Biology subjects

Nakata, T.

Publications and source records attributed to Nakata, T..

8 recordsLinked to original sources

bioRxiv2025

Molecular evolution of terpene synthase underlying the diversification of isoprene emission in Fagaceae

Plants emit a wide range of volatile organic compounds, among which isoprene is the most abundant and atmospherically influential. Although oak species are major contributors to isoprene emission, there is considerable variation in isoprene emission capacity within the Fagaceae family. To unravel the evolutionary origins of isoprene emission, we investigated the molecular evolution of terpene synthase (TPS) genes across eight species within the Fagaceae. We identified a Fagaceae-specific TPS-b subclade in which potential isoprene synthase (IspS) activity evolved independently in two gene lineages within subgenus Quercus. Ancestral sequence reconstruction revealed that the acquisition of a diagnostic amino acid residue for IspS function arose convergently in these lineages and was subject to positive selection, suggesting adaptive evolution. Ancestral-enzyme assays targeting the gene lineage with high gene expression revealed that the early protein primarily produced monoterpenes from geranyl diphosphate (GPP), whereas their descendants shifted substrate preference to dimethylallyl diphosphate (DMAPP), evolving into dedicated isoprene synthases. Our results indicate that IspS activity was not ancestral in Fagaceae, but evolved approximately 56 million years ago within the subgenus Quercus, and has been retained ever since. These findings emphasize the roles of enzyme structural innovation and regulatory shifts in the diversification of volatile terpenoid biosynthesis.

evolutionary biology↗

bioRxiv2024

Cryo-electron tomography of the actin cytoskeleton and membrane organization during lamellipodia formation using optogenetics

Lamellipodia are sheet-like protrusions essential for migration and endocytosis, yet the ultrastructure of the actin cytoskeleton during lamellipodia formation remains underexplored. Here, we combined the optogenetic tool PA-Rac1 with cryo-ET to enable ultrastructural analysis of newly formed lamellipodia. We successfully visualized lamellipodia at various extension stages, representing phases of their formation. In minor extensions, several unbundled actin filaments formed "Minor protrusions" at the leading edge. For moderately extended lamellipodia, cross-linked actin filaments formed small filopodia-like structures, termed "mini filopodia." In fully extended lamellipodia, filopodia matured at multiple points, and cross-linked actin filaments running nearly parallel to the leading edge increased throughout the lamellipodia. These observations suggest that actin polymerization begins in specific plasma membrane regions, forming mini filopodia that either mature into full filopodia or detach from the leading edge to form parallel filaments. This actin turnover likely drives lamellipodial protrusion, providing new insights into actin dynamics and cell migration.

cell biology↗

bioRxiv2024

Allopolyploidy enhances survival advantages for urban environments in the native plant genus Commelina

Background and AimsUrbanization-induced environmental changes have profound effects on the geographical distribution limits in natural plant species. Polyploidization, an influential dynamic genome change, may determine survival potentials of plant species in urban environments. This study focused on the native plants, Commelina communis L. (Cc) and closely related subspecies, C. communis f. ciliata (Masam.) Murata (Ccfc) which have different chromosome numbers (e.g. Cc: 2n = 88, Ccfc: 2n = 46). The aim is to investigate polyploidization effects on natural plant distribution in urban environments. MethodsThe geographical distribution across urban-rural gradients was investigated at a total of 218 sites in Japan. Stomata size and density were measured and compared between Cc and Ccfc. Flow cytometry was used to determine genome size and polyploidy. Chromosome karyotyping was performed by using the GISH method. Key resultsUrban areas were exclusively dominated by Cc, while Cc and Ccfc coexisted in rural areas. Cc had larger and fewer stomata and more than twice genome size than Ccfc. GISH results indicated that Cc possesses Ccfc and another unknown genome, suggesting allopolyploidy. ConclusionsThese results show that the ploidy difference affects the geographical distribution, the stomata traits, and genome size between Cc and Ccfc. In addition, GISH results indicate that Cc has Ccfc and another unknown genome, suggesting Cc is an allopolyploid and these two species of the genus Commelina are distinct. Therefore, not only polyploid but also allopolyploid contributes to Cc to enhance survival potentials in urban environments compared to Ccfc. This is the first investigation to clarify the distribution difference related to urban environments, the difference in stomata traits and genome size, and to conduct chromosome composition in Commelina species.

ecology↗

bioRxiv2023

CTRP7 as a Molecular Biomarker for Predicting Responsiveness to Pulmonary Vasodilators: Insights from Human and Animal Studies in Pulmonary Arterial Hypertension

BackgroundPulmonary arterial hypertension (PAH) is a life-threatening condition. Although pulmonary vasodilators have shown promise in managing PAH, the overall prognosis improvement is modest, partly because of the absence of a biomarker for guiding their selection. This study aimed to identify the molecular-based predictors of responsiveness to pulmonary vasodilators by combining human and basic studies. Methods and ResultsRNA sequencing was conducted on cultured pulmonary artery smooth muscle cells (PASMCs) from patients with and without PAH, identifying variations in 2,076 genes. The candidates were narrowed down to established clinical biomarkers, and their plasma levels were assessed in PAH (n = 123) and non-PAH patients (n = 25). C1q/TNF-related protein 7 (CTRP7) exhibited elevated expression in PASMCs (5.37 Log2 fold change, p < 0.01), and the plasma of patients with PAH compared to non-PAH (28.2 (9.3- 101.2) vs. 9.32 (4.6-18.6) ng/ml, p < 0.01). A plasma assessment revealed a significant correlation between CTRP7 and interleukin 6 (IL-6) levels (r = 0.544, p < 0.001). Chromatin immunoprecipitation demonstrated that IL-6 upregulated CTRP7 expression in PASMCs. Among pivotal factors, CTRP7 reduced prostacyclin analog receptor (PTGIR) expression through Rab5a-mediated internalization, resulting in diminished responsiveness to selexipag (a prostacyclin analog). Incorporating the human study, hypoxic PH mice demonstrated reduced PTGIR expression in the pulmonary arteries, which correlated with limited responses to selexipag treatment (low cardiac output and persistent pulmonary artery resistance), which was mitigated by silencing CTRP7 expression in the pulmonary arteries using adeno-associated virus 6. ConclusionsAmong the candidates with increased expression in patients with PAH based on RNA sequencing of PASMCs, CTRP7 demonstrated elevated plasma levels compared to non-PAH. CTRP7 regulated PTGIR internalization via inflammation and Rab5a, influencing responsiveness to selexipag. CTRP7 emerged as a compelling biomarker for predicting responsiveness to prostacyclin analogs, potentially advancing the development of innovative PAH treatment strategies. Novel and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIPulmonary arterial hypertension (PAH) is a life-threatening vascular disease with limited treatment options. C_LIO_LIAlthough pulmonary vasodilators have shown promise in managing PAH, the overall improvement in prognosis is modest. C_LIO_LIFurther research is needed to understand the molecular features of pulmonary vasodilators and identify biomarkers for appropriate selection. C_LI What New Information Does This Article Contribute?O_LIC1q/TNF-related protein 7 (CTRP7) was found to be elevated in the pulmonary artery smooth muscle cells (PASMCs) and plasma of patients with pulmonary arterial hypertension (PAH). C_LIO_LIThis study established a connection between inflammation and pulmonary vascular responsiveness via CTRP7-mediated internalization and degradation of the prostacyclin analog receptor. C_LIO_LIThis study indicated the possibility of using plasma CTRP levels as a clinical biomarker to monitor pulmonary responsiveness, which can contribute to the development of novel treatment strategies for patients with PAH. C_LI Pulmonary arterial hypertension (PAH) is a life-threatening and enigmatic lung vascular disorder characterized by progressive pulmonary vascular remodeling and vasoconstriction. Despite significant advancements in the treatment of PAH using pulmonary vasodilators, some individuals display limited responses, primarily due to a lack of understanding of the molecular features of each pulmonary vasodilator and the absence of a biomarker for appropriate selection. The present study aimed to identify promising biomarkers for predicting a patients responsiveness to pulmonary vasodilators through a two-step approach, involving RNA sequencing of PASMCs and the measurement of plasma levels. CTRP7 emerged as a prominent biomarker, displaying increased expression in both PASMCs and plasma obtained from patients with PAH. Mechanistically, IL-6 transcriptionally modulated CTPR7, leading to the internalization and degradation of PTGIR, resulting in a reduced response to selexipag (prostacyclin analog) in PASMCs. Integrating the human studies, a hypoxic mouse model exhibited lower PTGIR expression in pulmonary arteries, correlating with limited responses to selexipag treatment, which were ameliorated by silencing CTRP7 expression in pulmonary arteries using adeno-associated virus 6 transduction. This study collectively demonstrates that CTRP7 emerges as a compelling biomarker for predicting responsiveness to prostacyclin analogs, with the potential to advance the development of innovative PAH treatment strategies.

molecular biology↗

bioRxiv2023

Classification of Multiple Emotional States from Facial Expressions in Mice using a Deep Learning-Based Image Analysis

Facial expressions are widely recognized as universal indicators of underlying internal states in most species of animals, thereby presenting as a non-invasive measure for predicting physical and mental conditions. Despite the advancement of artificial intelligence-assisted tools for automated analysis of voluminous facial expression data in human subjects, the corresponding tools for mice still remain limited so far. Considering that mice are the most prevalent model animals for studying human health and diseases, a comprehensive characterization of emotion-dependent patterns of facial expressions in mice could extend our knowledge on the basis of emotions and the related disorders. Here, we present a framework for the development of a deep learning-powered tool for classifying mouse facial expressions. We demonstrate that our machine vision was capable of accurately classifying three different emotional states from mouse facial images. Moreover, we objectively determined how our classifier characterized the differences among the facial images through the use of an interpretation technique called Gradient-weighted Class Activation Mapping. Our approach is likely to facilitate the non-invasive decoding of a variety of emotions from facial images in mice.

neuroscience↗

bioRxiv2021

Horizontal orientation facilitates pollinator attraction and rain avoidance in radially symmetrical flowers

PremiseFloral angle, such as upward, horizontal, and downward orientation are known to evolve under both biotic and abiotic agents to enhance pollination success in zoophilious plants. Adaptive significance of horizontal orientation in radially symmetrical (actinomorphic) flowers under biotic and abiotic selection pressures were largely unknown, although those in bilaterally symmetrical flowers have been well studied. MethodsUsing experimentally angle changed flowers, we examined the effects of flower angle on pollinator behaviors, pollination success and rain avoidance in a population of insect-pollinated Platycodon grandiflorus. We further investigated the frequency and amount of precipitation in the flowering season and pollen damage by water in this species. Main results: Horizontally oriented flowers received more visitations and pollen grains on the stigma in male and/or female phases than downward and/or upward oriented flowers and avoided pollen damage by rainfall compared to upward oriented flowers. The pollen germination experiment showed that approximately 30% of pollen grains burst in distilled water, thus pollen damage by rainfall was potentially serious in P. garndiflorus. ConclusionIn this study, our field experiments revealed that upward flowers cannot avoid damage from rainfall during the flowering period whereas both upward and downward flowers suffered from pollinator limitation in female success. Thus, horizontal flower orientation is suggested to be adaptive in this insect-pollinated actinomorphic species which blooms in the rainy season.

ecology↗

bioRxiv2020

The schizophrenia-associated variant in SLC39A8 alters N-glycosylation in the mouse brain

A missense mutation (A391T) in the manganese transporter SLC39A8 is strongly associated with schizophrenia in genomic studies, though the molecular connection to the brain remains hypothetical. Human carriers of A391T have reduced serum manganese, altered plasma glycosylation, and brain MRI changes consistent with altered metal transport. Here, using a knock-in mouse model homozygous for A391T, we show that the schizophrenia-associated variant changes protein glycosylation in the brain. N-linked glycosylation was most significantly impaired, with effects differing between regions. RNAseq analysis showed negligible regional variation, consistent with changes in the activity of glycosylation enzymes rather than gene expression. Finally, nearly one third of detected glycoproteins were differentially N-glycosylated in the cortex, including members of several pathways previously implicated in schizophrenia such as cell adhesion molecules and neurotransmitter receptors. These findings provide a mechanistic link between a risk allele and biochemical changes in the brain, furthering our molecular understanding of the pathophysiology of schizophrenia.

neuroscience↗

bioRxiv2020

Optogenetic control of small GTPases reveals RhoA-mediated intracellular calcium signaling

Rho/Ras family small GTPases are known to regulate numerous cellular processes, including cytoskeletal reorganization, cell proliferation, and cell differentiation. These processes are also controlled by Ca2+, and consequently, crosstalk between these signals is considered likely. However, systematic quantitative evaluation is not yet reported. Thus, we constructed optogenetic tools to control the activity of small GTPases (RhoA, Rac1, Cdc42, Ras, Rap, and Ral) using an improved light-inducible dimer system (iLID). Using these optogenetic tools, we investigated calcium mobilization immediately after small GTPase activation. Unexpectedly, we found that a transient intracellular calcium elevation was specifically induced by RhoA activation in RPE1 and HeLa cells. These findings were confirmed in all the cell lines tested in this study. Of note, molecular mechanisms were identified to be different among cell types. In RPE1 and HeLa cells, RhoA activated phospholipase C epsilon (PLC{varepsilon}) at the plasma membrane, which in turn induced Ca2+ release from the endoplasmic reticulum (ER). The RhoA-PLC{varepsilon} axis induced calcium-dependent NFAT nuclear translocation, suggesting it does activate intracellular calcium signaling.

cell biology↗