Biology subjects

Miyawaki, A.

Publications and source records attributed to Miyawaki, A..

At least 19 recordsLinked to original sources

bioRxiv2026

OxyBLI: A Genetics-Based Approach for In Vivo Real-Time Visualization of Tissue Oxygenation Dynamics

Accurate measurement of cellular oxygen levels is essential for understanding the balance between oxygen demand and supply in tissues. However, conventional methods only yield compromised results. We harnessed the oxygen dependence of bioluminescence to develop OxyBLI--a noninvasive optical method that directly monitors oxygen levels in specific cell populations of intact experimental animals. We characterized OxyBLI signals across various critical situations associated with common interventions. Hypoxic breathing and subsequent systemic tissue hypoxia caused blood to be redistributed in a way that prioritized brain oxygenation. In contrast, hyperoxic breathing sharply increased tissue oxygenation, which promptly returned to the target level owing to a vasoconstrictor response. These findings are expected to help resolve the long-standing clinical issue regarding the risks and benefits of administering supplemental oxygen to acutely ill patients. Our multifaceted approach, which presents multiple challenges to individual animals over time, will advance our understanding of the delicate interaction between hypoxia and hyperoxia.

physiology↗

bioRxiv2025

FuChi: A cell cycle biosensor for investigating cell-cycle kinetics during avian development.

The ability to monitor the proliferative status of live cells both in vitro and in vivo over time has revolutionised our understanding of development, growth and disease. This was first made possible by fluorescent ubiquitination-based cell cycle indicator (Fucci) technology, which distinguishes specific cell cycle phases through the reciprocal degradation of fluorescently tagged, truncated forms of human CDT1 and GMNN proteins. Fucci genetic systems have been successfully implemented in transgenic mice, zebrafish, and axolotls. To date, no viable, stably expressing Fucci line has been developed in an avian species. Although a range of continuously improving Fucci constructs have been developed in recent years, existing in vivo Fucci models remain limited because they rely on older reporter technology that fails to distinguish cells in S, G2, and M phases or to label cells in early G1. As a result, these models can be challenging to interpret and their utility for continuous cell tracking and precise analysis of cell-cycle dynamics is limited. Here, we introduce FuChi, a multicistronic Fucci-expressing chicken line incorporating a newly optimised reporter construct composed of an mCerulean-tagged Histone H1.0 linker protein fused via a self-cleaving 2A peptide to the tandem Fucci(CA) cell cycle biosensor, with additional epitope tags included for detection in fixed tissues. We show that this system accurately discriminates and permits tracking of cells in G1, S, G2, and M phases both in vitro and in vivo, enabling faithful visualisation of cell cycle status in intact tissues and organs. Using FuChi embryos, we mapped proliferation dynamics across developing tissues, analysed cell cycle states of migrating cells, and performed live imaging of early embryos. These latter experiments revealed that transition from S phase may be a key morphogenetic event during gastrulation as mesendoderm cells egress from the primitive streak to form embryonic structures including the prechordal plate. Pairing this advanced reporter with the intrinsic experimental advantages of the chicken embryo positions FuChi as a premier in vivo system for studying cell-cycle kinetics in development, delivering clear technological improvements over current Fucci models. FuChi chickens provide a powerful new resource for studying embryonic development, organ growth, tissue homeostasis, disease processes, and infection responses.

developmental biology↗

bioRxiv2025

A bright synthetic near-infrared luciferin enhances the capabilities of deep-tissue bioluminescence imaging using firefly luciferases

Synthetic bioluminescence reactions exhibiting near-infrared (NIR)-shifted spectra have been explored to improve deep-tissue imaging through the design of firefly luciferin analogues. Although the NIR bioluminescence reactions improve the tissue penetration of bioluminescence signals from deep tissues, their photon output is markedly lower compared to the natural reaction with D-luciferin and firefly luciferase (Fluc), often by an order of magnitude or more. Consequently, in most instances, the sensitivity of NIR bioluminescence imaging (NIR-BLI) has not yet substantially surpassed that of BLI with the natural firefly reaction. Here, we present a synthetic firefly luciferin, named AkaSuke, that generates intense NIR bioluminescence ({lambda}max = 680 nm) in reaction with Fluc, greatly improving the detection sensitivity beyond that of the D-luciferin/Fluc reaction for targeting deep tissue. AkaSuke enables sensitive visualizations of ectopic hematogenesis through entire tissues of mice over time following transplantation of bone marrow stem cells labeled with Fluc. We additionally identify a Japanese firefly luciferase, DkumLuc1, that displays higher catalytic activities for bioluminescence emission of AkaSuke compared to typical Fluc, resulting in detection sensitivity comparable to that of AkaLumine/Akaluc reaction, one of the most sensitive bioluminescence systems for deep tissue imaging. We further propose the potential of the AkaSuke/DkumLuc1 reaction as an orthogonal pair with the AkaLumine/Akaluc for sensitive dual-target tracking in mice. Overall results suggest that AkaSuke enhances the capabilities of deep-tissue bioluminescence imaging using Fluc and its variant, and could serve as an emerging benchmark for the molecular design of NIR luciferin analogues.

cancer biology↗

bioRxiv2024

Direct Intercellular Vesicle Exchange between Adjacent Cells

Intercellular communication plays a central role in the development and integrity of multicellular organisms. Vesicle transfer, especially through extracellular space, has recently been highlighted as a critical intercellular communication modality, carrying nucleic acids, proteins, and others to distant cells. Previously, we demonstrated that extracellular vesicles induce "phenotypic synchronization of cells (PSyC)" during stem cell differentiation. While examining the mechanism underlying PSyC, we discovered a novel form of cellular communication mediated by direct intercellular vesicle exchange (DIVE) between adjacent cells across the plasma membrane (PM). By achieving cell-wide and high-spatiotemporal resolution imaging of vesicles labeled with fusion proteins of CD63 or CD81 to StayGold, a photostable fluorescent marker, we observed small vesicles (50-500 nm in diameter) directly transferred between adjacent cells. These vesicles moved at approximately 1 {micro}m/s and crossed PM in approximately 10-20 seconds. Furthermore, multiple vesicles traversed nearly identical sites of PM, suggesting the presence of specific routes or structures, potentially including a pore, mediating the vesicle transfer. Three-dimensional electron microscopy provided supportive observations for traversing vesicles with single membrane. These vesicles, named InterCellular Vesicles (InterCVs), were observed to colocalize with nucleic acids, including mRNA, microRNA, and DNA, suggesting the exchange of nucleic acid-mediated information, potentially inducing PSyC, between adjacent cells. Our discovery, DIVE, reveals a previously unknown modality of cell-cell communication, with the potential to reshape our understanding of cellular biology.

cell biology↗

bioRxiv2024

Single MAPT knock-in mouse models of frontotemporal dementia for sharing with the neurodegenerative research community

We recently reported development of human MAPT knock-in mice that carry single or double pathogenic mutations of frontotemporal dementia. However, it takes more than 14 months for the line with the most aggressive phenotypes to exhibit tau pathology without forming high-order tau oligomers, along with concomitant abnormal behavior. We thus generated MAPT knock-in mice carrying triple mutations, among which the MAPTP301S;Int10+3;S320F line exhibited robust pathology starting earlier than 6 months. Tau accumulation took place mainly in the thalamus, hypothalamus, amygdala and entorhinal cortex, but less so in the hippocampus, leading to synaptic loss, atrophy and behavioral abnormalities. Crossbreeding MAPTP301S;Int10+3;S320F with App knock-in mice AppNL-G-F resulted in the manifestation of tau pathology in the hippocampus and cortex. These mutant mice will be valuable tools for understanding the mechanisms of frontotemporal dementia, Alzheimers disease and other tauopathies.

neuroscience↗

bioRxiv2024

Epilepsy protein myoclonin1 interacts with inositol 1,4,5-trisphosphate (IP3) receptor and reduces Ca2+ store in endoplasmic reticulum

Mutations of EFHC1 gene have been identified in patients with epilepsies including juvenile myoclonic epilepsy (JME), and mice with Efhc1 deficiency exhibit epileptic phenotypes. Myoclonin1 protein encoded by EFHC1 is not expressed in neurons but in cells with motile cilia including those of choroid plexus and ependymal cells which form an epithelial layer lining brain ventricles. Detailed molecular basis of epilepsies caused by EFHC1 mutations, however, remain unclear. Here we report that myoclonin1 is well co-expressed with inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) at choroid plexus and ependymal cells and these two proteins bind each other. Endoplasmic reticulum (ER) of Efhc1-deficient mouse (Efhc1-/-) cells contains larger levels of calcium ions (Ca2+) than that of wild-type (WT) mice, and IP3-induced Ca2+ release (IICR) from ER is higher in Efhc1-/- cells than that of WT. Furthermore, myoclonin1 revealed to interact with PRKCSH, also known as a protein kinase C substrate 80K-H which interacts with IP3R1. Myoclonin1 further binds to IP3R2 and IP3R3. Thus, our results indicate that myoclonin1 modulates ER-Ca2+ homeostasis through interactions with IP3Rs and PRKCSH, and suggest that myoclonin1 dysfunctions cause impaired intracellular Ca2+ mobilization. Its relevance to the epileptic phenotypes of patients with EFHC1 mutations is now of interest.

physiology↗

bioRxiv2024

Plaque-associated endogenous IgG and its impact on immunohistochemical detection of mouse monoclonal IgG antibodies in mouse models of Alzheimer's disease

Experimental studies for Alzheimers disease (AD) have largely depended on transgenic mice with {beta}-amyloidosis. Here, we report plaque-associated endogenous immunoglobulin G (PA-IgG) and its impact on indirect immunohistochemical detection of mouse monoclonal IgG antibodies (Ms monoclonal IgG Abs) in the brain of AD mouse models. Immunostaining for Ms IgG in AD mouse models demonstrated endogenous IgG in the brain parenchyma accumulated on microglia associated with amyloid {beta} (A{beta}) plaques and/or A{beta} plaques themselves. This PA-IgG caused robust off-target binding of secondary Abs against Ms IgG (H+L) in indirect immunohistochemistry using Ms monoclonal IgG Abs. Blocking with Fab fragments of anti-Ms IgG (H+L) Ab was not effective against off-target binding. Unexpectedly, we found that secondary Abs that specifically recognize Ms IgG1, 2a, 2b, and 3 did not cause off-target binding on frozen brain sections of AppNL-G-F/NL-G-Fmice, and enabled specific labeling of Ms monoclonal IgG Abs in the AD mouse model brains. We further demonstrated that indirect detection with a conventional secondary Ab against Ms IgG (H+L) Ab could lead to erroneous conclusions regarding A{beta} plaque burden and phosphorylated tau accumulation in AppNL-G-F/NL-G-Fmice, and the use of Ms IgG subclass specific secondary Abs allowed to avoid the inevitable impediment caused by the endogenous IgG accumulation. Specific indirect detection of Ms monoclonal IgG Abs in AD mouse models by the use of secondary Abs against Ms IgG subclass would accelerate AD research by expanding the choice of Abs available for histochemical analysis in AD studies.

neuroscience↗

bioRxiv2024

Comparison of monomeric variants of StayGold

StayGold is a bright and highly photostable fluorescent protein (FP) that forms an obligate dimer, thereby limiting its application as a soluble marker. On the basis of the structural information of this FP, we disrupted the dimerization to generate a monomeric variant, mStayGold, which inherits both the extremely high photostability and the high practical brightness of StayGold, for molecular fusion and membrane-targeting applications. Meanwhile, two other research groups have independently monomerized StayGold using different strategies. As a result, multiple StayGold monomers are currently available, creating confusion in the research community. In the present study, we investigated the three basic properties--photostability, brightness, and dispersibility--of these monomers by performing detailed side-by-side comparisons. This study highlights the difficulties of StayGold monomerization with emphasis on the tradeoff between photostability and brightness in FP technology.

bioengineering↗

bioRxiv2023

StayGold Photostability under Different Illumination Modes

StayGold is a bright fluorescent protein (FP) that is over one order of magnitude more photostable than any of the currently available FPs across the full range of illumination intensities used in widefield microscopy and structured illumination microscopy, the latter of which is a widefield illumination-based technique. To compare the photostability of StayGold under other illumination modes with that of three other green-emitting FPs, namely EGFP, mClover3, and mNeonGreen, we expressed all four FPs as fusions to histone 2B in HeLa cells. Unlike the case of widefield microscopy, the photobleaching behavior of these FPs in laser scanning confocal microscopy (LSCM) is complicated. The outstanding photostability of StayGold observed in multi-beam LSCM was variably attenuated in single-beam LSCM, which produces intermittent and instantaneously strong illumination. We systematically examined the effects of different single-beam LSCM beam-scanning patterns on the photostability of the FPs. This study offers relevant guidelines for researchers who aim to achieve sustainable imaging by resolving problems related to FP photostability.

cell biology↗

bioRxiv2023

Development of two mouse strains conditionally expressing bright luciferases with distinct emission spectra as new tools for in vivo imaging

In vivo bioluminescence imaging (BLI) has been an invaluable noninvasive method to visualize molecular and cellular behaviors in laboratory animals. Bioluminescent reporter mice possessing luciferases for general use have been limited to a classical luciferase, Luc2, from Photinus pyralis, and have been extremely powerful for various in vivo studies. However, applicability of reporter mice for in vivo BLI could be further accelerated by increasing light intensity using other luciferases and/or improving the biodistribution of their substrates in animal body. Here, we created two Cre-dependent reporter mice incorporating luciferases: oFluc derived from Pyrocoeli matsumurai and Akaluc, both of which had been reported previously to be brighter than Luc2 when using appropriate substrates; we then tested their bioluminescence in neural tissues and other organs in living mice. When expressed throughout the body, both luciferases emitted an intense yellow (oFluc) or far-red (Akaluc) light easily visible to the naked eye. Moreover, oFluc and Akaluc were similarly bright in the pancreas for in vivo BLI. However, Akaluc was superior to oFluc for brain imaging, because its substrate, AkaLumine-HCl, was distributed to the brain more efficiently than the oFluc substrate, D-luciferin. We also demonstrated that the light produced by oFluc and Akaluc was sufficiently spectrally distinct for dual-color imaging in a single living mouse. Taken together, these novel bioluminescent reporter mice are an ideal source of cells with bright bioluminescence and may facilitate the in vivo BLI of various tissues/organs for preclinical and biomedical research in combination with a wide variety of Cre-driver mice.

bioengineering↗

bioRxiv2022

Spatial frequency-based correction of the spherical aberration in living brain imaging

We previously developed a fully automated spherical aberration compensation microscope system, Deep-C, to obtain spherical aberration-free images, but the contrast-based algorithm (Peak-C) may limit applications for low signal-to-noise ratio images. Herein we propose a new spatial frequency-based algorithm called Peak-F and compared its performance to Peak-C. Unlike Peak-C, Peak-F is robust to any noise level since it is independent of the dynamic range of the images, and it does not suffer from image saturation. Finally, Peak-F was implemented in a two-photon microscope to observe living aged and young mouse brains. Consequently, the average refractive index of brain tissue was higher in old mice than in young mice. The Peak-F algorithm determines high-resolution microscopic images stably and robustly.

biophysics↗

bioRxiv2021

Integrative analysis of scRNAs-seq and scATAC-seq revealed transit-amplifying thymic epithelial cells expressing autoimmune regulator

Medullary thymic epithelial cells (mTECs) are critical for self-tolerance induction in T cells via promiscuous expression of tissue-specific antigens (TSAs), which are controlled by transcriptional regulator AIRE. Whereas AIRE-expressing (Aire+) mTECs undergo constant turnover in the adult thymus, mechanisms underlying differentiation of postnatal mTECs remain to be discovered. Integrative analysis of single-cell assays for transposase accessible chromatin (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) suggested the presence of proliferating mTECs with a specific chromatin structure, which express high levels of Aire and co-stimulatory molecules CD80 (Aire+CD80hi). Proliferating Aire+CD80hi mTECs detected by using Fucci technology express a minimal level of Aire-dependent TSAs and are converted into quiescent Aire+CD80hi mTECs expressing high levels of TSAs after a transit amplification. These data provide evidence for the existence of transit amplifying Aire+mTEC precursors during Aire+mTEC differentiation process of the postnatal thymus.

immunology↗

bioRxiv2021

Novel System to Monitor In Vivo Neural Graft Activity After Spinal Cord Injury

Expectations for neural stem/progenitor cell (NS/PC) transplantation as a treatment for spinal cord injury (SCI) are increasing. However, whether and how grafted cells are incorporated into the host neural circuit and contribute to motor function recovery remain unknown. The aim of this project was to establish a novel non-invasive in vivo imaging system to visualize the activity of neural grafts by which we can simultaneously demonstrate the circuit-level integration between the graft and host, and the contribution of graft neuronal activity to host behaviour. We introduced Akaluc, a newly engineered luciferase, under control of a potent neuronal activity-dependent synthetic promoter, E-SARE, into NS/PCs and engrafted the cells into SCI model mice. Through the use of this system, we reveal that the activity of grafted cells was integrated with host behaviour and driven by host neural circuit inputs. This non-invasive system is expected to help elucidate the therapeutic mechanism of cell transplantation treatment for SCI and determine better therapy techniques that maximize the function of cells in the host circuit.

neuroscience↗

bioRxiv2021

Metastatic Single Tumor Cells Evade NK Cell-mediated Killing by Thrombin-mediated Loss of the Activating Ligand CD155/PVR/Necl-5

Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is not known. Here we have combined ultra-sensitive bioluminescence whole body imaging with intravital two-photon microscopy involving genetically-encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hrs of arrival, but not thereafter. Intravital dynamic imaging revealed that a disseminated tumor cell in a pulmonary capillary interacts with an NK cell every 2 hrs on average. In the first 4 hrs after tumor cell arrival, 50% of such encounters lead to tumor cell death but after 24 hrs of arrival, nearly 100% of the interactions result in the survival of the tumor cell. This evasion of NK cell surveillance is mediated by thrombin-dependent loss of cell surface CD155/PVR/Necl-5, a ligand for the NK cell activating receptor DNAM-1. This loss prevents the NK cell signaling needed for effective killing of tumor targets. By quantitatively visualizing the evasion of NK cell surveillance, we have uncovered a molecular mechanism for cancer evasion and provided an explanation for the anti-metastatic effect of anticoagulants. SUMMARYIntravital functional two-photon microscopy reveals that metastatic tumor cells lodged in pulmonary capillaries acquire resistance to patrolling NK cells. Protease-mediated loss of the activating ligand CD155/PVR/Necl-5 on tumor cells prevents NK cells from ERK activation and tumor cell killing.

immunology↗

bioRxiv2020

Distributed sensory coding by cerebellar complex spikes in units of cortical segments

Sensory processing is essential for motor control. Climbing fibers from the inferior olive transmit sensory signals to Purkinje cells, but how the signals are represented in the cerebellar cortex remains elusive. We examined the olivocerebellar organization of the mouse brain by optically measuring complex spikes (CSs) evoked by climbing fiber inputs over the entire dorsal surface of the cerebellum. We discovered that the surface was divided into approximately 200 segments each composed of [~]100 Purkinje cells that fired CSs synchronously. Our in vivo imaging of evoked responses revealed that whereas stimulation of four limb muscles individually similar global CS responses across nearly all segments, the timing and location of a stimulus were derived by Bayesian inference from coordinated activation and inactivation of multiple segments on a single trial basis. Our findings suggest that the cerebellum performs segment-based distributed population coding by assembling probabilistic sensory representation in individual segments.

neuroscience↗

bioRxiv2020

Fast scanning high optical invariant two-photon microscopy for monitoring a large neural network activity with cellular resolution

Fast and wide imaging with single-cell resolution, high signal-to-noise ratio and no optical aberration has the potential to open up new avenues of investigation in biology. However, this imaging is challenging because of the inevitable tradeoffs among those parameters. Here, we overcome the tradeoffs by combining a resonant scanning system, a large objective with low magnification and high numerical aperture, and highly sensitive large-aperture photodetectors. The result is a practically aberration-free, fast scanning high optical invariant two-photon microscopy (FASHIO-2PM) that enables calcium imaging from a large network composed of [~]16k neurons at 7.5 Hz in a 9 mm2 contiguous image plane including more than 10 sensory-motor and higher-order regions of the cerebral cortex in awake mice. Through a network analysis based on single-cell activities, we discover that the brain exhibits small-world-ness rather than scale-freeness. FASHIO-2PM will enable revealing biological dynamics by simultaneous monitoring of macroscopic activity and its composing elements.

neuroscience↗

bioRxiv2020

Two new coral fluorescent proteins of distinct colorsfor sharp visualization of cell-cycle progression

We cloned and characterized two new coral fluorescent proteins: h2-3 and 1-41. h2-3 formed an obligate dimeric complex and exhibited bright fluorescence. On the other hand, 1-41 formed a highly multimeric complex and exhibited dim red fluorescence. We engineered 1-41 into AzaleaB5, a practically useful red-emitting fluorescent protein for cellular labeling applications. We fused h2-3 and AzaleaB5 to the ubiquitination domains of human Geminin and Cdt1, respectively, to generate a new color variant of Fucci (Fluorescent Ubiquitination-based Cell-Cycle Indicator): Fucci5. We found Fucci5 provided brighter nuclear labeling for monitoring cell cycle progression than the 1st and 2nd generations that used mAG/mKO2 and mVenus/mCherry, respectively.

cell biology↗

bioRxiv2018

Moments reconstruction and local dynamic range compression of high order Superresolution Optical Fluctuation Imaging

AbstractSuper-resolution Optical Fluctuation Imaging (SOFI) offers a simple and affordable alternative to other super-resolution (SR) imaging techniques. The theoretical resolution enhancement of SOFI scales linearly with the cumulants order, while imaging conditions are less phototoxic to living samples as compared to other SR methods. High order SOFI could, therefore, be a method of choice for dynamic live cell imaging. However, due to cusp-artifacts and to dynamic range expansion of pixel intensities, this promise has not been materialized as of yet. Here we investigated and compared high order moments vs. high order cumulants SOFI reconstructions. We demonstrate that even-order moments reconstructions are intrinsically free of cusp artifacts, allowing for a subsequent deconvolution operation to be performed, hence enhancing the resolution even further. High order moments reconstructions performance was examined for various (simulated) conditions and applied to (experimental) imaging of QD labeled microtubules in fixed cells, and actin stress fiber dynamics in live cells.

bioengineering↗