Biology subjects

Kimura, H.

Publications and source records attributed to Kimura, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Nuclear wrinkles result from geometric adaptations to cellular and nuclear morphological changes in epithelial cells

Mechanical cues influence cell behavior and fate and are frequently accompanied by changes in nuclear shape; however, how epithelial nuclei accommodate such deformations remains incompletely understood. Here, we investigated the formation and regulation of nuclear wrinkles (NWs), inward folds of the nuclear envelope, in human epithelial cells. Using quantitative confocal imaging in 2.5D spheroid cultures and controlled 2D monolayers, we found that NWs formed frequently in MCF10A cells but rarely in hTERT-RPE1 cells, indicating pronounced cell-type specificity. NW frequency increased with cell density and was tightly associated with coordinated geometric changes consistent with nuclear rounding. Disruption of F-actin organization, but not microtubules, robustly induced NW formation, and acute cell rounding triggered by trypsinization was sufficient to induce widespread wrinkling across multiple cell types. Live-cell imaging revealed that NWs are dynamic and reversible at low cell density but become stabilized under sustained confinement. NW formation occurred without detectable nuclear envelope rupture, DNA damage, or stress-associated histone phosphorylation. Quantitative analysis supports a passive geometric model in which redistribution of excess nuclear surface area accommodates nuclear shape remodeling, allowing epithelial nuclei to buffer mechanical constraints while preserving nuclear integrity.

cell biology↗

bioRxiv2026

Dopamine vesicles are specified by mechanisms overriding canonical synaptic vesicle size constraints

Dopaminergic neurons contain distinct synaptic vesicle (SV) populations that segregate dopamine from glutamate, but the molecular basis of this segregation remains unclear. Using an established in vitro SV reconstitution system in fibroblasts, we show that synaptogyrin family proteins--synaptogyrin 1, synaptogyrin 2 and synaptogyrin 3--but not synaptophysin, selectively associate with VMAT2-positive dopamine vesicles in the presence of synapsin. Despite the known role of synaptophysin and synaptogyrin family proteins in generating small SVs (40-50 nm), VMAT2 vesicles retain a larger size (50-80 nm) when co-expressed with synaptogyrins, while remaining segregated from synaptophysin-containing vesicles. SV2 family proteins (SV2A, SV2B, and SV2C) localize to both VMAT2- and synaptophysin-positive vesicle populations, with SV2C preferentially enriched on VMAT2-positive dopamine vesicles. Consistent with the selective dystrophy of SV2C/VMAT2/DAT-positive dopaminergic terminals in parkinsonism-associated SJ1RQKI mice, iPSC-derived dopaminergic neurons carrying the same mutation accumulate small and enlarged SV-like organelles within autophagosomes, indicating defective SV clearance. Together, our findings suggest that dopamine vesicles are regulated by distinct trafficking mechanisms that override canonical SV size control, providing insight into the selective vulnerability of dopaminergic terminals in Parkinsons disease.

neuroscience↗

bioRxiv2026

Minute-scale coupling of chromatin marks and transcriptional bursts

Histone modifications are often described as stable epigenetic marks that contribute to maintaining gene-expression programs during development and environmental responses 1-5. However, transcription of many genes is intermittent, switching between transcriptionally active and inactive episodes within minutes 6-10. Whether chromatin marks around individual genes change on these rapid timescales remains unclear. Here we show that local chromatin modification signals around endogenous genes in mouse embryonic stem cells fluctuate reversibly with transcriptional state, using live imaging of individual genes together with fluorescent probes that report histone modifications11-16. Activation-associated acetylation and methylation marks increased in association with transcriptional activation and decreased with inactivation, whereas a Polycomb-associated repressive mark behaved oppositely. Transcriptional coactivators and both histone acetyltransferase and deacetylase complexes were enriched during transcriptionally active state, consistent with opposing enzymatic activities shaping local acetylation levels 17,18. Inhibiting histone deacetylases altered the durations of active and inactive events, supporting a role for deacetylation in regulating transcriptional state transitions. Thus, histone modifications undergo reversible, minute-scale changes coupled to transcriptional activity. This framework helps explain how stochastic transcriptional bursts can occur with stable gene regulation over longer timescales.

molecular biology↗

bioRxiv2025

Proximity of pre-mRNA 3' end processing and transcription termination predicts enhanced gene expression

Two key factors required for pre-mRNA 3 end cleavage and polyadenylation, CPSF73 and PCF11, exhibit oncogenic properties: their elevated expression is associated with poor cancer patient prognosis. However, both proteins are also transcription termination factors, and it is unclear which of the processes they promote might contribute to carcinogenesis. Here, we employ a cellular model of colorectal cancer (CRC) progression and find that cells from primary tumor are addicted to high levels of CPSF73 and PCF11, while metastatic cells become less sensitive to their levels. We find no association between alternative polyadenylation (APA) and cell dependence on CPSF73 and PCF11, and no impact of their downregulation on transcription-replication collisions. Instead, we uncover an uncoupling of changes in 3 cleavage and termination during CRC progression: primary tumor-derived cells display a global shift to more proximal termination, yet a tendency for distal APA, compared to normal cells. Metastatic cells display partial reversion toward termination patterns observed in normal cells, and opposite tendency favoring proximal APA. This prompts us to measure the distance between the site of 3 cleavage and transcription termination for active protein-coding genes and find it almost halved in cells from primary tumor compared to normal cells. Interestingly, this distance becomes critically short for oncogenes. Closer proximity of 3 cleavage to termination correlates with higher gene expression, both across genes within a cell line and when distance and expression change in parallel. This uncovers a new relationship of transcription termination with gene expression regulation. SIGNIFICANCE STATEMENTAt the 3 end of genes two processes occur, which are often treated as one: pre-mRNA 3 processing and transcription termination. In fact, they are promoted by a common set of protein factors, with oncogenic properties. Here, we employ a cellular model of colorectal cancer (CRC) progression, to study changes in both pre-mRNA 3 processing and transcription termination. Unexpectedly, we uncover that those changes occur in opposite directions: mRNA tends to be lengthened, while termination is accelerated in cells from primary tumors. We show that the cleavage-termination distance is strongly reduced in these cells compared to normal and metastatic cells, and further determine that cleavage-termination proximity correlates with enhanced gene expression. This uncovers a new layer of gene expression regulation.

molecular biology↗

bioRxiv2025

L-Phenylalanine restriction amplifies boron neutron capture therapy efficacy through increased L-boronophenylalanine uptake and induces activating transcription factor 4 stress response in tumor cell lines

Boron neutron capture therapy (BNCT) relies on the selective uptake of boron-10 compounds by tumor cells. L-boronophenylalanine (BPA) serves as a key carrier, and enhancing its accumulation is critical for improving BNCT efficacy. In this study, we investigated the effects of L-phenylalanine (Phe) restriction on the tumor cell lines SAS, U87-MG, PANC-1, and A375, and the immortalized keratinocyte line HaCaT on modulating BPA uptake and associated cellular responses. Quantitative analysis using inductively coupled plasma atomic emission spectroscopy (ICP-AES) showed that 24 h Phe restriction increased BPA uptake in the SAS, U87-MG, and PANC-1 cell lines. Colony formation assays confirmed enhanced sensitivity to neutron irradiation in these cells. RNA sequencing indicated that Phe restriction activated the integrated stress response downstream of activating transcription factor 4 (ATF4), although this pathway was not directly linked to increased BPA uptake. The L-type amino acid transporter 1 (LAT1)/4F2 heavy chain (4F2HC) complex was identified as the exclusive transport route for BPA. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that Phe restriction altered intracellular levels of amino acids that serve as LAT1/4F2HC exchange substrates, suggesting a metabolic basis for enhanced BPA transport. Our results reveal that Phe restriction enhances BPA uptake and BNCT efficacy in a cell line-dependent manner, likely through the modulation of amino-acid metabolism. Therefore, targeted amino-acid manipulation prior to BNCT may represent a promising strategy to improve therapeutic outcomes.

cancer biology↗

bioRxiv2025

Fragile nucleosomes are essential for RNA Polymerase II to transcribe in eukaryotes

Nucleosomes are barriers to RNA Polymerase II (Pol II) transcribing along gene bodies in eukaryotes. We found that a fragile "tailless nucleosome" could be generated to resolve the Pol II pausing caused by the +1 nucleosome in metazoans. We hypothesized that "pan-acetylated nucleosomes" along gene bodies are fragile and are generated to resolve the resistance of nucleosomes to the elongation of Pol II. To confirm this hypothesis, we systematically analyzed pan-acetylation and pan-phosphorylation of the entire human genome in HEK293T cells. Herein, we show that both pan-acetylation and pan-phosphorylation of histones in nucleosomes along gene bodies are coupled with the activities of Pol II. Our results show that there are four major types of fragile nucleosomes required for Pol II to transcribe along gene bodies: nucleosomes without tails, nucleosomes with dominant acetylation, nucleosomes with dominant phosphorylation, and nucleosomes with both acetylation and phosphorylation.

genetics↗

bioRxiv2025

Breast cancer identity is defined by specialized enhancer sets via lysine deacetylation

Breast cancer subtypes are defined by distinct transcriptional programs, yet the epigenetic mechanisms underlying subtype-specific gene regulation remain unclear. Enhancers, key regulators of gene expression and cell identity, are well positioned to define breast cancer subtypes. Here, we identify a previously unrecognized class of enhancers, termed hypoacetylation-defined (HD) enhancers, that regulate cancer-related genes in a luminal breast cancer cell line. HD enhancers are defined by RNA polymerase II dissociation upon lysine deacetylase inhibition, and bidirectional eRNA transcription. They are distinct from super-enhancers, require a specific Mediator subunit for gene-specific transcription, and form extensive chromatin interactions suggestive of a hub-like architecture. Analyses of clinical datasets further identified a subset of HD enhancers, termed HD cluster 1 enhancers, which classify patients into breast cancer subtypes and are associated with expression quantitative trait loci linked to subtype-specific gene expression. This study identifies the lysine deacetylation-regulated cell identity enhancers, which are potential therapeutic targets.

cancer biology↗

bioRxiv2025

ISRIB promotes aggregation of TIA-1 by modulating its phase separation in vitro

Stress granules (SGs) are non-membranous biomolecular condensates formed through liquid-liquid phase separation (LLPS) of RNA-binding proteins (RBPs) and RNA under stress conditions. T-cell intracellular antigen-1 (TIA-1), a major RBP of SGs, can undergo LLPS via its low-complexity domain, contributing to SG nucleation. Integrated stress response inhibitor (ISRIB), a small molecule that enhances eIF2B activity and inhibits the integrated stress response, has been widely studied for its therapeutic potential in neurodegenerative diseases. However, little is known about how ISRIB directly affects the behavior of SG proteins. Here, we show that ISRIB enhances TIA-1 phase separation and promotes its aggregation in vitro. Interestingly, this effect was mitigated in the presence of RNA or cell lysates, suggesting that RNA-binding plays a protective role against ISRIB-induced aggregation. These findings imply that ISRIB alters the physical properties of SG in an RNA-dependent manner, raising important considerations for their therapeutic application. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/663586v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@7e281org.highwire.dtl.DTLVardef@1a7863eorg.highwire.dtl.DTLVardef@15e89b6org.highwire.dtl.DTLVardef@1a33ca4_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Human fibrosarcoma cells selected for very-high doxorubicin resistance, acquire trabectedin and eribulin cross-resistance, remain sensitive to recombinant methioninase, and have increased c-MYC expression

Doxorubicin is first-line chemotherapy for soft tissue sarcoma; however, the development of drug resistance limits its efficacy. The purpose of the present study was to select very-high doxorubicin-resistant (VHDR) HT1080 fibrosarcoma cells, determine cross-resistance to second-line chemotherapy drugs, determine maintenance of sensitivity to recombinant methioninase (r-METase) alone and in combination with doxorubicin, and measure the level of c-MYC expression. VHDR-HT1080 cells were generated by cultivating HT-1080 cells in a series of step-wise progressively higher concentrations of doxorubicin, ranging from 8 nM to 15 {micro}M, an 1875-fold increase, over a five-month period. The WST-8 reagent was used to assess cell viability. Four groups of in vitro drug-sensitivity tests were conducted, which involved both parental HT1080 and VHDR-HT1080 cells: 1) doxorubicin alone; 2) rMETase alone; 3) a combination of doxorubicin and rMETase; and 4) untreated control. The cross-resistance of VHDR-HT1080 cells to eribulin, trabectedin, gemcitabine and docetaxel was determined. The c-MYC levels in HT1080 and VHDR-HT1080 cells were measured using Western blotting. Doxorubicin had an IC50 of 3.3 {micro}M against HT1080 cells and 38.2 {micro}M against VHDR-HT1080 cells an 11.6-fold increase. The rMETase IC50 value for HT1080 was 0.75 U/ml and 0.59 U/ml for VHDR-HT1080. rMETase sensitized VHDR-HT1080 cells to doxorubicin. VHDR-HT1080 cells were cross-resistant to trabectedin 8.9-fold and cross-resistant to eribulin 1.87-fold compared to parental HT1080 cells. c-MYC expression was 8.4 times higher in VHDR-HT1080 cells compared to HT-1080 cells. The present results suggest rMETase may be used as a future clinical strategy to overcome super-doxorubicin resistance in soft tissue sarcoma.

cancer biology↗

bioRxiv2025

Local, but not circulating, complement C3 governs immune checkpoint blockade efficacy

The roles of circulating complement C3, produced in hepatocytes, in immune defense are well defined in the literature. Interestingly, the C3 gene evolved before the circulatory system, suggesting an essential but yet unknown role of locally produced C3. Here, we investigated the effect of C3 on cancer immunotherapy and found that cancer-associated fibroblast (CAF)-derived C3, not hepatocyte-derived C3, determines the efficacy of immune checkpoint blockade (ICB). Tumors developed in CAF-specific C3 knockout mice exhibited resistance to anti-PD-1 therapy, with increased immunosuppressive M2-like macrophage infiltration. Mechanistically, the C3 degradation product iC3b suppressed myeloid cell infiltration via complement receptor 3 signaling, and the therapeutic targeting of this pathway restored ICB sensitivity of immunotherapy-resistant tumors. In human cancers, stromal C3 expression correlates with reduced M2-like macrophage infiltration and improved immunotherapeutic outcomes. These data demonstrate the role of locally produced C3 in the innate immune response, which may have been conserved across many organisms.

cancer biology↗

bioRxiv2025

Ab-trapping - a peripheral staining artifact in antibody-based microscopy and genomics

Antibodies (Ab) are essential for detecting specific epitopes in microscopy and genomics, but can produce artifacts leading to erroneous interpretations. Here, we characterize a novel artifact, Ab-trapping, in which antibodies bind at the periphery of a cellular structure and do not diffuse further into its interior. This causes anomalous peripheral staining for multiple critical targets, including endogenous or ectopically expressed nuclear proteins like nucleolar proteins, histone variants and their modifications like H3K9me2. Ab-trapping can affect any assay relying on Ab diffusion, including immunofluorescence microscopy and recent genomics approaches like CUT&Tag. Critically, computational modeling and experimental validation reveal that Ab-trapping is caused by high epitope abundance, high Ab affinity, and low diffusion rates. Consequently, its effects can be mitigated by using alternative Abs and optimizing incubation conditions. Ab-trapping is therefore a considerable artifact that should be considered when designing experiments and interpreting results.

cell biology↗

bioRxiv2025

AI-assisted protein design to rapidly convert antibody sequences to intrabodies targeting diverse peptides and histone modifications

Intrabodies are engineered antibodies that function inside living cells, enabling therapeutic, diagnostic, and imaging applications. While powerful, their development has been hindered by challenges associated with their folding, solubility, and stability in the reduced intracellular environment. Here, we present an AI-driven pipeline integrating AlphaFold2, ProteinMPNN, and live-cell screening to optimize antibody framework regions while preserving epitope-binding complementarity-determining regions. Using this approach, we successfully converted 19 out of 26 antibody sequences into functional single-chain variable fragment (scFv) intrabodies, including a panel targeting diverse histone modifications for real-time imaging of chromatin dynamics and gene regulation. Notably, 18 of these 19 sequences had failed to convert using the standard approach, demonstrating the unique effectiveness of our method. As antibody sequence databases expand, our method will accelerate intrabody design, making their development easier, more cost-effective, and broadly accessible for biological research.

cell biology↗

bioRxiv2024

Probing nanomechanics by direct indentation using Nanoendoscopy-AFM reveals the nuclear elasticity transition in cancer cells

The assessment of nuclear structural changes is considered a potential biomarker of metastatic cancer. However, accurately measuring nuclear elasticity remains challenging. Traditionally, nuclear elasticity has been measured by indenting the cell membrane with a bead-attached atomic force microscopy (AFM) probe or aspirating isolated nuclei with a micropipette tip. However, indentation using a bead-attached probe is influenced by the cell membrane and cytoskeleton, while measurements of isolated nuclei do not reflect their intact state. In this study, we used Nanoendoscopy-AFM, a technique in which a nanoneedle probe is inserted into a living cell to directly measure nuclear elasticity and map its distribution. Our findings show that nuclear elasticity increases under serum depletion but decreases when serum-depleted cells are treated with TGF-{beta}, which induces epithelial-mesenchymal transition (EMT). Furthermore, we found that changes in nuclear elasticity correlate positively with trimethylation levels of histone H4 at lysine 20, rather than with nuclear lamins expression levels. These findings suggest that alterations in chromatin structure underlie changes in nuclear elasticity during cancer progression.

biophysics↗

bioRxiv2024

PATROL1-mediated H+-ATPase translocation boosts plant growth under drought by optimizing root and leaf functions

Optimizing leaf photosynthesis and root water and mineral uptake in crops during drought is crucial for enhancing agricultural productivity under climate change. Although plasma membrane H+-ATPase plays a key role in plant physiological processes, its overexpression alone does not consistently improve growth. While PROTON ATPASE TRANSLOCATION CONTROL 1 (PATROL1) regulates H+-ATPase translocation in response to various environmental stimuli in leaves, its function in roots remains largely unknown. Here we show that H+-ATPase was coimmunoprecipitated with PATROL1 in roots of Arabidopsis thaliana. Micrografting between wild type and PATROL1 knockout or overexpression lines showed that PATROL1 are indispensable in both shoots and roots, indicating that root uptake and leaf photosynthesis are simultaneous limiting factors for plant growth under drought conditions. PATROL1 overexpression in whole plants resulted in a 41% increase above wild type in shoot dry weight under drought conditions. These findings highlight the potential of H+-ATPase regulation in roots as a new strategy to improve plant productivity, particularly under drought conditions.

plant biology↗

bioRxiv2024

Self-organization of spermatogenic wave coordinates sustained sperm production in the mouse testis

Spermatogenesis takes place in the testis, relying on the ordered turnover of differentiating cells supplied from stem cells. Classic histological analyses have revealed that this process shows hierarchical spatiotemporal patterning known as the spermatogenic cycle, wave, and descent of segmental order, indicative of currently underexplored mechanisms of tissue- and organ-scale homeostasis. Here, using mice, we conducted high-resolution, wide-field, and ultra long-term live imaging studies in vivo and ex vivo, combined with whole-organ mapping of differentiation stages. Such trans-scale measures demonstrate how stereotypic local cell turnover is coordinated into characteristic phase waves propagating along the seminiferous tubules, further organized into organ-scale patterning over the tubule loops. Minimal mathematical modeling shows that such higher-order dynamics can emerge from the local coupling of autonomous oscillators, which are rooted in delayed feedback interplay between stem and differentiating cells via retinoic acid signaling. These findings highlight a self-organization mechanism underpinning organ-scale homeostasis and constant sperm production.

developmental biology↗

bioRxiv2024

Local DNA compaction creates TF-DNA clusters that enable transcription

Transcription factor (TF) clusters have been suggested to facilitate transcription. The mechanisms driving the formation of TF clusters and their impact on transcription, however, remain largely unclear. This is mostly due to the lack of a tractable system. Here, we exploit the transcriptional activation of mir430 in zebrafish embryos to simultaneously follow the dynamic formation of a large Nanog cluster, the underlying DNA, and transcription output by live imaging at high temporal and spatial resolution. We find that the formation of a Nanog cluster that can support transcription requires local DNA compaction. This brings more Nanog-binding sites into the cluster, and therefore more Nanog. Importantly, we find that Nanog stabilizes this TF-DNA cluster, which emphasizes the interdependent relationship between TFs and DNA dynamics in cluster formation. Once the Nanog-DNA cluster at the mir430 locus reaches a maximum amount of Nanog, transcription begins. This maximum is a locus-intrinsic feature, which shows that the locus self-regulates the recruitment of an optimal amount of Nanog. Our study supports a model in which endogenous TF clusters positively impact transcription and form through a combination of DNA binding and local DNA compaction.

cell biology↗

bioRxiv2024

Reconstruction of artificial nuclei with nuclear import activity in living mouse oocytes

The cell nucleus is a dynamic structure repeating disassembly and reformation during mitosis. Reformation of the nucleus is essential for cell proliferation, and therefore, the factors required for nuclear reformation are fundamental for eukaryotes. Although various factors have been identified in in vitro systems using frog egg extracts and in vivo imaging of somatic cells, little is known about the factors required for the formation of functional nuclear structures in living mouse eggs. To identify such factors, we used a reconstruction approach to construct an artificial nucleus around DNA in mouse eggs. T4 phage DNA (166 kbp) was microinjected into living mouse oocytes. Amounts of DNA injected and injection timing were examined to determine the conditions appropriate for the formation of functional nuclei. Microinjection of 100-500 ng/{micro}l DNA during metaphase through telophase of the second meiosis, but not the subsequent interphase, was important for the formation of artificial nucleus. This T4 DNA-derived artificial nucleus had the structure of nuclear lamina and nuclear pore complexes, and nuclear transport activity, similar to natural nuclei. These results suggest that exogenous DNA can form a functional nucleus in mouse oocytes, regardless of the sequence or the source of DNA.

cell biology↗

bioRxiv2024

Visualizing histone H4K20me1 in knock-in mice expressing the mCherry-tagged modification-specific intracellular antibody

During development and differentiation, histone modifications dynamically change locally and globally, associated with transcriptional regulation, DNA replication and repair, and chromosome condensation. The level of histone H4 Lys20 monomethylation (H4K20me1) increases during the G2 to M phases of the cell cycle and is enriched in facultative heterochromatin, such as inactive X chromosomes in cycling cells. To track the dynamic changes of H4K20me1 in living cells, we have developed a genetically encoded modification-specific intracellular antibody (mintbody) probe that specifically binds to the modification. Here, we report the generation of knock-in mice in which the coding sequence of the mCherry-tagged version of the H4K20me1-mintbody is inserted into the Rosa26 locus. The knock-in mice, which ubiquitously expressed the H4K20me1-mintbody, developed normally and were fertile, indicating that the expression of the probe does not disturb the cell growth, development, or differentiation. Various tissues isolated from the knock-in mice exhibited nuclear fluorescence without the need for fixation. The H4K20me1-mintbody was enriched in inactive X-chromosomes in developing embryos and in XY bodies during spermatogenesis. The knock-in mice will be useful for the histochemical analysis of H4K20me1 in any cell types.

cell biology↗