similarity region is derived from transposons, and even the 44-bp region could correspond to the boundary of the two transposons, gypsy and satellite DNA.

genetics↗

bioRxiv2023

Physicochemical properties of the vacuolar membrane and cellular factors determine formation of vacuolar invaginations

Vacuoles change their morphology in response to stress. In yeast exposed to chronically high temperatures, vacuolar membranes get deformed and invaginations are formed. We show that phase-separation of vacuolar membrane occurred after heat stress leading to the formation of the invagination. In addition, Hfl1, a vacuolar membrane-localized Atg8-binding protein, was found to suppress the excess vacuolar invaginations after heat stress. At that time, Hfl1 formed foci at the neck of the invaginations in wild-type cells, whereas it was efficiently degraded in the vacuole in the atg8{Delta} mutant. Genetic analysis showed that the endosomal sorting complex required for transport (ESCRT) machinery was necessary to form the invaginations irrespective of Atg8 or Hfl1. In contrast, a combined mutation with the vacuole BAR domain protein Ivy1 led to vacuoles in hfl1{Delta}ivy1{Delta} and atg8{Delta}ivy1{Delta} mutants having constitutively invaginated structures; moreover, these mutants showed stress-sensitive phenotypes. Our findings suggest that vacuolar invaginations result from the combination of changes in the physiochemical properties of the vacuolar membrane and other cellular factors. Summary statementVacuolar invaginations occur through a combination of the changes of physiochemical properties of the vacuolar membrane and other cellular factors in yeast. Particularly, Hfl1 suppresses the excess invaginations.

cell biology↗

bioRxiv2022

Novel phosphorylation of Rab29 that regulates its localization and lysosomal stress response in concert with LRRK2

Rab proteins are small GTPases that regulate a myriad of intracellular membrane trafficking events. Rab29 is one of the Rab proteins phosphorylated by leucine-rich repeat kinase 2 (LRRK2), a Parkinsons disease-associated kinase. Recent studies suggest that Rab29 regulates LRRK2, whereas the mechanism by which Rab29 is regulated remained unclear. Here we report a novel phosphorylation in Rab29 that is not mediated by LRRK2 and occurs under lysosomal overload stress. Mass spectrometry analysis identified the phosphorylation site of Rab29 as Ser185, and cellular expression studies of phosphomimetic mutants of Rab29 at Ser185 unveiled the involvement of this phosphorylation in counteracting lysosomal enlargement. PKC was deemed to be responsible for this phosphorylation and control the lysosomal localization of Rab29 in concert with LRRK2. These results implicate PKC in the lysosomal stress response pathway comprised of Rab29 and LRRK2, and further underscore the importance of this pathway in the mechanisms underlying lysosomal homeostasis.

molecular biology↗

bioRxiv2022

On-grid labeling method for freeze-fracture replicas

Sodium dodecyl sulfate-treated freeze-fracture replica labeling (SDS-FRL) is an electron microscopic (EM) method that can define the two-dimensional distribution of membrane proteins and lipids in a quantitative manner. Despite its unsurpassed merit, SDS-FRL has been adopted in a limited number of labs, probably because it requires a laborious labeling process as well as equipment and technique for freeze-fracture. Here, we present a method that reduces the manual labor significantly by mounting freeze-fracture replicas on EM grids prior to labeling. This was made possible by the discovery that freeze-fracture replicas invariably adhere to the carbon-coated formvar membrane with their platinum-carbon side, ensuring that the membrane molecules retained in replicas are accessible to labeling solutions. The replicas mounted on EM grids can be stored dry until labeling, checked by light microscopy before labeling, and labeled in the same manner as tissue sections. This on-grid method will make SDS-FRL easier to access for many researchers.

cell biology↗

bioRxiv2020

Phosphatidylserine prevents the generation of a protein-free giant plasma membrane domain in yeast

Membrane phase separation accompanied with micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large phase separation has not been reported in the plasma membrane of the living cells. We demonstrate here that a stable micron-scale protein-free region is generated in the plasma membrane of the yeast mutants lacking phosphatidylserine. We named this region the "void zone". Transmembrane proteins, peripheral membrane proteins, and certain phospholipids are excluded from the void zone. The void zone is rich in ergosterol and requires ergosterol and sphingolipids for its formation. These characteristics of the void zone are similar to the properties of the cholesterol-enriched domain in phase-separated artificial membranes. We propose that phosphatidylserine prevents the formation of the void zone by preferentially interacting with ergosterol. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site. Summary statementYeast cells lacking phosphatidylserine generate protein-free plasma membrane domains, and vacuoles contact with this domain. This is the first report of micron-scale plasma membrane domains in living cells.

cell biology↗

bioRxiv2019

ESCRT machinery mediates selective microautophagy of endoplasmic reticulum

ER-phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER-phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro-ER-phagy has been discovered, the molecules and mechanisms mediating micro-ER-phagy remain unknown. Here, we first show that micro-ER-phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1-Spo7 phosphatase complex and ESCRT proteins as key components for micro-ER-phagy. Third, we demonstrate that macro- and micro-ER-phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that ESCRT proteins directly function in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro-ER-phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis.

cell biology↗

bioRxiv2018

Mitochondrial Fusion Suppresses Pancreatic Cancer Growth via Reduced Oxidative Metabolism

Pancreatic cancer is a highly lethal disease whose aggressive biology that is driven by mitochondrial oxidative metabolism. Mitochondria normally form a network of fused organelles, but we find that patient-derived and genetically engineered murine pancreatic cancer cells exhibit highly fragmented mitochondria with robust oxygen consumption rates (OCR). When mitochondrial fusion was activated by the genetic or pharmacological inhibition Drp1, the morphology and metabolism of human and murine pancreatic cancer cells more closely resembled that of normal pancreatic epithelial cells. This reduced metabolism was correlated with slower tumor growth, fewer metastases, and enhanced survival in a syngeneic orthotopic model. Similarly, directly activating mitochondrial fusion by overexpression of Mfn2 also reduced tumor growth and metastases. Mitochondrial fusion in pancreatic cancer cells was associated with reduced mitochondrial mass and Complex I expression and function. Thus, these data suggest that enhancing mitochondrial fusion through Drp1 inhibition or enhanced Mfn2 expression or function has strong tumor suppressive activity against pancreatic cancer and may thus represent a highly novel and efficacious therapeutic target.

cancer biology↗

bioRxiv2017

Syntaxin 17 promotes lipid droplet formation by regulating the distribution of acyl-CoA synthetase 3

Lipid droplets (LDs) are ubiquitous organelles that contain neutral lipids and are surrounded by a phospholipid monolayer. How proteins specifically localize to the phospholipid monolayer of the LD surface has been a matter of extensive investigations. Here we show that syntaxin 17 participates in LD biogenesis by regulating the distribution of acyl-CoA synthetase 3 (ACSL3), a key enzyme for LD biogenesis that redistributes from the endoplasmic reticulum to LDs during LD formation. Time course experiments revealed that syntaxin 17 binds to ACSL3 in the initial stage of LD formation, and that ACSL3 is released as a consequence of competitive binding of SNAP23 to syntaxin 17 in the maturation stage. We propose a model in which ACSL3 redistributes from the endoplasmic reticulum to LDs through association with syntaxin 17 and SNAP23-mediated dissociation from syntaxin 17. We also provide evidence that lipid raft-like structures are important for LD formation and SNAREs-ACSL3 interactions.

cell biology↗