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Biology subjects

Ishikawa, Y.

Publications and source records attributed to Ishikawa, Y..

At least 19 recordsLinked to original sources

Distinct associative learning abilities for colour and odour in the flower-feeding Drosophila elegans and the fruit-feeding Drosophila melanogaster

Animal behaviour is both innately constrained and shaped by learning. This mosaic organization has evolved in response to species-specific ecological demands and may differ between sensory modalities. Flower-visiting animals are a particularly useful system for investigating the relationship between sensory ecology and learning because they rely on multiple floral cues, particularly odour and colour, to locate food sources. However, it remains largely unexplored whether specialization on floral resources entails divergence in learning abilities across sensory modalities. Drosophila elegans is a flower-feeding species that depends heavily on floral resources throughout its life; adults spend much of their time on flowers and larvae develop on fallen flower leaves. Here, we compared odour-reward and colour-reward associative learning between the flower-feeding D. elegans and the fruit-feeding D. melanogaster. We found that, under conditions of equilibrated motivation, odour- and colour-preference, and using the same sugar reward, D. elegans exhibited poorer odour-reward learning performance but better colour-reward learning performance than D. melanogaster. These results suggest that the modality-specific eligibility of sensory information to enter into associations, known as the 'Garcia-effect' in experimental psychology, can evolve oppositely between species. This highlights the relationship between ecological specialization and mnemonic processing, and shows that biological 'intelligence' is not general.

evolutionary biology↗

Biomechanical regulation of Ca2+ dynamics during muscle stem cell activation

Muscle satellite cells (MuSCs) are muscle-resident stem cells that are responsible for myofiber regeneration. Although the importance of calcium ions (Ca2+) in muscle physiology has been well established, the mechanism by which Ca2+ mobilization governs MuSC function remains poorly understood. In this study, we aimed to systematically characterize Ca2+ dynamics in MuSCs and to define the mechanisms regulating these signals during muscle regeneration. By employing modified protocols for mouse MuSC isolation and Ca2+ measurement, we observed spontaneous Ca2+ fluctuations in MuSCs isolated from regenerating muscle after cardiotoxin-induced myofiber injury. Our detailed analysis using chemical Ca2+ indicators and a genetically encoded Ca2+ indicator revealed that the frequency and amplitude of Ca2+ fluctuations increased significantly during the activated and proliferative stages of MuSCs in muscle regeneration. This effect was more pronounced in MuSCs isolated from dystrophic and aged mice. Mechanistically, these Ca2+ fluctuations were at least partially mediated by mechanosensitive ion channels, including PIEZO1 and TRPM7, which promote MuSC migration. Collectively, our findings demonstrate that Ca2+ fluctuations through mechanosensitive ion channels act as a key regulator of MuSC activation during muscle regeneration and may provide new insights into the role of Ca2+ influx in muscle biology and the pathogenesis of muscle diseases.

cell biology↗

Synthetic activation of gibberellin signaling reveals spatial coordination of root growth

Gibberellins (GAs) influence cell division and elongation, profoundly shaping plant architecture and yield. GA perception occurs when bioactive GAs bind the receptor GID1, promoting DELLA degradation and activating transcriptional programs. While GA signaling in the root endodermis is essential for promoting root elongation, functions of other layers in spatial control of GA responses have not been explored. Here, we developed a synthetic GA (sGA) that does not bind endogenous GID1, together with a modified GID1 (mGID1) engineered to selectively recognize sGA, enabling cell-specific activation of GA signaling in vivo. Using this system in Arabidopsis, we demonstrate that coordinated action of GA signaling in the endodermis, epidermis, and other layers is required for full root elongation. Moreover, cell type-specific expression of GA biosynthetic enzymes indicates the existence of intercellular GA transport. The sGA-mGID1 system provides a versatile platform for spatially precise reprogramming of hormone signaling, enabling synthetic control of developmental processes such as root-shoot growth balance, thereby advancing applications in plant synthetic biology and sustainable crop improvement.

plant biology↗

PRSS56 acts as an intrinsic retinal signal driving postnatal ocular axial growth and myopia susceptibility

Myopia is a leading cause of visual impairment worldwide, and high myopia markedly increases the risk of irreversible vision loss. Although visual experience guides postnatal ocular elongation, the role of intrinsic retinal growth signals remains poorly defined. Here we identify the serine protease PRSS56 as a retinal factor that promotes ocular axial growth beyond early development. Using genetic mouse models, we show that conditional inactivation of Prss56 in Muller glia reduces axial length and causes hyperopia even under dark-rearing conditions, demonstrating that PRSS56 drives axial elongation independently of light-evoked visual input during emmetropization. Conversely, Muller glia-specific overexpression of Prss56 induces axial elongation in a proteolysis-dependent manner, supporting its role as an autonomous retinal growth signal. In concordance, human genetic analyses reveal that the common PRSS56 variant rs2853447 is associated with increased axial length in individuals with myopia and high myopia, but not in non-myopes, suggesting that this variant confers a selective growth advantage in individuals predisposed to ocular elongation. Functional genomic analyses further identify a myopia-associated variant, rs2741297, within a retinal enhancer in intron 4 of PRSS56 marked by open chromatin and transcription factor occupancy. Together, these findings establish PRSS56 as an intrinsic retinal growth factor that functions beyond early eye development and support a model in which genetic and environmental factors converge on retinal pathways to modulate myopia susceptibility.

genetics↗

2-Mercaptoethanol/DMSO workflow enables highly reproducible quantitative proteomics

Proteomics provides a systematic and high-throughput approach to comprehensively characterize protein networks, enabling insights into cellular functions and disease mechanisms. Carbamidomethylation using iodoacetamide (IAA), a common method for cysteine alkylation, is known to cause nonspecific modifications that increase MS spectral complexity and reduce quantitative accuracy. Here, we extended our 2-mercaptoethanol (2-ME) adduction method, enhanced by dimethyl sulfoxide (DMSO), from single-protein applications to a proteome-wide workflow. Mouse liver proteomes were processed using either 2-ME/DMSO or conventional IAA treatment, followed by LC-MS/MS analysis. The optimized 2-ME treatment increased the number of cysteine-modified peptides by 1.6-1.9-fold. Although total protein identifications were comparable, 77% of proteins exhibited improved sequence coverage. Quantitative reproducibility was also enhanced, with peptides quantified coefficient of variation (CV) [≤] 20% increasing from 61.5% (IAA) to 86.1% (2-ME), and proteins from 80.6% to 93.5%. Application of the workflow to an ovarian clear cell carcinoma reliably detected cisplatin-induced alterations. The 2-ME/DMSO workflow offers a simple and highly reproducible proteomics strategy for accurate quantitative proteomics.

biochemistry↗

Evidence for rapid hydrolysis of shoot-derived sucrose using an ultrasensitive ratiometric Matryoshka-type MGlucoMeter sensor

To enable sensitive in vivo monitoring of the glucose transport and metabolism, we developed a series of ultrasensitive and ratiometric genetically encoded nanosensors by inserting a Matryoshka dual fluorophore cassette consisting of cpsfGFP and LSSmApple into the glucose binding protein ttGBP from Thermus thermophilus. The initial MGlucoMeter1.0 was subjected to an alanine scan of the hinge region producing more sensitive MGlucoMeter2.6 with a glucose-induced {Delta}F/F0 change of 3.0, an affinity for glucose of 15 {micro}M, and an approximate detection range of 1.1-216 {micro}M. To generate variants suitable for in vivo measurements, a series of affinity mutants was generated by mutating two histidines predicted to be involved in substrate binding. MGlucoMeter2.6-353n, MGlucoMeter2.6-15{micro}, MGlucoMeter2.6-700{micro}, MGlucoMeter2.6-1m, and MGlucoMeter2.6-7m cover a detection range between [~]40 nM - 55 mM. When expressed from a ubiquitous promoter in the cytosol of the Arabidopsis gene silencing mutant rdr6, MGlucoMeter2.6-1m reports time- and concentration-dependent accumulation of glucose in seedling roots. The sensor also detects rapid hydrolysis of shoot-derived sucrose in the root tip.

plant biology↗

Evidence for the major role of PH4EFB in the prolyl 4-hydroxylation of Drosophila collagen IV

Collagens are fundamental components of extracellular matrices, requiring precise intracellular post-translational modifications for proper function. Among the modifications, prolyl 4-hydroxylation is critical to stabilise the collagen triple helix. In humans, this reaction is mediated by collagen prolyl 4-hydroxylases (P4Hs). While humans possess three genes encoding these enzymes (P4Hs), Drosophila melanogaster harbour at least 26 candidates for collagen P4Hs despite its simple genome, and it is poorly understood which of them are actually working on collagen in the fly. In this study, we addressed this question by carrying out thorough bioinformatic and biochemical analyses. We demonstrate that among the 26 potential collagen P4Hs, PH4EFB shares the highest homology with vertebrate collagen P4Hs. Furthermore, while collagen P4Hs and their substrates must exist in the same cells, our transcriptomic analyses at the tissue and single cell levels showed a global co-expression of PH4EFB but not the other P4H-related genes with the collagen IV genes. Moreover, expression of PH4EFB during embryogenesis was found to precede that of collagen IV, presumably enabling efficient collagen modification by PH4EFB. Finally, biochemical assays confirm that PH4EFB binds collagen, supporting its direct role in collagen IV modification. Collectively, we identify PH4EFB as the primary and potentially constitutive prolyl 4-hydroxylase responsible for collagen IV biosynthesis in Drosophila. Our findings highlight the remarkably simple nature of Drosophila collagen IV biosynthesis, which may serve as a blueprint for defining the minimal requirements for collagen engineering.

biochemistry↗

Adolescent morphine exposure does not alter low-dose lipopolysaccharide-induced sickness behavior in adult mice

Adolescent opioid use in the United States commands attention: millions of twelve- to nineteen-year-olds are exposed to opioids each year by prescription and misuse. Recent findings show that opioids bind not only to canonical opioid receptors but also interact with receptors on immune cells within both the central and peripheral nervous systems. The potential for early life opioid exposure to induce long-term changes in the neuroimmune system is not fully understood, particularly given the adolescent brains high susceptibility to neuroplastic changes. The goal of this study was to investigate the hypothesis that adolescent opioid use potentiates physiological and behavioral responses to pathogen-induced sickness later in life. To achieve this, we treated adolescent mice (PND 35-42) with bi-daily escalating doses of morphine to model dependence and then administered a low dose of lipopolysaccharide (LPS, 0.1 mg/kg) in adulthood (PND 60-76) to induce an immune response. In contrast to our hypotheses, we found that adolescent morphine exposure had no additive effect on low-dose LPS-induced sickness measures when assessed in adulthood. These data suggest that adolescent opioid exposure may have minimal effects on future immune challenges, although further research is needed to confirm this.

neuroscience↗

Mating status-dependent dopaminergic modulation of auditory sensory neurons in Drosophila

Mating status often modulates responses to courtship sounds in animals. The neural mechanisms underlying this modulation, however, have not been well clarified. Here, we show that dopaminergic signals are involved in modulating the responses of auditory sensory neurons in Drosophila melanogaster females depending on their mating status. These neurons abundantly express three types of dopamine receptors, with some having direct synaptic connections with dopaminergic neurons. Of these receptors, suppressing the expression of Dop1R2 reduces sound responses of auditory sensory neurons in unmated but not mated females. Moreover, expression of Dop1R2 in auditory sensory neurons enhances the song response behavior of unmated females, manifested by copulation receptivity when exposed to songs. Our research suggests that dopaminergic modulation via Dop1R2 is involved in mating state-dependent regulation of auditory sensory processing.

neuroscience↗

STT3A is required for recognition of pathogen-derived sphingolipids in Arabidopsis

Plants recognize pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors, leading to the activation of pattern-triggered immunity in response to pathogen attack. Phytophthora infestans ceramide D (Pi-Cer D) is a sphingolipid from the oomycete pathogen P. infestans. Pi-Cer D is cleaved by the plant extracellular ceramidase NEUTRAL CERAMIDASE 2 (NCER2), and the resulting 9-methyl-branched sphingoid base is recognized by the plant receptor RESISTANT TO DFPM-INHIBITION OF ABSCISIC ACID SIGNALING 2 (RDA2) at the plasma membrane to transduce a defense signal. However, additional components are likely involved in sphingolipid recognition, which remain to be identified. Here, we employed a screen based on Lumi-Map technology to look for Arabidopsis (Arabidopsis thaliana) mutants with altered defense responses to Pi-Cer D. We identified three mutants showing diminished responses to Pi-Cer D and elf18, each carrying mutations in STAUROSPORIN AND TEMPERATURE SENSITIVE 3-LIKE A (STT3A), which encodes an oligosaccharyltransferase. The stt3a mutants exhibited higher susceptibility to the pathogen Colletotrichum higginsianum than the wild type and displayed alterations in NCER2 protein modifications. These findings suggest that STT3A contributes to plant immunity via post-translational modification of proteins including NCER2.

plant biology↗

Predicted constrained accessible regions mark regulatory elements and causal variants

Open chromatin regions (OCRs) define cell-type-specific regulatory elements across the genome, yet their functional significance varies, making it challenging to pinpoint biologically essential regions. Here, we introduce CAMBUS (Chromatin Accessibility Mutation Burden Score), a machine-learning framework that identifies active and evolutionarily constrained OCRs by leveraging surrounding DNA sequences. Applying CAMBUS to 29 immune cell types, we identified 66,043 constrained OCRs, which were substantially enriched in the known constraint genome (odds ratio=11.45 (95% confidence interval 9.33-14.05), P=4.7 x 10-68), while 90% of these OCRs were not prioritized by existing constraint metrics. These OCRs were highly enriched for known enhancers and super-enhancers, independent of known epigenetic markers and annotated regions, and overlapped with regulatory elements implicated in immune-mediated diseases and experimentally validated functional variants, including rare variants, particularly in leukocyte-related traits. Furthermore, CAMBUS revealed cell-type-specific transcriptional regulatory landscapes, linking genetic constraint with gene regulation in immune cells and identifying plausible connections between 1,533 causal variants and 70 complex traits. By defining biologically constrained regulatory elements at high resolution, CAMBUS provides a framework for understanding the selective pressures shaping the non-coding genome and its role in human health and disease.

genetics↗

Renalase inhibition regulates β cell metabolism to defend against acute and chronic stress

Renalase (Rnls), annotated as an oxidase enzyme, is a GWAS gene associated with Type 1 Diabetes (T1D) risk. We previously discovered that Rnls inhibition delays diabetes onset in mouse models of T1D in vivo, and protects pancreatic {beta} cells against autoimmune killing, ER and oxidative stress in vitro. The molecular biochemistry and functions of Rnls are entirely uncharted. Here we find that Rnls inhibition defends against loss of {beta} cell mass and islet dysfunction in chronically stressed Akita mice in vivo. We used RNA sequencing, untargeted and targeted metabolomics and metabolic function experiments in mouse and human {beta} cells and discovered a robust and conserved metabolic shift towards glycolysis, amino acid abundance and GSH synthesis to counter protein misfolding stress, in vitro. Our work illustrates a function for Rnls in mammalian cells, and suggests an axis by which manipulating intrinsic properties of {beta} cells can rewire metabolism to protect against diabetogenic stress.

physiology↗

Investigating EMT-mediated resistance to EGFR tyrosine kinase inhibitors in NSCLC using innovative organoid models

BackgroundEpithelial-mesenchymal transition (EMT) has emerged as a key mechanism underlying resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC). However, the intricacies of EMT-mediated resistance, driven by tumor microenvironment (TME) interactions, remain enigmatic. This study aimed to probe EMT-induced resistance in NSCLC using innovative in vitro organoid models. MethodsWe generated organoids by co-culturing EGFR-mutant NSCLC cells (HCC827, H1975), mesenchymal stem cells and endothelial cells. Drug susceptibility was compared between organoids and spheroids (cancer cells only) using EGFR TKIs - Gefitinib, Afatinib, Osimertinib. EMT marker (E-cadherin, ZEB1) expression was analyzed via immunofluorescence and western blotting. The effects of Bevacizumab and miR200c on overcoming resistance were also investigated. ResultsThe study identified a significant link between EMT and EGFR-TKI resistance. Notable findings included the decrease of E-cadherin and an increase in ZEB1, both of which influenced EMT and resistance to treatment. Bevacizumab showed promise in improving drug resistance and mitigating EMT, suggesting an involvement of the VEGF cascade. Transfection with miR200c was associated with improved EMT and drug resistance, further highlighting the role of EMT in TKI resistance. ConclusionsThis study offered vital insights into EMT-driven EGFR TKI resistance, highlighting the utility of organoid models in evaluating resistance modulated by TME interactions. Our findings reveal promising directions for overcoming EMT-mediated resistance involving Bevacizumab and miR200c, warranting further in vivo validation.

cancer biology↗

Sequential Transitions of Male Sexual Behaviours Driven by Dual Acetylcholine-Dopamine Dynamics

The neural mechanisms regulating sequential transitions of male sexual behaviors, such as mounting, intromission, and ejaculation, in the brain remain unclear. Here, we report that dopamine (DA) and acetylcholine (ACh) dynamics in the ventral shell of the nucleus accumbens (vsNAc) closely aligns with serial transitions of sexual behaviors in male mice. During intromission, the vsNAc exhibits dual ACh-DA rhythms generated by reciprocal regulation between ACh and DA signaling via nicotinic acetylcholine (nAChR) and dopamine D2 (D2R) receptors. Knockdown of choline acetyl transferase (ChAT) or D2R in the vsNAc diminished the likelihood of intromission and ejaculation. Optogenetic manipulations reveal that DA signaling sustains male sexual behaviors by suppressing activities of D2RvsNAc neurons. Moreover, ACh signaling promotes the initiation of mounting and intromission, but also induces the intromission-to-ejaculation transition by triggering a slowdown of DA rhythm. Therefore, dual ACh-DA dynamics harmonize in the vsNAc to drive sequential transitions of male mating behaviors.

neuroscience↗

Neural-circuit basis of song preference learning in fruit flies

As observed in human language learning and song learning in birds, the fruit fly Drosophila melanogaster changes its auditory behaviors according to prior sound experiences. Female flies that have heard male courtship songs of the same species are less responsive to courtship songs of different species. This phenomenon, known as song preference learning in flies, requires GABAergic input to pC1 neurons in the central brain, with these neurons playing a key role in mating behavior by integrating multimodal sensory and internal information. The neural circuit basis of this GABAergic input, however, has not yet been identified. Here, we find that pCd-2 neurons, totaling four cells per hemibrain and expressing the sex-determination gene doublesex, provide the GABAergic input to pC1 neurons for song preference learning. First, RNAi-mediated knockdown of GABA production in pCd-2 neurons abolished song preference learning. Second, pCd-2 neurons directly, and in many cases mutually, connect with pC1 neurons, suggesting the existence of reciprocal circuits between pC1 and pCd-2 neurons. Finally, GABAergic and dopaminergic inputs to pCd-2 neurons are necessary for song preference learning. Together, this study suggests that reciprocal circuits between pC1 and pCd-2 neurons serve as a sensory and internal state-integrated hub, allowing flexible control over female copulation. Consequently, this provides a neural circuit model that underlies experience-dependent auditory plasticity. SignificanceTo find a suitable mate, an organism must adapt its behavior based on past experiences. In the case of Drosophila, female assessments of male song signals, which contain information about the status and species of the sender, are experience dependent. Here, we show that reciprocal circuits in the central brain modulate the females song response depending on her previous auditory experiences. These circuits exhibit feedback and lateral inhibition motifs, and are regulated by dopaminergic and GABAergic inputs. While the effects of prior auditory experiences on sound responsiveness have been extensively studied in other species, our research advances the use of Drosophila as a model for dissecting the circuitry underlying experience-dependent auditory plasticity at single-cell resolution.

neuroscience↗

A monochromatically excitable green-red dual-fluorophore fusion incorporating a new large Stokes shift fluorescent protein

Genetically encoded sensors enable quantitative imaging of analytes in live cells. State-of-the-art sensors are commonly constructed by combining ligand-binding domains with one or more sensitized fluorescent protein (FP) domains. Sensors based on a single FP are susceptible to artifacts caused by differing expression levels or sensor distribution in vivo. Hence, our lab developed dual-FP Matryoshka technology introduced by a single cassette that contains a stable large Stokes shift (LSS) reference FP nested within a reporter FP (cpEGFP), allowing simple construction of intensiometric sensors with the capacity for ratiometric quantification. The first-generation Green-Orange (GO) Matryoshka cassette established proof of concept but required custom optical setups to maximize achievable dynamic range. Here, we present a genetically encoded calcium sensor that employs optimized second-generation Green-Apple (GA) Matryoshka technology that incorporates a newly designed red LSSmApple fluorophore. LSSmApple provides improved excitation spectrum overlap with cpEGFP, allowing for monochromatic co-excitation with blue light. The exceptionally large Stokes shift of LSSmApple results in improved emission spectrum separation from cpEGFP, which minimizes fluorophore bleed-through and facilitates imaging using standard dichroics and red fluorescent protein (RFP) emission filters. We developed an image analysis pipeline for yeast (Saccharomyces cerevisiae) timelapse imaging that utilizes LSSmApple to segment and track cells for high-throughput quantitative analysis. In summary, we engineered a new fluorescent protein, constructed a genetically encoded calcium indicator (GA-MatryoshCaMP6s), and performed calcium imaging in yeast as a demonstration.

bioengineering↗

Covalent Targeting of Glutamate Cysteine Ligase to Inhibit Glutathione Synthesis

Dysregulated oxidative stress plays a major role in cancer pathogenesis and some types of cancer cells are particularly vulnerable to inhibiting cellular antioxidant capacity. Glutamate-cysteine ligase (GCL) is the first and rate-limiting step in the synthesis of the major cellular antioxidant glutathione (GSH). Developing a GCL inhibitor may be an attractive therapeutic strategy for certain cancer types that are particularly sensitive to oxidative stress. In this study, we reveal a cysteine-reactive covalent ligand EN25 that covalently targets an allosteric cysteine C114 on GCLM, the modifier subunit of GCL, leading to inhibition of GCL activity, lowering of cellular GSH levels, and impaired cell viability in ARID1A-deficient cancer cells that are particularly vulnerable to glutathione depletion, but not in ARID1A-positive cancer cells. Our studies uncover a novel potential ligandable site within GCLM that can be targeted to inhibit the GSH synthesis in cancer cells to target vulnerable cancer cell types.

biochemistry↗

Low-dose cytokine immunotherapy of solid cancers enabled by phagocytic-competent protein co-crystals

Protein therapeutics are often compromised by sub-optimal biodistribution contributing to poor efficacy and adverse events. Drug delivery mechanisms better able to target protein drugs to the disease site and provide localized, sustained release have the potential to transform therapeutic standards. PODS(R) crystals (PODS) are natural-mimetic, micron-scale protein co-crystals engineered to incorporate a protein cargo that can be sustainably released under the action of resident proteases. PODS are efficiently taken up by phagocytic cells with the cargo protein subsequently released in a bioactive form. Since blood-circulating phagocytic cells, including monocytes, are actively recruited into diseased and inflamed tissue, such as the tumour microenvironment, we postulated that monocyte/macrophage-mediated PODS delivery could be used as a molecular "Trojan horse" to efficiently deliver therapeutic proteins to target cells. This could improve the pharmacodynamics and pharmacokinetics of protein drug delivery to treat systemic and disseminated diseases. Interleukin-2 (IL-2) is notoriously toxic at the high doses required for therapeutic efficacy. Here, we demonstrate the therapeutic efficacy and tolerability of low doses of PODS containing IL-2 cargo (PODS-IL-2) administered intravenously in a mouse model of melanoma. We further demonstrate the therapeutic benefit of PODS delivering IL-2, interleukin-15 (IL-15) and interferon gamma (IFN-{gamma}) in a mouse model of renal cell carcinoma at two doses. Efficacy was seen in both doses with the higher dose generating rapid and complete rejection of the tumour in some of the mice treated with each cytokine. This study provides proof-of-concept for the utility of intravenously administered PODS to provide a generalised and widely applicable mechanism to effectively deliver protein drugs for the therapy of cancer and potentially other diseases.

cancer biology↗