Biology subjects

Yoshikawa, H.

Publications and source records attributed to Yoshikawa, H..

10 recordsLinked to original sources

bioRxiv2025

CaMKII-RNMT axis directs activity-dependent control of RNA dynamics in neurons

In neurons, mRNAs are transported into axons and dendrites for local translation at synapses. Diverse proteins support the transport of specific mRNAs, however, mechanisms co-ordinating mRNA translocation are unclear. We report that that CaMKII (Ca2+/calmodulin-dependent protein kinase II) is a key regulator of RNA dynamics through activity-dependent phosphorylation of the RNA cap guanine-7 methyltransferase, RNMT. Following RNA cap methylation in the nucleus, RNMT is retained on specific mRNAs. On stimulation, RNMT translocates into the cytoplasm, increasing levels of locally translated mRNAs. In the cytoplasm, CaMKII phosphorylates RNMT Thr317 on the active site, which inhibits methyltransferase activity and targets RNMT for degradation, thus limiting cytoplasmic RNMT function. RNMT mutants protected from CaMKII-dependent degradation increase locally translated, cytoplasmic mRNAs and accelerate neuronal morphogenesis. RNMT Thr317 phospho-mimic co-ordinately decreases locally translated mRNAs in the cytoplasm and reduces differentiation. The CaMKII-RNMT relationship links neuronal activity to the spatial regulation of gene expression in neurons. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/685591v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@b97c4borg.highwire.dtl.DTLVardef@18f38eaorg.highwire.dtl.DTLVardef@1e1bcb0org.highwire.dtl.DTLVardef@12ac7c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

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↗

bioRxiv2024

Early development of the mineralized external skeleton of the polyplacophoran mollusk, with insight into the evolutionary history of shell plates and spicules.

Recent molecular phylogenetic studies have raised two questions about the evolutionary history of the calcified exoskeleton of mollusks. The first question concerns the homology of the two types of skeleton; whether spicules and shell plates share an evolutionary origin. The second question is the homology of the shell plates between chitons and other mollusks, including gastropods and bivalves. To gain insight into these questions, we examined the early development of shell plates and spicules in chitons. We identified several developmental genes that are involved in both shell plates and spicules, suggesting that spicules and shell plates share a common evolutionary origin. We also found that subpopulations of the dorsal shell field (the ridge and the plate field) have specific gene expression profiles. The differential gene expression of the ridge and plate field is not identical to the profiles of the zones of the gastropod shell field. This observation may suggest an independent evolutionary origin of the shell plates in chitons and gastropods.

evolutionary biology↗

bioRxiv2024

Development of shell field populations in gastropods

The embryonic shell field of mollusks first appears during gastrulation of the dorsal ectoderm and subsequently develops into the shell-secreting mantle in adult animals. Although several lines of evidence have revealed that this shell field lineage is exclusively derived from the second quartet (2q) of the 16-cell embryos, it is generally believed that the establishment of the shell field fate would be accomplished only after receiving inductive signals from the invaginated endoderm. Despite being accepted as a comprehensive model for molluskan shell field specification, the validity of this induction hypothesis remains questionable owing to the lack of clear experimental evidence and contradictory results. Here, we attempted to re-investigate the inductive role of the endoderm in shell field fate establishment in the limpet Nipponacmea fuscoviridis by experimentally disrupting cell-cell contacts between cell lineages after the 16-cell stage. First, we characterized the shell field cell population by performing two-color in situ hybridization. We characterized at least three cell populations in the developing shell field. Using single-cell transcriptome analysis, we identified several specific effector genes for each population, as well as transcription factor genes. Differentiation of each shell field population was inspected in 2q blastomeres isolated from other cells of the 16-cell embryos. Despite the absence of any interlineage interactions (including ectoderm-endoderm contacts), the expression of marker genes for each shell field population was observed in the isolated 2q fragments. In addition, the expression of several shell field genes was detected in embryos in which cytokinesis was blocked at the 16-cell stage. We concluded that the early process of shell field differentiation in the 2q lineage occurs mostly independently of the interactions with other lineages.

developmental biology↗

bioRxiv2022

Optimized Workflow for Enrichment and Identification of Biotinylated Peptides using Tamavidin 2-REV for BioID and Cell Surface Proteomics

Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein-protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the low abundance of protein biotinylation in cells required large amounts of cellular proteins to detect enough biotinylated peptides. Here we optimized the workflow for efficient enrichment and identification of biotinylated peptides. The most efficient recovery was achieved by heat inactivation of trypsin, prewashing Tamavidin 2-REV beads, clean-up of biotin solution, mock elution, and the optimal temperature and salt concentration for elution. Using the optimized workflow, over 2-fold more biotinylated peptides were identified with higher purity from RAW264.7 macrophages expressing TurboID-fused STING. In addition, sequential digestion with Glu-C and trypsin led to the identification of biotinylation sites that were not identified by trypsin digestion alone. Furthermore, the combination of this workflow with TMT labeling enabled large-scale quantification of cell surface proteome changes upon EGF stimulation. This workflow would be useful not only for BioID and cell surface proteomics but also for various other applications based on protein biotinylation.

biochemistry↗

bioRxiv2021

A quantitative comparison of human embryonic and induced stem cell proteomes

Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling, thereby avoiding many of the ethical issues arising from the use of embryo-derived cells. However, despite clear similarities between the two cell types, it is likely they are not identical. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors. We find that while hESCs and hiPSCs express a near identical set of proteins, they show consistent quantitative differences in the expression levels of a wide subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs. The proteomic data show hiPSCs have significantly increased abundance of vital cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins, which correlated with phenotypic differences between hiPSCs and hESCs. Thus, higher levels of glutamine transporters correlated with increased glutamine uptake, while higher levels of proteins involved in lipid synthesis correlated with increased lipid droplet formation. Some of the biggest metabolic changes were seen in proteins involved in mitochondrial metabolism, with corresponding enhanced mitochondrial potential, shown experimentally using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including ECM components and growth factors, some with known tumorigenic properties, as well as proteins involved in the inhibition of the immune system. Our data indicate that reprogramming of human fibroblasts to iPSCs effectively restores protein expression in cell nuclei to a state comparable to hESCs, but does not similarly restore the profile of cytoplasmic and mitochondrial proteins, with consequences for cell phenotypes affecting growth and metabolism. The data improve understanding of the molecular differences between induced and embryonic stem cells, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.

biochemistry↗

bioRxiv2021

The Epiallelic Nature of Mouse rDNA

BackgroundRibosomal DNA (rDNA) displays substantial inter-individual genetic variation in human and mouse. A systematic analysis of how this variation impacts epigenetic states and expression of the rDNA has thus far not been performed. ResultsUsing a combination of long- and short-read sequencing, we establish that 45S rDNA units in the C57BL/6J mouse strain exist as distinct genetic haplotypes that influence the epigenetic state and transcriptional output of any given unit. DNA methylation dynamics at these haplotypes are dichotomous and life-stage specific: at one haplotype, the DNA methylation state is sensitive to the in utero environment, but refractory to post-weaning influences, whereas other haplotypes entropically gain DNA methylation during ageing only. On the other hand, individual rDNA units in human show limited evidence of genetic haplotypes, and hence little discernible correlation between genetic and epigenetic states. However, in both species, adjacent units show similar epigenetic profiles, and the overall epigenetic state at rDNA is strongly positively correlated with total rDNA copy number. Analysis of different mouse inbred strains reveals that in some strains, such as 129S1/SvImJ, rDNA copy number is only approximately 150 copies per diploid genome and DNA methylation levels are <5%. ConclusionsOur work demonstrates that rDNA-associated genetic variation has a considerable influence on rDNA epigenetic state and consequently rRNA expression outcomes. In the future, it will be important to consider the impact of inter-individual rDNA (epi)genetic variation on mammalian phenotypes and diseases.

genomics↗

bioRxiv2020

Erosion of human X chromosome inactivation alters proteome expression from genes across the X chromosome and all autosomes

X chromosome inactivation (XCI) is a dosage compensation mechanism in female mammals whereby genes from one X chromosome are repressed. Analysis of human induced pluripotent stem cell (iPSC) lines using proteomics, RNAseq and polysome profiling showed a major change in the proteome upon XCI erosion. This resulted in amplified RNA and protein expression from X-linked genes. However, increased protein expression was also detected from autosomal genes without a corresponding mRNA increase, altering the protein-RNA correlation between genes on the X chromosome and autosomes. Eroded iPSC lines display ~13% increase in cell protein content, along with increased expression of ribosomal proteins, ribosome biogenesis and translation factors. They also showed significantly increased levels of active polysomes within the eroded lines. We conclude that erosion of XCI causes a major remodelling of the proteome, with translational mechanisms affecting the expression of a much wider range of proteins and disease-linked loci than previously realised.

biochemistry↗

bioRxiv2019

Differences in splicing defects between the grey and white matter in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is a multi-system disorder caused by CTG repeats in the myotonic dystrophy protein kinase (DMPK) gene. This leads to sequestration of the splicing factor, muscleblind-like 2 (MBNL2), and aberrant splicing, mainly in the central nervous system. We investigated the splicing patterns of MBNL1/2 and genes controlled by MBNL2 in several regions of the brain and between the grey matter (GM) and white matter (WM) in DM1 patients using RT-PCR. Compared with the control, the percentage of spliced-in parameter (PSI) for most of the examined exons were significantly altered in most of the brain regions of DM1 patients, except for the cerebellum. The splicing of many genes was differently regulated between the GM and WM in both DM1 and control. The level of change in PSI between DM1 and control was higher in the GM than in the WM. The differences in alternative splicing between the GM and WM may be related to the effect of DM1 on the WM of the brain. We hypothesize that in DM1, aberrantly spliced isoforms in the neuronal cell body of the GM may not be transported to the axon. This might affect the WM as a consequence of Wallerian degeneration secondary to cell body damage. Our findings may have implications for analysis of the pathological mechanisms and exploring potential therapeutic targets.

molecular biology↗

bioRxiv2019

Characteristics of University Students Supported by Counseling Services: Analysis of psychological tests and pulse rate variability

ObjectiveThe number of students who use counseling services is increasing in universities. We clarified the characteristics of the students who access counseling services using both psychological tests and pulse rate variability (PRV).\n\nMethodsWe recruited the participants for this study from the students who had counseling sessions at Kanazawa University (Group S). As a control group, we also recruited students who had no experience in counseling sessions (Group H). We obtained health information from the database of annual health check-ups. Participants received the Wechsler Adult Intelligence Scale (WAIS) III, Autism-Spectrum Quotient (AQ), Sukemune-Hiew (S-H) Resilience Test, and State-Trait Anxiety Inventory-JYZ (STAI). We also studied the SF-12v2 Health Survey. We examined the spectral analyses of pulse rate variability (PRV) by accelerating plethysmography. We performed linear analysis of PRV for LF, HF, and LF/HF as indexes of autonomic nervous function. We also performed non-linear analysis of PRV for the largest Lyapunov exponent (LLE). We examined participants blood for autoantibodies against glutamate decarboxylase (GAD) 65.\n\nResultsA total of 105 students participated in this study. Group S had 37 participants (Male: 26, Female: 11) and Group H had 68 participants (Male: 27, Female 41). There were 12 males and two females who had been diagnosed with autism spectrum disorder (ASD) in Group S. Group S had 1) lower Working Memory Index (WMI) despite relatively high Full-Scale IQ, 2) higher AQ scores, 3) lower resilience scores, 4) higher anxiety scores, 5)lower QOL in social health, and 6) shifting of autonomic nervous balance toward higher sympathetic activity. There were only two participants who had the anti-GAD65 antibody in Group S, and one participant had the anti-GAD65 antibody in Group H.\n\nConclusionThe results of our study revealed valuable information about the students who seek mental support in our university. Our results provide information that can be used in interventions for the university students who need counseling services.

neuroscience↗