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Biology subjects

Shibata, Y.

Publications and source records attributed to Shibata, Y..

At least 19 recordsLinked to original sources

The Role of Environmental Information and the Limitations of Predictability with Transformer-based Time-Series Models for complex Astrocyte Trajectories

Morphogenesis is the process by which an organism develops from a single cell into a complex organism, and one factor contributing to this complexity is the interaction between cells and their environment. In heterogeneous environments comprising differing cell types and polymeric structures, the observed complexity appears to approach an infinite value. Recent developments in imaging tools and deep learning for image analysis enable the extraction of meaning from terabyte-scale images at a realistic computational cost. In this paper, we used a Transformer-based model to predict the migration trajectories of astrocytes during mouse brain development, aiming to quantitatively elucidate (i) what makes prediction difficult, i.e., the structure of predictability and its theoretical limit, and (ii) to what extent environmental information (vascular structure) and multimodal modeling of the predictive distribution can improve prediction. Based on a bivariate Gaussian distribution ($\mu, \sigma, \rho$) at each time point, we predicted future positions while accounting for uncertainty and evaluate the quality of the predictive distribution. We report three main findings. First, point-prediction accuracy reached a fundamental limit set by the irreducibility of the movement direction; the error curve was described with high accuracy by the mean-squared-displacement law of a persistent random walk. Second, extending the predictive distribution from a single bivariate Gaussian to a continuous mixture (mixture density network, MDN) captured the run/rest bimodality without discretization and substantially improved the quality of the predictive distribution (negative log-likelihood, NLL) while preserving point-prediction accuracy. Third, a model that integrated vascular environmental information into this multimodal model achieved the best performance in point-prediction accuracy, consistently across three training seeds, showing that environmental information contributes to prediction in combination with multimodal output. This study presents both an improvement of the predictive distribution for complex cell migration and a framework for quantifying the limit of predictability based on movement statistics.

bioinformatics↗

Standardizing image-derived fish length-frequency distributions to reference measurements using bin-specific error matrices

Artificial intelligence (AI)-based image analysis can efficiently estimate fish length, but differences in devices, imaging conditions, operators, and AI models limit comparability among surveys. We propose a standardization framework that estimates a bin-specific error matrix from paired reference measurements and AI-derived lengths and applies it to standardize (correct) AI-derived length-frequency distributions. The Richardson-Lucy expectation-maximization algorithm was used, with the number of iterations selected via cross-validation. Simulations based on empirical length-frequency data from 110 species showed that standardization reduced relative bias and distributional discrepancy; median relative-bias and root mean square error ratios were below 1, and the performance was more affected by the amount of paired data than by the number of cross-validation folds. In real data from 957 Japanese jack mackerel, standardized AI-derived distributions approached human-observer histograms, although discrepancies remained in the range of 160-230 mm. The proposed framework provides a practical approach for improving the comparability of image-derived length-frequency data using paired calibration data, without retraining the underlying AI model.

ecology↗

Estimation of the curved body length of tuna larvae from microscope images using a zero-shot model and image processing techniques

Fixed larval specimens often shrink and curve, making length measurement labor-intensive. Although recent studies have demonstrated efficient fish-length estimation from images using deep learning, methods for estimating curved length remain limited. Furthermore, although deep learning is a powerful method for object detection in images, an essential step for length measurement, it requires preparing large amounts of training data, which can hinder practical implementation. In this study, we used a zero-shot model that requires no training to detect fish in an image. The curved length was then estimated using an image-processing approach that combines image thinning with Bezier curve approximation, and its accuracy was evaluated. We analyzed 1,040 larvae from five tuna species captured in stereomicroscope images. Manual measurements (notochord length or standard length; 1.5-8.5 mm) were conducted by two measurers and served as reference values. Fish regions were detected using GroundedSAM, and curved body centerlines were extracted through image thinning and approximated with Bezier curves. The curve length was used as the estimated body length. Estimation accuracy was assessed using bias and standard deviation between estimated and manual measurements. GroundedSAM detected all 1,040 fish, although there were 49 overdetections. Overdetection was caused by the double-detection of a single fish or by the misidentification of debris and light reflections as fish. Although the standard deviation of the differences between manual measurements and the image analysis-based (IAB) method was larger than the inter-measurer differences, the bias for [≤]5 mm was comparable to or smaller than the inter-measurer bias. According to the strength-of-agreement criteria for the concordance correlation coefficient, the IAB method demonstrated substantial agreement in the [≤]5.0-mm range. The IAB method accurately measured most curved tuna larvae without prior training, particularly in the [≤]5.0-mm range. Combining the IAB method with manual remeasuring can improve the efficiency of curved-length measurement tasks.

ecology↗

tRF-3021a, a tRNA-Ala-TGC derived 3' fragment, promotes glioblastoma cell invasion, suppresses apoptosis, and is required for normal levels of protein synthesis

tRNA-derived fragments (tRFs) are a relatively recently discovered class of small RNAs implicated in gene-regulatory processes in diverse biological contexts but there have been very few reports of a clear phenotypic role of these small RNAs in cancer progression. To select tRFs that should receive priority for mechanistic experiments, we analyzed small RNA-seq data from The Cancer Genome Atlas (TCGA) and found that high expression of three 3' tRFs (tRF-3a), tRF-3009a, tRF-3021a or tRF-3030a, is significantly associated with poor overall survival in low-grade glioma (LGG). In glioblastoma cells, tRF-3009a, tRF-3021a and tRF-3030a enhance cell invasion and migration but tRF-3021a was uniquely required for cell proliferation and suppression of apoptosis. Interestingly, tRF-3021a knockdown decreases global protein synthesis prior to and independent of apoptosis in a number of cancer cell lines, both from and outside the glioblastoma lineage. RNA-seq reveals that the results cannot be explained by microRNA-like functions of tRF-3021a. These data indicate that tRF-3021a supports cancer cell survival and particularly protein synthesis while promoting cellular invasion and migration and suggests that strategies to downregulate this short RNA could be a potential therapeutic approach in cancers. ImplicationtRF-3021a promotes malignant cell phenotypes, sustains global protein synthesis and prevents spontaneous apoptosis, motivating efforts to evaluate it as a biomarker and therapeutic target.

cancer biology↗

A safer fluorescent in situ hybridization protocol for cryosections

Fluorescent in situ hybridization (FISH) enables highly sensitive, high-resolution detection of gene transcripts. Moreover, by employing multiple probes, this technique allows for multiplexed, simultaneous detection of distinct gene expression patterns spatiotemporally, making it a valuable spatial transcriptomics approach. Owing to these advantages, FISH techniques are rapidly being adopted across diverse areas of basic biology. However, conventional protocols often rely on volatile, toxic reagents such as formalin or methanol, posing potential health risks to researchers. Here, we present a safer protocol that replaces these chemicals with low-toxicity alternatives, without compromising the high detection sensitivity of FISH. We validated this protocol using both in situ hybridization chain reaction (HCR) and signal amplification by exchange reaction (SABER)-FISH in frozen sections of various model organisms, including mouse (Mus musculus), amphibians (Xenopus laevis and Pleurodeles waltl), and medaka (Oryzias latipes). Our results demonstrate successful multiplexed detection of morphogenetic and cell-type marker genes in these model animals using this safer protocol. The protocol has the additional advantage of requiring no proteolytic enzyme treatment, thus preserving tissue integrity. Furthermore, we show that this protocol is fully compatible with EGFP immunostaining, allowing for the simultaneous detection of mRNAs and reporter proteins in transgenic animals. This protocol retains the benefits of highly sensitive, multiplexed, and multimodal detection afforded by integrating in situ HCR and SABER-FISH with immunohistochemistry, while providing a safer option for researchers, thereby offering a valuable tool for basic biology.

molecular biology↗

HSF1 remodels mitochondrial biogenesis and function in cancer cells via TIMM17A

Mitochondria play critical roles in energy production and cellular metabolism. Despite the Warburg effect, mitochondria are crucial for the survival and proliferation of cancer cells. Heat Shock Factor 1 (HSF1), a key transcription factor in the cellular heat shock response, promotes malignancy and metastasis when aberrantly activated. To understand the multifaceted roles of HSF1 in cancer, we performed a genome-wide CRISPR screen to identify epistatic interactors of HSF1 in cancer cell proliferation. The verified interactors of HSF1 include those involved in DNA replication and repair, transcriptional and post-transcriptional gene expression, and mitochondrial functions. Specifically, we found that HSF1 promotes cell proliferation, mitochondrial biogenesis, respiration, and ATP production in a manner dependent on TIMM17A, a subunit of the inner membrane translocase. HSF1 upregulates the steady-state level of the short-lived TIMM17A protein via its direct target genes, HSPD1 and HSPE1, which encode subunits of the mitochondrial chaperonin complex and are responsible for protein refolding once imported into the matrix. The HSF1- HSPD1/HSPE1-TIMM17A axis remodels the mitochondrial proteome to promote mitochondrial translation and energy production, thereby supporting robust cell proliferation. Our work reveals a mechanism by which mitochondria adjust protein uptake according to the folding capacity in the matrix by altering TIM complex composition.

cell biology↗

Impaired oxidative phosphorylation drives primary tumor escape and metastasis

Metastasis causes most cancer deaths and reflects transitions from primary tumor escape to seeding and growth at metastatic sites. Epithelial-to-mesenchymal transition (EMT) is important early in metastasis to enable cancer cells to detach from neighboring cells, become migratory, and escape the primary tumor. While different phases of metastasis expose cells to variable nutrient environments and demands, the metabolic requirements and plasticity of each step are uncertain. Here we show that EMT and primary tumor escape are stimulated by disrupted oxidative metabolism. Using Renal Cell Carcinoma (RCC) patient samples, we identified the mitochondrial electron transport inhibitor NDUFA4L2 as upregulated in cells undergoing EMT. Deletion of NDUFA4L2 enhanced oxidative metabolism and prevented EMT and metastasis while NDUFA4L2 overexpression enhanced these processes. Mechanistically, NDUFA4L2 suppressed oxidative phosphorylation and caused citric acid cycle intermediates to accumulate, which modified chromatin accessibility of EMT-related loci to drive primary tumor escape. The effect of impaired mitochondrial metabolism to drive EMT appeared general, as renal cell carcinoma patient tumors driven by fumarate hydratase mutations with disrupted oxidative phosphorylation were highly metastatic and also had robust EMT. These findings highlight the importance of dynamic shifts in metabolism for cell migration and metastasis, with mitochondrial impairment driving early phases of this process. Understanding mitochondrial dynamics may have important implications in both basic and translational efforts to prevent cancer deaths.

cancer biology↗

Revealing the diversity of light-harvesting antenna of in vivo photosystem I

The photosynthetic reaction is driven by the cooperation of two light-excited pigment-protein supercomplexes: photosystem II (PSII) and photosystem I (PSI). The efficiency of the excitation of the two PSs relies on the exquisite organization of their light-harvesting antenna under environmental fluctuations. However, since the antenna-protein composition within cells remains elusive, the in vivo events arising from antenna variations cannot be accurately explored. Here, we implemented the single-pixel excitation-emission spectroscopy of Chlamydomonas cells under 80 K using a cryogenic optical microscope. The antenna variations of in vivo PSI can be exclusively evaluated via this low-temperature spectro-imaging method. The simultaneous acquisition of two types of fluorescence spectra enables the analysis of the intracellular correlation between the PSII/PSI intensity ratio and the chlorophyll-b/a (Chl-b/a) concentration ratio. We found that the Chl-b/a ratio hardly correlated with the PSII/PSI intensity ratio in most cases, suggesting that the in vivo PSI intensity ratio reflects the relative PSI stoichiometry rather than their antenna sizes. More importantly, the analysis of the PSI antenna-related Chl-b concentration within cells reveals a mega-antenna system that is much larger than the antenna sizes of the PSI supercomplexes whose structures have been resolved so far. Such PSI megacomplexes tended to be enriched in the region surrounding the pyrenoids of Chlamydomonas cells. We anticipate the present investigation to be a starting point for directly estimating the arrangements of antenna systems of photosystems at the single-cell scale, which is necessary for a deeper understanding of dynamic in vivo events related to the photosynthetic light-harvesting process.

biophysics↗

Regulation of epigenetics and chromosome structure by human ORC2

We report a multi-omics study in a human cell line with mutations in three subunits of Origin Recognition Complex (ORC). The ORC subunits bind to DNA independent of each other in addition to as part of a common six-subunit ORC. DNA-bound ORC2 compacts chromatin and attracts repressive histone marks to focal areas of the genome, but ORC2 also activates chromatin at many sites and protects the genes from repressive marks. The epigenetic changes regulate hundreds of genes, including some epigenetic regulators, adding an indirect mechanism by which ORC2 regulates epigenetics without local binding. DNA-bound ORC2 also prevents the acquisition of CTCF at focal sites in the genome to regulate chromatin loops and indirectly affect epigenetics. Thus, our study reveals the genes and ORC1 regions bound by individual ORC subunits and suggests their role as epigenetics and chromosome structure regulators, independent of the role of the six-subunit ORC in DNA replication.

genomics↗

Specific origin selection and excess functional MCM2-7 loading in ORC-deficient cells.

The six subunit Origin Recognition Complex (ORC) loads excess MCM2-7 on chromosomes to promote initiation of DNA replication and is believed to be important for origin specification. Mapping of origins in cancer cell lines engineered to delete three of the subunits, ORC1, ORC2 or ORC5 shows that specific origins are still used and are mostly at the same sites in the genome as in wild type cells. The few thousand origins that were up-regulated in the absence of ORC suggest that GC/TA skewness and simple repeat sequences facilitate, but are not essential for, origin selection in the absence of the six-subunit ORC. Despite the lack of ORC, excess MCM2-7 is still loaded at comparable rates in G1 phase to license dormant origins and is also repeatedly loaded in the same S phase to permit re-replication. Thus, origin specification and excess MCM2-7 loading on origins do not require the six-subunit ORC in human cancer cell lines.

molecular biology↗

Time- Space Resolved Fluorescence Spectroscopy in Live Chlamydomonas Cells under Light-Harvesting Regulation

We report here a technical advancement that enables time-resolved fluorescence spectroscopy in spatially resolved domains of a living cell at low temperatures. The technique is based on a combination of the self-developed cryo-confocal microscope system and the streak-camera technology. An instrumental response time of ca. 24 ps was achieved. This technique was applied to reveal the light-harvesting dynamics in local domains within single Chlamydomonas reinhardtii cells. Organisms performing oxygenic photosynthesis, like Chlamydomonas, have evolved a regulation mechanism called state transitions (ST), which maintains the excitation balance between PSI and PSII. ST relies on the shuttling of light-harvesting chlorophyll protein complex II (LHCII) between the two PSs. In the present experiment, cells were induced either to state1, where LHCII is bound to PSII, or state2, where LHCII moved and is bound to PSI. After the induction of ST, cells were immediately cooled to ca. 80 K, where PSI and PSII show clearly separated fluorescence emission bands, enabling the visualization of these components separately. Based on kinetic analyses of the time-resolved fluorescence spectra in both PSI-rich and PSII-rich local domains, we concluded that (1) the intracellular inhomogeneity in the PSII/PSI fluorescence ratio comes from that in the PSII/PSI stoichiometry, not from that in the antenna sizes of the PSs, and (2) the antenna size of PSI in state2 cells may larger in intact cells than that of the isolated PSI-LHCI-LHCII super-complex reported so far.

biophysics↗

GPRC6A as a novel kokumi receptor responsible for enhanced taste preferences by ornithine

The concept of "kokumi," which refers to an enhanced and more delicious flavor of food, has recently generated considerable interest in food science. However, kokumi has not been well studied in gustatory physiology, and the underlying neuroscientific mechanisms remain largely unexplored. Our previous research demonstrated that ornithine (L-ornithine), which is abundant in shijimi clams, enhanced taste preferences in mice. The present study aimed to build on these findings and investigate the mechanisms responsible for kokumi in rats. In two-bottle preference tests, the addition of ornithine, at a low concentration that did not increase the favorability of this substance alone, enhanced the animals preferences for umami, sweet, fatty, salty, and bitter solutions, with the intake of monosodium glutamate showing the most significant increase. Additionally, a mixture of umami and ornithine synergistically induced significant responses in the chorda tympani nerve, which transmits taste information to the brain from the anterior part of the tongue. The observed preference enhancement and increase in taste-nerve response were abolished by antagonists of the G-protein-coupled receptor family C group 6 subtype A (GPRC6A). Furthermore, immunohistochemical analysis indicated that GPRC6A was expressed in a subset of type II taste cells in rat fungiform papillae. These results provide new insights into flavor-enhancement mechanisms, confirming that ornithine is a kokumi substance and GPRC6A is a novel kokumi receptor.

neuroscience↗

Pemphigus vulgaris autoantibodies induce an ER stress response

Desmosomes are intercellular junctions that mediate cell-cell adhesion and are essential for maintaining tissue integrity. Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease caused by autoantibodies (IgG) targeting desmoglein 3 (Dsg3), a desmosomal cadherin. PV autoantibodies cause desmosome disassembly and loss of cell-cell adhesion, but the molecular signaling pathways that regulate these processes are not fully understood. Using high- resolution time-lapse imaging of live keratinocytes, we found that ER tubules make frequent and persistent contacts with internalizing Dsg3 puncta in keratinocytes treated with PV patient IgG. Biochemical experiments demonstrated that PV IgG activated ER stress signaling pathways, including both IRE1 and PERK pathways, in cultured keratinocytes. Further, ER stress transcripts were upregulated in PV patient skin. Pharmacological inhibition of ER stress protected against PV IgG-induced desmosome disruption and loss of keratinocyte cell-cell adhesion, suggesting that ER stress may be an important pathomechanism and therapeutically targetable pathway for PV treatment. These data support a model in which desmosome adhesion is integrated with ER function to serve as a cell adhesion stress sensor that is activated in blistering skin disease.

cell biology↗

Endo-reduplication in mouse liver after conditional mutation of ORC2 and combined mutation of ORC1 and ORC2

The six subunit ORC is essential for initiation of DNA replication in eukaryotes. Cancer cell-lines in culture can survive and replicate DNA replication after genetic inactivation of individual ORC subunits, ORC1, ORC2 or ORC5. In primary cells, ORC1 was dispensable in the mouse liver for endo-reduplication, but this could be explained by the ORC1 homolog, CDC6, substituting for ORC1 to restore functional ORC. Here, we have created mice with a conditional deletion of ORC2, which does not have a homolog. Although mouse embryo fibroblasts require ORC2 for proliferation, mouse hepatocytes synthesize DNA in cell culture and endo-reduplicate in vivo without ORC2. Mouse livers endo-reduplicate after simultaneous deletion of ORC1 and ORC2 both during normal development and after partial hepatectomy. Since endo-reduplication initiates DNA synthesis like normal S phase replication these results unequivocally indicate that primary cells, like cancer cell lines, can load MCM2-7 and initiate replication without ORC.

genetics↗

Effects of input image size on the accuracy of fish identification using deep learning.

The length composition of catches by species is important for stock assessment. However, length measurement is performed manually, jeopardizing the future of continuous measurement because of likely labor shortages. We focused on applying deep learning to estimate length composition by species from images of fish caught for sustainable management. In this study, input image sizes were varied to evaluate the effect of input image size on detection and classification accuracy, as a method for improving the accuracy. The images (43,226 fish of 85 classes) were captured on conveyor belts to sort set-net catches. Fish detection and classification were performed using Mask R-CNN. The effect of input image size on accuracy was examined using three image sizes of 1333x888, 2000x1333, and 2666x1777 pixels, achieving an mAP50-95 of 0.580 or higher. The accuracy improved with increasing image size, attaining a maximum improvement of 4.3% compared to the smallest size. However, increasing the image size too far from the default size may not improve the accuracy of models with fine-tuning. Improvements in accuracy were primarily observed for the species with low accuracy at the smallest image size. Increasing image size would be a useful and simple way to improve accuracy for these species.

ecology↗

The membrane curvature inducing REEP1 proteins generate a novel ER-derived vesicular compartment

The endoplasmic reticulum (ER) is shaped by abundant, membrane curvature-generating proteins that include the REEP family member REEP5. The REEP1 subfamily, consisting of REEP1-4 in mammals, differs in abundance and topology from REEP5. Mutations in REEP1 and REEP2 cause Hereditary Spastic Paraplegia, but REEP1-4s function remains enigmatic. Here we show that the REEP1 proteins reside in a novel vesicular compartment and identify features that determine their localization. Mutations in REEP1 proteins that compromise curvature-inducing activity, including those that cause disease, relocalize the proteins to the bulk ER. These mutants interact with wildtype proteins to retain them in the ER, consistent with their autosomal-dominant disease inheritance. REEP1vesicles contain the fusogen atlastin-1, but not general ER proteins. We propose that REEP1 proteins generate these vesicles themselves by budding directly from the ER, and that they cycle back to the ER by atlastin-mediated fusion. The vesicles may serve to regulate ER tubule dynamics.

cell biology↗

Integrative analysis of DNA replication origins and ORC binding sites in human cells reveals a lack of overlap between them

Based on experimentally determined average inter-origin distances of [~]100 kb, DNA replication initiates from [~]50,000 origins on human chromosomes in each cell cycle. The origins are believed to be specified by binding of factors like the Origin Recognition Complex (ORC) or CTCF or other features like G-quadruplexes. We have performed an integrative analysis of 113 genome-wide human origin profiles (from five different techniques) and 5 ORC-binding profiles to critically evaluate whether the most reproducible origins are specified by these features. Out of [~]7.5 million union origins identified by all datasets, only 0.27% were reproducibly obtained in at least 20 independent SNS-seq datasets and contained in initiation zones identified by each of three other techniques (20,250 shared origins), suggesting extensive variability in origin usage and identification. 21% of the shared origins overlap with transcriptional promoters, posing a conundrum. Although the shared origins overlap more than union origins with constitutive CTCF binding sites, G-quadruplex sites and activating histone marks, these overlaps are comparable or less than that of known Transcription Start Sites, so that these features could be enriched in origins because of the overlap of origins with epigenetically open, promoter-like sequences. Only 6.4% of the 20,250 shared origins were within 1 kb from any of the [~]13,000 reproducible ORC binding sites in human cancer cells, and only 4.5% were within 1 kb of the [~]11,000 union MCM2-7 binding sites in contrast to the nearly 100% overlap in the two comparisons in the yeast, S. cerevisiae. Thus, in human cancer cell lines, replication origins appear to be specified by highly variable stochastic events dependent on the high epigenetic accessibility around promoters, without extensive overlap between the most reproducible origins and currently known ORC- or MCM-binding sites.

genomics↗

Fgf10 mutant newts can regenerate normal limbs despite severe developmental hindlimb defects

In the amniote limb, FGF10 is essential for limb bud initiation and outgrowth. However, whether this function is broadly conserved in tetrapods and/or involved in adult limb regeneration remains unknown. To tackle this question, we established an Fgf10 null mutant line in the newt Pleurodeles waltl which have amazing regenerative ability. While Fgf10 mutant forelimbs develop normally, the hindlimbs exhibit severe digit reduction, fail to ossify the zeugopod, and downregulate FGF target genes like Sall1, Runx1 and Hoxa11/d11. Despite these developmental defects, Fgf10 mutants were able to regenerate near-normal hindlimbs. Together, our results suggest an important role for Fgf10 in hindlimb digit formation and zeugopod ossification during development, but little or no function in regeneration, suggesting that different mechanisms operate during limb regeneration versus development.

developmental biology↗