Biology subjects

Nagai, T.

Publications and source records attributed to Nagai, T..

36 records · Page 2Linked to original sources

bioRxiv2024

Creating coveted bioluminescence colors for simultaneous myriad-color bioimaging

Bioluminescence, an optical marker that does not require excitation by light, allows researchers to simultaneously observe multiple targets, each exhibiting a different color. Notably, the colors of the bioluminescent proteins must sufficiently vary to enable simultaneous detection. Here, we aimed to introduce a method that can be used to expand the color variation by tuning dual-acceptor bioluminescence resonance energy transfer (BRET). Using this approach, we could visualize multiple targets with up to 20 colors through single-shot acquisition using a color CMOS camera. Overall, this method enables simple and simultaneous observation of numerous biological targets and phenomena.

biophysics↗

bioRxiv2024

Autonomous multicolor bioluminescence imaging in bacteria, mammalian, and plant hosts

Bioluminescence imaging has become a valuable tool in biological research, offering several advantages over fluorescence-based techniques, including the absence of phototoxicity and photobleaching, along with a higher signal-to-noise ratio. Common bioluminescence imaging methods often require the addition of an external chemical substrate (luciferin), which can result in a decrease in luminescence intensity over time and limit prolonged observations. Since the bacterial bioluminescence system is genetically encoded for luciferase-luciferin production, it enables autonomous bioluminescence (auto-bioluminescence) imaging. However, its application to multiple reporters is restricted due to a limited range of color variants. Here, we report five colors auto-bioluminescence system named Nano-lanternX (NLX), which can be expressed in bacterial, mammalian, and plant hosts, thereby enabling auto-bioluminescence in various living organisms. We have also expanded the applications of the NLX system, such as multiplexed auto-bioluminescence imaging for gene expression, protein localization, and dynamics of biomolecules within living mammalian cells.

bioengineering↗

bioRxiv2023

NMDAR Phosphoproteome Controls Synaptic Growth and Learning

In the mammalian brain, NMDA receptors (NMDARs) activation triggers a calcium-dependent signal transduction cascade resulting in postsynaptic remodeling and behavioral learning. However, the phosphoprotein signal flow through this transduction network is poorly understood. Here, we show that NMDAR-dependent phosphorylation drives the assembly of protein signaling complexes that regulate synaptic morphology and behavior. We performed large-scale phosphoproteomic analyses of protein kinase target proteins in successive layers of the signaling network in mouse striatal/accumbal slices. NMDARs activation resulted in the phosphorylation of 194 proteins, including Rho GTPase regulators. CaMKII-mediated phosphorylation of ARHGEF2 increased its RhoGEF activity, thereby activating the RhoA-Rho-kinase pathway. Subsequent phosphoproteomics of Rho-kinase revealed 221 protein targets, including SHANK3. Experimental validation revealed a pathway from NMDAR-dependent calcium influx through CaMKII, ARHGEF2, Rho-kinase, and SHANK3 to coordinate assembly of an actin-tethered postsynaptic complex of SHANK3/NMDAR/PSD95/DLGAP3 for spine growth and aversive learning. These findings show that NMDARs initiate metabolic phosphorylation for learning.

neuroscience↗

bioRxiv2023

Volumetric trans-scale imaging of massive quantity of heterogeneous cell populations in centimeter-wide tissue and embryo

We established a volumetric trans-scale imaging system with an ultra-large field-of-view (FOV) that enables simultaneous observation of millions of cellular dynamics in centimeter-wide three-dimensional (3D) tissues and embryos. Using a custom-made giant lens system with a magnification of 2x and a numerical aperture (NA) of 0.25, and a CMOS camera with more than 100 megapixels, we built a trans-scale scope AMATERAS-2, and realized fluorescence imaging with a transverse spatial resolution of approximately 1.1 {micro}m across an FOV of approximately 1.5 x 1.0 cm2. The 3D resolving capability was realized through a combination of optical and computational sectioning techniques tailored for our low-power imaging system. We applied the imaging technique to 1.2 cm-wide section of mouse brain, and successfully observed various regions of the brain with sub-cellular resolution in a single FOV. We also performed time-lapse imaging of a 1-cm-wide vascular network during quail embryo development for over 24 hours, visualizing the movement of over 4.0 x 105 vascular endothelial cells and quantitatively analyzing their dynamics. Our results demonstrate the potential of this technique in accelerating production of comprehensive reference maps of all cells in organisms and tissues, which contributes to understanding developmental processes, brain functions, and pathogenesis of disease, as well as high-throughput quality check of tissues used for transplantation medicine.

developmental biology↗

bioRxiv2023

Time-deterministic cryo-optical microscopy

We developed a technique to rapidly freeze biological systems during observation under an optical microscope, which allows for detailed optical observation at a precise time in the biological dynamics at high spatial resolution and quantifiability under low-temperature conditions. Our study further found that ion distributions and molecular conformations can be fixed in space and time, demonstrated by visualization of calcium waves rapid-frozen at an instant using fluorescent ion indicators. These results show that time-deterministic cryo-optical microscopy can suspend cellular dynamics while maintaining molecular and ionic states, allowing us to view cellular dynamics in a way that has not been possible before. One-Sentence SummaryRapid freezing of dynamic biological systems during optical observation presents time information to the fixed samples

biophysics↗

bioRxiv2023

Selective plane activation structured illumination microscopy

Resolution enhancement in structured illumination microscopy is hindered when volumetric samples are observed owing to background signals from out-of-focus planes. In this study, we utilized selective plane illumination and reversibly photoswitchable fluorescent proteins to realize structured illumination within the focal plane and eliminate the out-of-focus background. We demonstrated high-resolution fluorescence imaging of volumetric samples, including dense fluorescent objects in a cell spheroid.

biophysics↗

bioRxiv2023

Coordinated multiple cellular processes in tongue development

Dysfunction of primary cilia leads to genetic disorder, ciliopathies, which shows various malformations in many vital organs such as brain. Multiple tongue deformities including cleft, hamartoma and ankyloglossia are also seen in ciliopathies, which yield difficulties in fundamental functions such as mastication and vocalization. Here, we found these tongue anomalies in mice with mutation of ciliary protein. Abnormal cranial neural crest-derived cells (CNCC) failed to evoke Hh signal for differentiation of mesoderm-derived cells into myoblasts, which resulted in abnormal differentiation of mesoderm-derived cells into adipocytes. The ectopic adipose subsequently arrested migration of other mesoderm-derived cells and CNCC. Some aberrant CNCC abnormally differentiated into osteoblasts due to the lack of Hh signal, which migrated into tongue to form ectopic bone. Ankyloglossia was caused by aberrant cell migration due to lack of non-canonical Wnt signaling. In addition to ciliopathies, these tongue anomalies are often observed as non-familial condition in human. We found that these tongue deformities could be reproduced in wild-type mice by simple mechanical manipulations in CNCC to disturb cellular processes which were disrupted in mutant mice. Thus, tongue development requires coordinated multiple cellular processes (cell-cell contact, migration and differentiation). Our results provide hints for possible future treatment in ciliopathies.

developmental biology↗

bioRxiv2022

Negative legacy effects of past forest use on plant diversity in semi-natural grasslands on ski slopes

Over the past century, grassland and forest ecosystems globally have been heavily influenced by land use changes driven by diverse socioeconomic activities. Ski resorts are a modern land-use type associated with biodiversity loss in mountain ecosystems worldwide. Below the treeline, by contrast, some ski slopes have been shown to provide suboptimal semi-natural habitats for native grassland plants and animals, depending on specific construction and management practices. We compared environmental factors and grassland vegetation between two types of ski slopes in central Japan with different land-use histories: slopes constructed on old pastures (pasture slopes) and slopes constructed by clearing secondary forests or Larix kaempferi plantations established on abandoned pastures during the 1940s-1990s (forest slopes). We examined the effects of land use history and machine grading as well as other environmental factors on ski slope vegetation, including total species richness and the richness of native, endangered, and exotic plants, using a total of 108 plots of 2 m x 10 m. Compared to pasture slopes, forest slopes exhibited significantly lower richness of total plants and native grassland species, including endangered species. Forest slopes were more graded than pasture slopes, resulting in lower native and higher exotic grassland species richness. A significantly lower duration of direct sunlight on forest slopes than on pasture slopes possibly decreased endangered species diversity. The lower species richness on forest slopes may be partly caused by seed dispersal limitations. Our findings demonstrate that ski slopes have good potential to support numerous native grassland plant species, including endangered species, but this potential is significantly and negatively affected by forest use history and concomitant environmental changes. The conservation of semi-natural conditions on pasture slopes as habitats for native grassland species can be promoted through the maintenance of annual mowing practices, avoidance of machine grading, and wider ski courses. NomenclatureYonekura & Kajita (2003) BG Plants: index of Japanese names and scientific names (YList; http://ylist.info/index.html)

plant biology↗

bioRxiv2022

High salt induces cognitive impairment via the angiotensin II-AT1 and prostaglandin E2-EP1 systems

High salt (HS) intake is a known risk factor for hypertension and dementia. Clinical studies have shown that antihypertensive drugs can decrease the incidence of dementia. Accordingly, a strong relationship can be suggested between hypertension and cognitive impairment. It is well-known that angiotensin II (Ang II)-AT1 and prostaglandin E2 (PGE2)-EP1 systems are involved in hypertension and neurotoxicity. However, the involvement of these systems in HS-mediated hypertension and emotional and cognitive impairments remains unclear. Herein, we demonstrated that hypertension and impaired social behavior and object recognition memory following HS intake could be associated with tau hyperphosphorylation, decreased phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII), and postsynaptic density protein 95 (PSD95) expression in the prefrontal cortex and hippocampus of mice. These changes were blocked by pharmacological treatment with losartan, an Ang II receptor blocker (ARB), or EP1 gene knockout. Our findings suggest that Ang II-AT1 and PGE2-EP1 systems could be novel therapeutic targets for hypertension-induced dementia.

neuroscience↗

bioRxiv2022

Isoform-specific mutation in Dystonin-b gene causes late-onset protein aggregate myopathy and cardiomyopathy

Dystonin (DST), which encodes cytoskeletal linker proteins, express three tissue-selective isoforms: neural DST-a, muscular DST-b, and epithelial DST-e. DST mutations cause different disorders including hereditary sensory and autonomic neuropathy 6 (HSAN-VI) and epidermolysis bullosa simplex; however, etiology of the muscle phenotype in DST-related diseases has been unclear. Because DST-b contains all of the DST-a-encoding exons, known HSAN-VI mutations could affect both DST-a and DST-b isoforms. To investigate the specific function of DST-b in striated muscles, we generated a Dst-b-specific mutant mouse model harboring a nonsense mutation. Dst-b mutant mice exhibited late-onset protein aggregate myopathy and cardiomyopathy without neuropathy. We observed desmin aggregation, focal myofibrillar dissolution, and mitochondrial accumulation in striated muscles, which are common characteristics of myofibrillar myopathy. We also found nuclear inclusions containing p62, ubiquitin, and SUMO proteins with nuclear envelope invaginations as a unique pathological hallmark in Dst-b mutation-induced cardiomyopathy. RNA sequencing analysis revealed changes in expression of genes responsible for cardiovascular functions. In silico analysis identified DST-b alleles with nonsense mutations in populations worldwide, suggesting that some unidentified hereditary myopathy and cardiomyopathy are caused by DST-b mutations. Here, we demonstrate that the Dst-b isoform is essential for long-term maintenance of striated muscles.

pathology↗

bioRxiv2021

Optical and computational dissection of emergent prefrontal rewiring to encode fear memory

Associative learning is crucial for adapting to environmental changes. The encoding of associative learning involves the dorso-medial prefrontal cortex (dmPFC), and is underpinned by interactions within the resident neuronal population. However, the nature of this population coding is poorly understood. Here we developed a pipeline for computational dissection and longitudinal two-photon imaging of neural population activities in the mouse dmPFC during fear-conditioning procedures, enabling us to detect learning-dependent changes in the dmPFC topology. Through regularized regression methods and graphical modeling, we found fear conditioning organized neuronal ensembles encoding conditioned responses (CR), with enhancing their coactivity, functional connectivity, and association with conditioned stimuli (CS). This suggests that fear conditioning drives dmPFC reorganization to generate novel associative circuits for CS-to-CR transformation. Importantly, neurons strongly responding to unconditioned stimuli (US) during conditioning anterogradely became a hub of the CR ensemble. Altogether, we demonstrate learning-dependent dynamic modulation of population coding structured on an activity-dependent hub-network formation within the dmPFC. TeaserOptical and computational dissection uncovered how prefrontal cortical networks are rewired to encode new associative memory Significance statementAnimals learn to adapt to changing environments. Associative learning is one of the simplest types of learning that has been intensively studied over the past century. Recent development in molecular, genetic, and optogenetic methods has enabled the identification of a neural population encoding the associative memory in the brain. However, it remains unclear how information is stored and processed by the neural population to encode and retrieve the associative memory. To investigate the nature of this population coding, we developed an optical and computational dissection method, demonstrating how associative learning drives reorganization of the neural network in the dorso-medial prefrontal cortex and generates novel circuits for associative memory and signal transformation.

neuroscience↗

bioRxiv2020

Trans-scale-scope to find rare cellular activity in sub-million cells

In many phenomena of biological systems, not a majority, but a minority of cells act on the entire multicellular system causing drastic changes in the system properties. To understand the mechanisms underlying such phenomena, it is essential to observe the spatiotemporal dynamics of a huge population of cells at sub-cellular resolution, which is difficult with conventional tools such as microscopy and flow cytometry. Here, we describe an imaging system named AMATERAS that enables optical imaging with an over-one-centimeter field-of-view and a-few-micrometer spatial resolution. This trans-scale-scope has a simple configuration, composed of a low-power lens for machine vision and a hundred-megapixel image sensor. We demonstrated its high cell-throughput, capable of simultaneously observing more than one million cells. We applied it to dynamic imaging of calcium ions in HeLa cells and cyclic-adenosine-monophosphate in Dictyostelium discoideum, and successfully detected less than 0.01% of rare cells and observed multicellular events induced by these cells.

cell biology↗

bioRxiv2019

AUTS2 regulation of synapses for proper synaptic inputs and social communication.

Impairments in synapse development are thought to cause numerous psychiatric disorders. Autism susceptibility candidate 2 (AUTS2) gene has been associated with various psychiatric disorders, such as autism and intellectual disabilities. Although roles for AUTS2 in neuronal migration and neuritogenesis have been reported, its involvement in synapse regulation remains unclear. In this study, we found that excitatory synapses were specifically increased in the Auts2-deficient primary cultured neurons as well as Auts2 mutant forebrains. Electrophysiological recordings and immunostaining showed increases in excitatory synaptic inputs as well as c-fos expression in Auts2 mutant brains, suggesting that an altered balance of excitatory and inhibitory inputs enhances brain excitability. Auts2 mutant mice exhibited autistic-like behaviors including impairments in social interaction and altered vocal communication. Together, these findings suggest that AUTS2 regulates excitatory synapse number to coordinate E/I balance in the brain, whose impairment may underlie the pathology of psychiatric disorders in individuals with AUTS2 mutations.

neuroscience↗

bioRxiv2019

Biophysical properties of intermediate states of EMT outperform both epithelial and mesenchymal states.

Potential metastatic cells can dissociate from a primary breast tumor by undergoing an epithelial-to-mesenchymal transmission (EMT). Recent work has revealed that cells in intermediate states of EMT acquire an augmented capacity for tumor-cell dissemination. These states have been characterized by molecular markers, but the structural features and the cellular mechanisms that underlie the acquisition of their invasive properties are still unknown. Using human mammary epithelial cells, we generated cells in intermediate states of EMT through the induction of a single EMT-inducing transcription factor, ZEB1, and cells in a mesenchymal state by stimulation with TGF{beta}. In stereotypic and spatially-defined culture conditions, the architecture, internal organization and mechanical properties of cells in the epithelial, intermediate and mesenchymal state were measured and compared. We found that the lack of intercellular cohesiveness in epithelial and mesenchymal cells can be detected early by microtubule destabilization and the repositioning of the centrosome from the cell-cell junction to the cell center. Consistent with their high migration velocities, cells in intermediate states produced low contractile forces compared with epithelial and mesenchymal cells. The high contractile forces in mesenchymal cells powered a retrograde flow pushing the nucleus away from cell adhesion to the extracellular matrix. Therefore, cells in intermediate state had structural and mechanical properties that were distinct but not necessarily intermediate between epithelial and mesenchymal cells. Based on these observations, we found that a panel of triple-negative breast cancer lines had intermediate rather than mesenchymal characteristics suggesting that the structural and mechanical properties of the intermediate state are important for understanding tumor-cell dissemination.

cell biology↗

bioRxiv2018

Bioluminescent Multi-Characteristic Opsin for monitoring visual cortical activity upon optical stimulation

Non-invasive detection of neural activity is important for the diagnosis of neurological diseases, evaluation of therapeutic outcomes, and collection of real-time feedback for stimulation based therapeutic approaches. In the case of vision loss, due to retinal degeneration, optic neuropathy, or enucleation of the eye, there is a need to map changes in visual cortical activity during disease progression and subsequent vision restoration by retinal, optic nerve or cortical stimulation. Existing technologies allow interrogation of neuronal circuits by both read and write activity, albeit with inherent limitations. Here, we present the development of bioluminescent multi-characteristic opsin (bMCO-11), which is comprised of a highly photosensitive ambient light-activatable domain and a Ca2+-sensitive bioluminescence reporter. The high quantum efficiency of bMCO-11 enables light activation and recording of cellular activity upon local as well as wide area optical stimulation. Furthermore, persistent Ca2+ influx was achieved by bioluminescence based cyclic activation of the opsin-domain of bMCO-11 by transporting the ions only into bMCO expressing cells. This allowed us to continuously monitor visual cortical activity in wild type and retinal degenerated mice, without requiring any additional external excitation source.

neuroscience↗

bioRxiv2017

In vivo brain activity imaging of interactively locomoting mice

Electrophysiological field potential dynamics have been widely used to investigate brain functions and related psychiatric disorders. Conversely, however, various technical limitations of conventional recording methods have limited its applicability to freely moving subjects, especially when they are in a group and socially interacting with each other. Here, we propose a new method to overcome these technical limitations by introducing a bioluminescent voltage indicator called LOTUS-V. Using our simple and fiber-free recording method, named \"SNIPA,\" we succeeded in capturing brain activity in freely-locomotive mice, without the need for complicated instruments. This novel method further allowed us to simultaneously record from multiple independently-locomotive animals that were interacting with one another. Further, we successfully demonstrated that the primary visual cortex was activated during the interaction. This methodology will further facilitate a wide range of studies in neurobiology and psychiatry.

neuroscience↗

bioRxiv2017

A spontaneously blinking fluorescent protein for simple single laser super-resolution live cell imaging

Super-resolution imaging techniques based on single molecule localization microscopy (SMLM) broke the diffraction limit of optical microscopy in living samples with the aid of photoswitchable fluorescent probes and intricate microscopy systems. Here, we developed a fluorescent protein, SPOON, which can be switched-off by excitation light illumination and switched-on by thermally-induced dehydration resulting in an apparent spontaneous blinking behavior. This unique property of SPOON provides a simple SMLM-based super-resolution imaging platform which requires only a single 488 nm laser.

biophysics↗

bioRxiv2017

Concept and in-silico assessment of an algorithm for monitoring cytosolic fluorescent aggregates in cells.

Autophagy is an evolutionary conserved pathway, by which eukaryotic cells degrade long-living cellular proteins and intracellular organelles, to maintain a pool of available nutrients. Impaired autophagy has been associated to important pathophysiological conditions, and this is the reason why several techniques have been developed for its correct assessment and monitoring. Fluorescence microscopy is one of these tools, which relies on the detection of specific fluorescence changes of targeted GFP-based reporters in dot-like organelles in which autophagy is executed. Currently, several procedures exist to count and segment this punctate structures in the resulting fluorescence images, however, they are either based on subjective criteria, or no information is available related to them. Here we present the concept of an algorithm for a semi-automatic detection and segmentation in 2D fluorescence images of spot-like structures similar to those observed under induction of autophagy. By evaluating the algorithm on more than 20000 simulated images of cells containing a variable number of punctate structures of different sizes and different levels of applied noise, we demonstrate its high robustness of puncta detection, even on a high noise background. We further demonstrate this feature of our algorithm by testing it in experimental conditions of a high non-specific background signal. We conclude that our algorithm is a suitable tool to be tested in biologically-relevant contexts.

bioinformatics↗