Biology subjects

Fujita, N.

Publications and source records attributed to Fujita, N..

16 recordsLinked to original sources

bioRxiv2026

Sarcoplasmic reticulum remodeling through autophagosome biogenesis during Drosophila metamorphosis

The sarcoplasmic reticulum (SR) is an elaborate membrane network and the primary intracellular Ca2+ store for muscle contraction. SR abundance, morphology, and spatial organization must be remodeled during muscle atrophy and hypertrophy, yet the mechanisms underlying this remodeling remain poorly understood. Autophagy has been implicated in SR homeostasis and may contribute to this process. We previously showed that a subset of Drosophila larval muscles remodels into adult dorsal internal oblique muscles through an autophagy-dependent process accompanied by extensive SR reorganization. Here, we show that autophagy is essential for maintaining SR abundance and architecture during muscle remodeling. Blocking autophagosome formation caused the accumulation of large aggregates of SR membranes, whereas blocking autophagosome-lysosome fusion led to the accumulation of autophagosomes with SR transmembrane proteins localized to their membranes. Moreover, disrupting LC3-interacting region (LIR)-dependent cargo recognition did not impair SR remodeling, arguing against a major contribution from selective autophagic degradation of the SR. Instead, our findings suggest that SR membrane components are incorporated into autophagosomal membranes during muscle remodeling. We propose that this membrane flow couples the removal of excess SR membrane to autophagosome biogenesis during atrophy, thereby facilitating large-scale SR reorganization during muscle remodeling.

cell biology↗

bioRxiv2026

Loss of starch synthase IIa alleviates the negative impact of high temperature on rice starch during grain filling

High temperatures during grain filling stage are becoming increasingly frequent, compromising both grain and eating quality and thereby driving demand for heat-resilient cultivars. Such conditions are known to reduce the expression of granule-bound starch synthase I (GBSSI) and starch branching enzyme IIb (BEIIb), which are involved in starch biosynthesis, resulting in a decrease in amylose content and an increase in long-chain amylopectin. Thus, the present study introduced functional mutation in starch synthase IIa (SSIIa) that increases the proportion of short amylopectin chains to genetically compensate for the high-temperature-induced increase in amylopectin long chain. Rice lines carrying the ss2a mutation were grown at two locations with cooler (Akita) and warmer (Okayama) temperatures. Their grain traits, starch structure, and eating quality were compared. The ss2a mutant lines showed an increased proportion of short amylopectin chains as well as an increased apparent amylose content. Furthermore, these alterations in starch structure varied with the grain-filling temperature of the cultivation sites, ultimately affected eating quality. These results suggest that enriching short amylopectin chain via the ss2a mutation can counteract the increase in long amylopectin chain caused by high temperatures during grain filling, thereby maintaining a desirable starch structure and eating quality.

plant biology↗

bioRxiv2025

Proteomic and in silico dissection of MetaAggregates in amyotrophic lateral sclerosis brains

RNA-binding proteins (RBPs), key translation regulators, are thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). The pathological entities associated with ALS are known as "MetaAggregates": heterogeneous coaggregates composed of amyloids, RBPs, and RNA G-quadruplexes (rG4s). In this study, to explore the molecular constituents of ALS-associated MetaAggregates, we developed a proteomic approach using a psoralen-conjugated RBP and crosslinked it with a biotinylated rG4 to enable the isolation of MetaAggregates from ALS brain extracts. Single-cell RNA-seq using in vitro ALS models identified ELAVL4 as a cytoplasmic RBP and revealed the enrichment of an IGFBP2-derived rG4 structure in ALS-specific neurons. Mass spectrometry and amyloidogenicity-based principal component analysis revealed 79 candidate proteins with roles in RNA processing, metabolism, trafficking, and stress responses. Docking simulations highlighted a subset of proteins with potential pro-aggregation characteristics, diverse cytosolic associations and functional links to RNA processing relevant to ALS. Through proteomic and in silico dissection of ALS-associated MetaAggregates, the findings of this study establish a conceptual framework for the exploration of unrecognized amyloidogenic drivers of neurodegeneration.

neuroscience↗

bioRxiv2025

Genotype-first approach revealed unrecognized breed-specific genetic diseases in dogs

Numerous dogs are affected by genetic diseases because of their unique breeding situation. The identification of pathogenic variants in dogs not only has veterinary benefits but also leads to the development of new human disease models. However, fewer pathogenic variants have been identified in dogs than in humans, and standard genetic approaches, such as large-scale case/control studies and family analyses, often fail to identify novel causative variants in dogs. To address this, we applied a genotype-first approach by developing a multiplex polymerase chain reaction (PCR)-based targeted sequencing method for dogs, analyzing 203 genes--most of which are related to human disease--from 6,333 dogs collected from a veterinary hospital, and characterizing pathogenic variant carriers. In total, 120 pathogenic variants in 83 genes were identified. Frequent pathogenic variants were enriched in specific dog breeds. Dogs homozygous for a pathogenic CHEK2 variant, frequently identified in French Bulldogs (5.74%, homozygous), showed a five-fold increased risk of cancer (P = 3.45 x 10-2). A pathogenic NOD2 variant was detected in half of the Italian Greyhounds. However, their medical records did not document the typical clinical symptoms observed in humans with NOD2 deficiency. Some pathogenic variants were present in three or fewer dogs, and 11.0% of these dogs exhibited symptoms potentially associated with gene defects. These rare variants could become common within a short period due to the canine breeding system to induce breed-specific genetic diseases. These findings suggest that the genotype-first approach successfully identified unrecognized breed-specific genetic diseases. Further characterization of dogs with pathogenic variants enable to define clinical diagnosis, providing an important spontaneous model for human genetic diseases.

genomics↗

bioRxiv2025

Linear ubiquitination triggers Amph-mediated T-tubule biogenesis

T-tubules are specialized invaginations of the plasma membrane essential for muscle contraction. While their physiological importance is well established, the mechanisms underlying T-tubule formation remain elusive. Here, we identify LUBEL/RNF31, a ubiquitin E3 ligase responsible for linear (M1-linked) ubiquitination, as a key regulator of T-tubule biogenesis through proximity proteomics and RNAi screening in Drosophila. Loss of LUBEL leads to the formation of Amphiphysin (Amph)-positive membrane sheets instead of tubular networks in muscle cells. Mechanistically, the ubiquitin ligase activity of LUBEL, and direct interaction with Amph, a BAR domain protein involved in membrane tubule extension, are crucial for proper T-tubule morphology. LUBEL and M1-linked ubiquitin chains assemble into condensates on membranes, facilitating Amph-mediated membrane tubulation. Notably, the Amph-LUBEL/RNF31 interaction is evolutionarily conserved across a broad range of species, underscoring a fundamental role for linear ubiquitination in membrane remodeling. Our findings uncover a previously unrecognized role for linear ubiquitination in membrane deformation driven by BAR domain proteins.

cell biology↗

bioRxiv2025

LM-GlycoRepo Version 1.0: A novel repository system for mouse tissue glycome mapping data

Lectin microarray (LMA) is a high-sensitive profiling method of protein glycosylation. The increasing use of this method in many studies has led to a growing demand for a repository system that meets the FAIR data principles (Findable, Accessible, Interoperable, and Reusable). Herein, we present a novel repository system, "LM-GlycoRepo," for lectin-based multimodal (LM) data, including LMA data, in accordance with the international guideline MIRAGE (Minimum Information Required for a Glycomics Experiment). As a first step in our efforts to provide a general repository for storing various types of LM data, LM-GlycoRepo Version 1.0 is specialized for mouse tissue glycome mapping data obtained using standardized laser microdissection (LMD)-assisted LMA procedures. This system allows users to deposit datasets containing LMD images, LMA data, and high-resolution lectin staining images as LM data. In addition, this repository adopted an "embargo" system that allows users to specify the release date of datasets, allowing compatibility with an article peer review system. Notably, after the release date, the deposited data were visualized using an existing web tool called LM-GlycomeAtlas. LM-GlycoRepo will evolve into a comprehensive tool for lectin-based multimodal data for various biospecimens, including human samples. LM-GlycoRepo is freely available at the GlyCosmos portal (https://lm-glycorepo.glycosmos.org/lm_glycorepo/).

bioinformatics↗

bioRxiv2024

Transcriptional Dynamics Uncover the Role of BNIP3 in Mitophagy during Muscle Remodeling in Drosophila

Differentiated muscle cells contain myofibrils and well-organized organelles, enabling powerful contractions. Muscle cell reorganization occurs in response to various physiological stimuli; however, the mechanisms behind this remodeling remain enigmatic due to the lack of a genetically trackable system. Previously, we reported that a subset of larval muscle cells is remodeled into adult abdominal muscle through an autophagy-dependent mechanism in Drosophila. To unveil the underlying mechanisms of this remodeling, we performed a comparative time-course RNA-seq analysis of isolated muscle cells with or without autophagy. It revealed both transcriptional dynamics independent of autophagy and highlighted the significance of BNIP3-mediated mitophagy in muscle remodeling. Mechanistically, we found that BNIP3 recruits autophagic machinery to mitochondria through its LC3-interacting (LIR) motif and minimal essential region (MER), which interact with Atg8a and Atg18a, respectively. Loss of BNIP3 leads to a substantial accumulation of larval mitochondria, ultimately impairing muscle remodeling. In summary, this study demonstrates that BNIP3-dependent mitophagy is critical for orchestrating the dynamic process of muscle remodeling. Impact statementA time-course RNA-seq analysis of muscle remodeling reveals transcriptional dynamics independent of autophagy and highlights the significance of BNIP3 in mitochondrial degradation in vivo.

cell biology↗

bioRxiv2024

Explainable AI-guided identification of a novel protein-RNA interactive frame for selective siRNA accumulation in plants

Small interference RNA (siRNA) selectively accumulates and acts in RNA interference (RNAi). Although the components involved in siRNA production have long been the focus of studies to elucidate RNAi processes, the mechanism(s) for selectivity of siRNA (or RNAi effectivity) remains unclear. In a novel approach, we developed a progressive deep learning (DL) framework integrating Transformer and convolutional neural networks to predict the sequences of selectively accumulated siRNAs across various land plant species. These approaches achieved high-accuracy prediction of selectively accumulated 21-nt siRNAs and further identified their key signals, which are positionally and linguistically flexible sequences surrounding the target siRNA. We experimentally validated the contribution of these flexible key signal sequences to siRNA accumulation selectivity using virus-induced gene silencing (VIGS) in Nicotiana benthamiana, and identified RNA-binding proteins that directly recognize the key signal sequences to act for selective siRNA accumulation. These insights provide a novel framework for investigating RNAi mechanisms in plants. One sentence summaryWe discovered a novel mechanism centered on RNA-protein interactions involving the selective accumulation of small-RNAs in plants by applying advanced deep learning frames.

plant biology↗

bioRxiv2024

Forelimb motor recovery by modulating extrinsic and intrinsic signaling as well as neuronal activity after the cervical spinal cord injury

Singular strategies for promoting axon regeneration and motor recovery after spinal cord injury (SCI) have been attempted with limited success. Here, we propose the combinatorial approach of deleting extrinsic and intrinsic factors paired with neural stimulation, will enhance adaptive axonal growth and motor recovery after SCI. We previously showed the deletion of RhoA and Pten in corticospinal neurons inhibits axon dieback and promotes axon sprouting after lumbar SCI. Here, we examined the effects of RhoA;Pten deletion coupled with neural stimulation after cervical SCI. This combinatorial approach promoted more boutons on injured corticospinal neurons in the spinal cord compared to sole RhoA;Pten deletion. Although RhoA;Pten deletion does not promote motor recovery in the forelimb after SCI, stimulating corticospinal neurons in those mice results in partial motor recovery. These results demonstrate that a combinatorial approach that pairs genetic modifications with neuronal stimulation can promote axon sprouting and motor recovery following SCI.

neuroscience↗

bioRxiv2024

PI(4,5)P2 role in Transverse-tubule membrane formation and muscle function

Transverse (T)-tubules - vast, tubulated domains of the muscle plasma membrane - are critical to maintain healthy skeletal and heart contractions. How the intricate T-tubule membranes are formed is not well understood, with challenges to systematically interrogate in muscle. We established the use of intact Drosophila larval body wall muscles as an ideal system to discover mechanisms that sculpt and maintain the T-tubule membrane network. A muscle-targeted genetic screen identified specific phosphoinositide lipid regulators necessary for T-tubule organization and muscle function. We show that a PI4KIII-Skittles/PIP5K pathway is needed for T-tubule localized PI(4)P to PI(4,5)P2 synthesis, T-tubule organization, calcium regulation, and muscle and heart rate functions. Muscles deficient for PI4KIII or Amphiphysin, the homolog of human BIN1, similarly exhibited specific loss of transversal T-tubule membranes and dyad junctions, yet retained longitudinal membranes and the associated dyads. Our results highlight the power of live muscle studies, uncovering distinct mechanisms and functions for sub-compartments of the T-tubule network relevant to human myopathy. SummaryT-tubules - vast, tubulated domains of the muscle plasma membrane - are critical to maintain skeletal and heart contractions. Fujita et al. establish genetic screens and assays in intact Drosophila muscles that uncover PI(4,5)P2 regulation critical for T-tubule maintenance and function. Key FindingsO_LIPI4KIII is required for muscle T-tubule formation and larval mobility. C_LIO_LIA PI4KIII-Sktl pathway promotes PI(4)P and PI(4,5)P2 function at T-tubules. C_LIO_LIPI4KIII is necessary for calcium dynamics and transversal but not longitudinal dyads. C_LIO_LIDisruption of PI(4,5)P2 function in fly heart leads to fragmented T-tubules and abnormal heart rate. C_LI

cell biology↗

bioRxiv2023

Rapid and dynamic evolution of a giant Y chromosome in Silene latifolia

To test hypotheses about the evolution of massive sex-linked regions in plants, we sequenced the genome of Silene latifolia, whose giant heteromorphic sex chromosomes were first discovered in 1923. It has long been known that the Y consists mainly of a male-specific region which does not recombine with the X in male meiosis, and that this region carries the primary sex-determining genes, and other genes contributing to male functioning. However, only with a whole Y chromosome assembly can the candidates be validated experimentally, as we describe. Our new results also illuminate the genomic changes as the ancestral chromosome evolved into the current XY pair, testing ideas about why large regions of sex-linkage evolve, and the mechanisms creating the present recombination pattern. One sentence summaryBased on the whole genome sequences of Silene latifolia, a model species for plant sex chromosome evolution, we describe discovery of genes underlying male-female flower differences, and relate the results to ideas about the evolution of the vast non-recombining regions of the Y chromosome.

genetics↗

bioRxiv2023

Fungal hacking of the plant sex-determination pathway via interference with AGL24 in Silene latifolia

Plants have evolved lineage-specific sex-determination systems that is determined not only by genetic factors, but also the surrounding environmental conditions, including interactions with pathogens. Silene latifolia is a model dioecious plant whose sexuality is genetically regulated by X/Y chromosomes; however, anther smut fungus mimics the plant Y chromosome and forcibly converts female plants to male. Here, transcriptome analyses of healthy or fungus-infected S. latifolia inflorescence meristems suggested that an orthologue of AGL24 (SlAGL24), a flowering activator, is a key factor in sex conversion via fungus infection. Overexpression of SlAGL24 in Arabidopsis thaliana suppressed stamen development, whereas knock-down of SlAGL24 in S. latifolia converted males into hermaphrodites. Furthermore, SlAGL24 expression affected sexual dimorphisms in S. latifolia. Our results propose an adaptive scenario wherein the anther smut fungus targets SlAGL24, as a master regulator connecting the fungal signal to sex determination, to confer male and potentially male-beneficial traits, effectively transmitting its teliospores.

plant biology↗

bioRxiv2022

Ongoing rapid evolution of a post-Y region revealed by chromosome-scale genome assembly of a hexaploid monoecious persimmon (Diospyros kaki)

Sex chromosomes have evolved independently in many plant lineages. They have often undergone rapid structural degeneration and extension of non-recombining regions, which is conventionally considered to be strongly associated with the expression of sexually dimorphic traits. In this study, we assembled a monoecious hexaploid persimmon (Diospyros kaki) in which the Y chromosome had lost its function in male determination. Comparative genomic analysis among D. kaki and its dioecious relatives revealed that the non-functional Y chromosome (Ym) via silencing of the sex-determining gene, OGI, arose approximately two million years ago. Comparative analyses of the whole X and Ym chromosomes suggested that the non-functional male-specific region of the Y-chromosome (MSY), or post-MSY, retained certain conserved characteristics of the original functional MSY. Specifically, comparison of the functional MSY in D. lotus and the non-functional post-MSY in D. kaki indicated that the post-MSY had been rapidly rearranged mainly via ongoing transposable element bursts, as well as in the functional MSY. These results suggest a novel interpretation that the rapid evolution of the post-MSY (and possibly also MSYs in dioecious Diospyros species) might reflect the ancestral genomic properties of these regions, rather than the evolution of male-determining functions and/or sexually dimorphic traits.

plant biology↗

bioRxiv2021

A Drosophila Toolkit for Imaging of HA-tagged Proteins Unveiled a Block in Autophagy Flux in the Last Instar Larval Fat Body

For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with POI harboring different tags are needed but generation of these strains is still labor-intensive work. To overcome this, we developed a versatile Drosophila toolkit with a genetically encoded single-chain variable fragment for the HA epitope tag: "HA Frankenbody". This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Autophagy was dispensable for the swelling of lysosomes, indicating that lysosomal activity is downregulated at this stage. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, we propose that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila.

cell biology↗

bioRxiv2020

An Autophagy-Dependent Tubular Lysosomal Network Synchronizes Degradative Activity Required for Muscle Remodeling

Previously, we reported that autophagy is critical for Drosophila muscle remodeling during metamorphosis (Fujita et al., 2017). However, little is known about how lysosomes meet increased degradative demand upon cellular remodeling. Here, we found an extensive tubular autolysosomal network in remodeling muscle. The tubular network transiently appeared and exhibited the capacity to degrade autophagic cargoes. The tubular autolysosomal network was uniquely marked by the autophagic SNARE protein, Syntaxin 17, and its formation depended on both autophagic flux and degradative function, with the exception of the Atg12 and Atg8 ubiquitin-like conjugation systems. Among ATG-deficient mutants, the efficiency of lysosomal tubulation correlated with the phenotypic severity in muscle remodeling. The lumen of the tubular network was continuous and homogeneous across a broad region of the remodeling muscle. Altogether, we revealed that the dynamic expansion of a tubular autolysosomal network synchronizes the abundant degradative activity required for developmentally regulated muscle remodeling. Impact StatementAnalysis of developmentally-regulated Drosophila muscle remodeling revealed autophagy-dependent formation of an extensive, Syntaxin 17-marked, tubular network that synchronizes the abundant degradative activity across a broad region of the remodeling muscle

cell biology↗

bioRxiv2019

Aberrant expression of the COX2/PGE2 axis is induced by activation of the RAF/MEK/ERK pathway in BRAFV595E canine urothelial carcinoma

Cancer-promoting inflammation is an important event in cancer development. The cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) axis is a prominent inducer of inflammation. Canine urothelial carcinoma (cUC) uniquely overexpresses PGE2 and is often managed well with COX inhibitor monotherapy. In most cases, cUC tissue harbours homologous human BRAFV600E mutation, which causes aberrant activation of the RAF/MEK/ERK pathway in human cancer. However, mechanisms underlying aberrant PGE2 production and the importance of the BRAF mutation remain unclear. We show that activation of the RAF/MEK/ERK pathway in BRAF mutant cUC cells leads to COX2 overexpression and PGE2 production. Drug screening revealed that treatment with inhibitors of the arachidonic acid cascade (FDR<0.086), RAF/MEK/ERK pathway (FDR<0.067), and p38/JNK pathway (FDR<0.067) significantly reduced PGE2 production in cUC cells. We also validated the association between RAF/MEK/ERK pathway activation and COX2/PGE2 overexpression in BRAF mutant cUC cells using protein detection techniques. In histochemical analysis, BRAF mutant cUC tissue showed higher COX2 expression. Therefore, the driver mutation in the BRAF gene probably promotes tumour-promoting inflammation. These findings would benefit dogs suffering from cUC and can be extrapolated to human cancer. Finally, cUC can serve as a valuable model to elucidate the association between driver mutations and tumour-promoting inflammation.

cancer biology↗