Biology subjects

Arai, S.

Publications and source records attributed to Arai, S..

14 recordsLinked to original sources

bioRxiv2026

A Nanoheater-Integrated Fluorescence Lifetime Thermometer for Investigating Subcellular Heat Shock Factor 1 Responses

Subcellular thermal engineering provides a powerful approach for investigating and manipulating biological processes. However, existing subcellular heating platforms capable of combining spatially confined heating, quantitative thermometry and simultaneous imaging of cellular responses remain limited. We developed a quantitative nanoheater-thermometer (qNanoHT), a polymeric nanoparticle integrating a temperature-sensitive fluorescent, dye and a photothermal dye. qNanoHT determines local temperature from fluorescence lifetime using fluorescence lifetime imaging microscopy (FLIM), thereby reducing susceptibility to photobleaching, focal drift and variations in probe concentration compared with intensity-based methods. The platform enabled real-time measurement at a subcellular heat spot while the dynamics of heat shock factor 1 (HSF1) were monitored in living cells. Heating at a single intracellular site was sufficient to induce HSF1 foci. Foci induced by mild heating at approximately 38 {degrees}C dissolved after heating ceased, whereas those induced by stronger heating at approximately 41 {degrees}C persisted and were associated with caspase-3/7 activation and apoptosis. Notably, qNanoHT-mediated subcellular heating induced HSF1 foci at a lower measured temperature than uniform whole-cell heating approximately 38 {degrees}C versus 39 {degrees}C indicating that the spatial extent of heating influences the HSF1 activation threshold. qNanoHT therefore provides a quantitative platform for relating local intracellular temperature to cellular stress responses and subsequent cell fate.

bioengineering↗

bioRxiv2026

Protein-independent light harvesting governed by structural heterogeneity

Chlorosomes are the largest known photosynthetic light-harvesting antennas, yet unlike protein-based antennas, they lack protein scaffolds that organize pigment molecules and instead contain self-assembled tubular and lamellar bacteriochlorophyll aggregates. How these antennas achieve directional and efficient energy transfer has remained unresolved. By applying ultrafast transient absorption spectroscopy to individual wild-type and mutant chlorosomes, we resolved six kinetic components obscured by ensemble averaging and assigned each to an excitation process associated with either lamellar or tubular aggregates. Lamellar aggregates tend toward exciton localization and expand light-harvesting capacity, whereas tubular aggregates retain a relatively delocalized excitonic character and serve as the primary energy donors to the baseplate. Structural heterogeneity therefore emerges as a functional feature that balances light-harvesting capacity with robust directional energy transfer. These findings reveal a division of labor among self-assembled pigment aggregates for efficient light harvesting without protein scaffolds.

biophysics↗

bioRxiv2026

Electroporation-mediated delivery of protein biosensors for metabolic imaging in differentiated myotubes

Understanding skeletal muscle metabolism involves real-time monitoring of key cellular parameters, such as calcium ions (Ca2+), adenosine triphosphate (ATP), cyclic adenosine monophosphate (cAMP), and intracellular temperature. Fluorescent protein (FP)-based biosensors are used for live-cell imaging of these signals with high spatiotemporal resolution. Differentiated myotubes are in vitro models used for physiological muscle metabolism research. However, efficient transfection of FP-based biosensors into these cells is challenging. Here, we developed an electroporation-based strategy for delivering recombinant protein biosensors into fully differentiated myotubes. Biosensors for Ca2+, ATP, cAMP, and temperature were recombinantly produced using Escherichia coli and introduced into myotubes using electroporation. Electroporation conditions were optimised to maximise delivery efficiency, preserve cell viability, and minimise cellular damage. We established a robust intracellular delivery system that effectively demonstrated Ca2+, ATP, and temperature dynamics. Furthermore, we achieved the successful co-delivery of two biosensors that enabled dual imaging of Ca2+ and cAMP in response to stimulation.

bioengineering↗

bioRxiv2026

Heterogeneity-Resolved Ultrafast Transient Absorption Spectroscopy of Single Supramolecular Light-Harvesting Antennas

Photosynthetic light harvesting proceeds rapidly and robustly through precisely arranged pigment molecules. However, the molecular arrangements are not uniform because they exhibit structural heterogeneity among individual complexes and across spatial regions, and undergo dynamic fluctuations. Such static and dynamic disorder can substantially perturb excitation dynamics. Here, we report a highly sensitive transient absorption microscope that integrates single-objective absorption microscopy, balanced detection, and lock-in amplification, enabling quantitative analysis of heterogeneity and temporal fluctuations in excitation dynamics. By analyzing individual chlorophyll-derivative aggregates mimicking photosynthetic light-harvesting antennas, we demonstrated that two kinetic components with nearly identical time constants can be resolved based on differences in their time-constant distributions. We further quantified the photophysical properties of each component, including absorbance change, fluorescence intensity, fluorescence efficiency, and fluorescence peak intensity ratio. These results establish an analytical framework for excitation dynamics that leverages not only the mean values of time constants but also their distribution profiles.

biophysics↗

bioRxiv2025

High-Speed AFM Reveals IDR-Mediated Structural Plasticity of EML4-ALK Modulated by ALK Inhibitors

EML4-ALK is a key oncogenic driver in lung cancer, but variant-specific chemoresistance limits the efficacy of current ALK inhibitors. Because the N-terminus contains an intrinsically disordered region (IDR), how ALK inhibitors affect the structure and dynamics of full-length EML4-ALK remains unclear. Here, using high-speed atomic force microscopy (HS-AFM), we visualize the overall structures of three full-length EML4-ALK variants (v1, v3, and v5) at the single-molecule level. We identified a transient globular subdomain (residues 191-217) within the v3 IDR that contributes to distinct oligomerization patterns. Notably, ALK inhibitors compact the IDR subdomain and reduce oligomerization, whereas this effect is abolished by the resistance mutation ALKG1202R, suggesting that ALK inhibitors not only affect kinase activity but also modulate IDR dynamics. Our findings shed new light on the structural regulation of EML4-ALK and provide a foundation for developing next-generation targeted therapies that account for IDR-driven molecular behavior. TeaserHigh-speed AFM revealed the full-length EML4-ALK structures and their IDR-mediated plasticity caused by ALK kinase inhibitors

cancer biology↗

bioRxiv2024

Androgen deprivation-mediated activation of AKT is enhanced in prostate cancer with TMPRSS2:ERG fusion

TMPRSS2:ERG gene fusion (T:E fusion) in prostate adenocarcinoma (PCa) puts ERG under androgen receptor (AR) regulated TMPRSS2 expression. T:E fusion is associated with PTEN loss, and is highly associated with decreased INPP4B expression, which together may compensate for ERG-mediated suppression of AKT signaling. We confirmed in PCa cells and a mouse PCa model that ERG suppresses IRS2 and AKT activation. In contrast, ERG downregulation did not increase INPP4B, suggesting its decrease is indirect and reflects selective pressure to suppress INPP4B function. Notably, INPP4B expression is decreased in PTEN-intact and PTEN-deficient T:E fusion tumors, suggesting selection for a nonredundant function. As ERG in T:E fusion tumors is AR regulated, we further assessed whether AR inhibition increases AKT activity in T:E fusion tumors. A T:E fusion positive PDX had increased AKT activity in vivo and response to AKT inhibition in vitro after androgen deprivation. Moreover, two clinical trials of neoadjuvant AR inhibition prior to radical prostatectomy showed greater increases in AKT activation in the T:E fusion positive versus negative tumors. These findings indicate that AKT activation may mitigate the efficacy of AR targeted therapy in T:E fusion PCa, and that these patients may most benefit from combination therapy targeting AR and AKT.

cancer biology↗

bioRxiv2024

Harnessing Dye-induced Photothermal Confinement in Lipid Membranes: A Path to NIR-modulated Artificial Synaptic Vesicles

Optical heating coupled with near-infrared (NIR) light and photothermal materials enables thermal confinement within biospecimens, minimizing undesirable thermal damage. Here, we demonstrated that photothermally heating lipid bilayers embedded with a unique phthalocyanine dye (VPc) efficiently perturbs the bilayers, resulting in increased permeability. Notably, microscopic studies revealed that the mechanism causing changes in membrane permeability may not follow the conventional temperature-sensitive liposome model. Furthermore, the heat generated by NIR laser illumination rarely diffused into the surrounding environment, and the dye was located within the bilayers at the molecular level, where it effectively transferred heat to the lipid bilayer. We prepared VPc-embedded liposomes encapsulating acetylcholine (ACh) and demonstrated the NIR laser-triggered release of ACh, creating a concentration jump across a few cells or within a limited single cell region. This method induced Ca2+ flux through ACh receptor stimulation in thermally delicate biospecimens such as C2C12 myotubes and the Drosophila brain.

bioengineering↗

bioRxiv2024

Acute BRCAness Induction and AR Signaling Blockage through CDK12/7/9 Degradation Enhances PARP Inhibitor Sensitivity in Prostate Cancer

Current treatments for advanced prostate cancer (PCa) primarily target the androgen receptor (AR) pathway. However, the emergence of castration-resistant prostate cancer (CRPC) and resistance to AR pathway inhibitors (APSIs) remains ongoing challenges. Here, we present BSJ-5-63, a novel proteolysis-targeting chimera (PROTAC) targeting cyclin-dependent kinases (CDKs) CDK12, CDK7, and CDK9, offering a multi-pronged approach to CRPC therapy. BSJ-5-63 degrades CDK12, diminishing BRCA1 and BRCA2 expression and inducing a sustained "BRCAness" state. This sensitizes cancer cells to PARP inhibitors (PARPis) regardless of their homologous recombination repair (HRR) status. Furthermore, CDK7 and CDK9 degradation attenuates AR signaling, enhancing its therapeutic efficacy. Preclinical studies, including both in vitro and in vivo CRPC models, demonstrate that BSJ-5-63 exerts potent anti-tumor activity in both AR-positive and AR-negative setting. This study introduces BSJ-5-63 as a promising therapeutic agent that addresses both DNA repair and AR signaling mechanisms, with potential benefits for a board patient population.

cancer biology↗

bioRxiv2024

A versatile platform for single fluorescent protein-based fluorescence lifetime biosensors

Single fluorescent protein (FP)-based FLIM (fluorescence lifetime imaging) biosensors are potent tools for quantitatively imaging intracellular processes with high spatial and temporal resolution. They only require a single wavelength for detection, which facilitates multi-color imaging. However, the development of single FP-based FLIM biosensors has been limited by the absence of a general design framework and the complexity of the screening process. In this study, we engineered FLIM biosensors capable of detecting ATP (adenosine triphosphate), cAMP (cyclic adenosine monophosphate), citrate, and glucose by inserting each sensing domain into the mTurquoise2 fluorescent protein between Tyr-145 and Phe-146 using peptide linkers. Through efficient linker screening, we successfully developed FLIM biosensors exhibiting an effective dynamic range from 0.5 to 1.0 ns upon analyte binding. This demonstrates that the qmTQ2-ATP-0 backbone is a universal platform for developing mTQ2-based biosensors. As a proof-of-concept, we demonstrated the capabilities of these FLIM biosensors in monitoring the intracellular dynamics of ATP and cAMP alongside dual-color imaging. Therefore, our work presents an accessible methodology for establishing a single FP-based FLIM biosensor platform for quantitative imaging.

molecular biology↗

bioRxiv2023

qMaLioffG: A single green fluorescent protein FLIM indicator enabling quantitative imaging of endogenous ATP

The widespread use of fluorescence lifetime imaging microscopy (FLIM) for quantitative imaging is hindered by the limited availability of a FLIM-based genetically encoded indicator using a conventional 488 nm laser. Here, we present qMaLioffG, a single green fluorescent protein FLIM indicator showing a fluorescence lifetime change in ATP concentration within the physiological range. This allows quantitative imaging of endogenous ATP to investigate cellular energy status of different cell types.

biophysics↗

bioRxiv2021

Photothermal Dye-based Subcellular-sized Heat Spot Enabling the Modulation of Local Cellular Activities

Thermal engineering at microscale such as the control and measurement of temperature is a key technology in basic biological research and biomaterials development, which remains challenge yet. Here, we engineered the polymeric nanoparticle, in which a fluorescent temperature sensory dye and a photothermal dye were embedded in its polymer matrices, termed nanoHT. When a near infrared laser at 808 nm is illuminated to the particle, it enables to create the subcellular-sized heat spot in a live cell, where fluorescence thermometry allows the read out of the temperature increment concurrently at individual heat spots. Owing to the controlled local heating, we found that the cell death of HeLa cells was induced at the certain temperature at rate of a few seconds. It should be also noted that the cell death was triggered from the very local heat spot at subcellular level. Furthermore, nanoHT was applied for the induction of muscle contraction of the C2C12 myotube by heat. We successfully showed that the heat-induced contraction took place at the limited area of a single myotube according to the alteration of protein-protein interactions related to the contraction event. These studies demonstrated that even a single heat spot provided by a photothermal material could be very effective in altering cellular functions, paving the way for novel photothermal therapies.

bioengineering↗

bioRxiv2021

Whole-genome sequencing analysis reveals the population history of Mus musculus in Madagascar

In Madagascar, the house mouse (Mus musculus) is thought to have colonized with human activities and is now one of the most abundant rodents on the island. In this study, we determined the whole-genome sequences of five Madagascar house mice captured from the wild. We examined the evolutionary history of the population by analyzing the mitochondrial and autosomal genomes. We confirmed that the mitochondrial genome lineages of Madagascar house mice formed a monophyletic clade placed at one of the most basal positions in the species. An analysis of autosomal genomic sequences indicates that the Madagascar house mice are genetically members of M. m. castaneus (CAS), but also contain genetic elements of M. m. domesticus (DOM) resulting from hybridization between subspecies. The signature of a strong population bottleneck 1000-3000 years ago was observed in both mitochondrial and autosomal genomic data. All samples showed strong genetic affinity to many CAS samples across a wide range of Indian Ocean coastal and island regions, with divergence time estimated around 4000 years ago. These findings support that the Madagascar house mice started to colonize the island with human agricultural activity, and experienced complex history for the establishment.

genetics↗

bioRxiv2020

Ubiquitin Ligase MARCH5 Regulates Apoptosis through Mediation of Stress-Induced and NOXA-Dependent MCL1 Degradation

MCL1 has critical antiapoptotic functions and its levels are tightly regulated by ubiquitylation and degradation, but mechanisms that drive this degradation, particularly in solid tumors, remain to be established. We show here in prostate cancer cells that increased NOXA, mediated by activation of an integrated stress response, drives the degradation of MCL1, and identify the mitochondria-associated ubiquitin ligase MARCH5 as the primary mediator of this NOXA-dependent MCL1 degradation. Therapies that enhance MARCH5-mediated MCL1 degradation markedly enhance apoptosis in response to a BH3 mimetic agent targeting BCLXL, which may provide for a broadly effective therapy in solid tumors. Conversely, increased MCL1 in response to MARCH5 loss does not sensitize to BH3 mimetic drugs targeting MCL1, but instead also sensitizes to BCLXL inhibition, revealing a codependence between MARCH5 and MCL1 that may also be exploited in tumors with MARCH5 genomic loss.

cancer biology↗

bioRxiv2019

Disease-associated mutations hyperactivate KIF1A motility and anterograde axonal transport of synaptic vesicle precursors

KIF1A is a kinesin-family motor involved in the axonal transport of synaptic vesicle precursors (SVPs) along microtubules. In humans, more than ten point mutations in KIF1A are associated with the motor neuron disease, hereditary spastic paraplegia (SPG). However, not all of these mutations appear to inhibit the motility of the KIF1A motor, and thus, a clear molecular explanation for how KIF1A mutations lead to neuropathy is not available. In this study, we established in vitro motility assays with purified full-length human KIF1A and found that KIF1A mutations associated with the pure form of spastic paraplegia hyperactivate motility of the KIF1A motor. Introduction of the corresponding mutations into Caenorhabditis elegans KIF1A homologue unc-104 revealed abnormal accumulation of SVPs at the tips of axons and increased anterograde axonal transport of SVPs. Our data reveal that hyper-activation of kinesin motor activity, rather than its loss-of-function, is a novel cause of motor neuron disease in humans.\n\nSignificance StatementAnterograde axonal transport supplies organelles and protein complexes throughout axonal processes to support neuronal morphology and function. It has been observed that reduced anterograde axonal transport is associated with neuronal diseases. In contrast, here we show that particular disease-associated mutations in KIF1A, an anterograde axonal motor for synaptic vesicle precursors, induce hyperactivation of KIF1A motor activity and increased axonal transport of synaptic vesicle precursors. Our results advance the knowledge of the regulation of motor proteins and axonal transport and cell biology of motor neuron diseases.

cell biology↗