Biology subjects

Nakata, R.

Publications and source records attributed to Nakata, R..

4 recordsLinked to original sources

bioRxiv2025

Graded regulation of microtubule-binding of Tau by the phosphorylation state of the proline-rich region in living neurons

Tau protein is a microtubule-associated protein that plays a crucial role in maintaining neuronal morphology and axonal transport. While phosphorylation is known to regulate Tau-microtubule interactions, the contribution of specific phosphorylation patterns in situ remains poorly understood due to the complexity of the intracellular environment. In this study, we combined fluorescence recovery after photobleaching (FRAP) in primary cultured rat hippocampal neurons with dephosphorylation-mimetic mutations and computational modeling to analyze the effects of phosphorylation on Tau-microtubule interaction. We particularly focused on the proline-rich region, of which phosphorylation has been studied in physiological and pathological perspectives, and generated a dephosphorylation-mimetic Tau mutant by substituting key phosphorylation sites with alanine residues and compared its microtubule-binding dynamics to those of WT-Tau in FRAP experiments. Experimental data, together with simulation-based parameter exploration, revealed that the overall number of phosphorylated sites plays a more dominant role than their specific locations in modulating Tau-microtubule affinity. These findings provide new insights into the post-translational regulation of Tau and establish a computational-experimental framework for interrogating intracellular protein dynamics.

neuroscience↗

bioRxiv2025

Vertical rooting caused by enhanced functional allele of qSOR1 improves rice yield under drought stress

Drought considerably affects crop productivity, and its severity is being intensified by climate change. Therefore, enhancing drought resistance is a crucial priority in crop breeding for ensuring sustainable agriculture. The root system architecture (RSA) influences the efficiency of water acquisition from land; therefore, a deep RSA is advantageous for avoiding drought stress. Here, we demonstrated that deeper RSA promoted by the qSOR1-v mutant allele (an enhanced functional allele of the quantitative trait locus for SOIL SURFACE ROOTING 1) significantly improves rice yield under drought when compared to the deep RSA achieved through the functional qSOR1 allele that originated from natural variation. The qSOR1-v mutant exhibited stronger root gravitropism than the wild type. This was characterized by a more pronounced polarization of auxin on the lower side during root curvature, leading to a robust vertical rooting phenotype that was consistently expressed across different soil-water environments. Additionally, the qSOR1-v mutation site was well conserved among angiosperm orthologs, and the corresponding mutation in LZY3 of Arabidopsis (qSOR1 ortholog) resulted in a steeper root growth angle. The rice introgression line, which was substituted from the functional qSOR1 allele to qSOR1-v through marker-assisted selection, showed vertical rooting, resulting in increased grain yield in an upland field under drought stress. No yield penalty was observed for this line under well-watered upland conditions than the original variety. These findings highlight the potential of qSOR1-v and corresponding mutations in angiosperm orthologs to promote vertical rooting across plant species, which can help sustain crop yields in drought-prone areas. Significance StatementGenetic modification of the root system architecture in crops represents a viable strategy for the development of climate-resilient crops. This study identified the qSOR1-v allele that consistently demonstrates a vertical rooting phenotype in rice across diverse growth conditions, from dry to wet. The preservation of the qSOR1 and LZY3 orthologs in angiosperms provides opportunities for the development of genotypes characterized by vertical rooting. The introgression of the qSOR1-v allele enhanced drought resistance under upland conditions. These findings underscore the potential of these genetic modifications to improve crop resilience in an era characterized by water scarcity.

plant biology↗

bioRxiv2024

Beta-Glucanase superfamily identified by sequential, functional, and structural analyses

{beta}-1,2-Glucans are natural glucose polymers that play important physiological roles, including as symbiotic or pathogenic factors and in osmoregulation. Phylogenetically new glycoside hydrolase (GH) families have recently been identified from {beta}-1,2-glucanase (SGL) sequences from bacteria (GH144) and a fungus (GH162). In this study, we identified four phylogenetically new groups (Groups 1-4), and determined that these families, together with GH144, GH162, and GH189, a family of transglycosylase domains in cyclic {beta}-1,2-glucan synthases, form a superfamily. Biochemical analysis of six proteins in these groups revealed that the proteins in Groups 1-3 showed hydrolytic activity specific to {beta}-1,2-glucan. The kinetic parameters of the enzymes of Groups 1-3 were similar to GH144 and GH162 SGLs, indicating that these enzymes were SGLs. Optical rotation analysis revealed that the SGLs followed an anomer-inverting mechanism. Structural analysis and prediction of the proteins in Groups 1-4, GH144, GH162, and GH189 suggested that Groups 1-3 and GH144 had the same reaction mechanism. Nevertheless, Groups 1-3 were dispersed irregularly in the superfamily. Overall, we determined that Groups 1-3 were new GH families, GHxxx, GHyyy, and GHzzz, respectively, and proposed that this superfamily be called an SGL superfamily because of the phylogenetical, functional, and structural relationships within the superfamily. HighlightsWide variety of glycoside hydrolases is far beyond our understanding. Functional and structural analysis identified three new glycoside hydrolase families. Molecular evolution with irregular changes in reaction mechanism was revealed.

biochemistry↗

bioRxiv2023

Active Transport by Cytoplasmic Dynein Maintains the Localization of MAP-2 in Developing Neurons

MAP2 has been widely used as a marker of neuronal dendrites because of its extensive restriction in the somatodendritic region of neurons. Despite that, how the precise localization of such a soluble protein is established and maintained against thermal forces and diffusion has been elusive and long remained a mystery in neuroscience. In this study, we aimed to uncover the mechanism behind how MAP2 is retained in the somatodendritic region. Using GFP-tagged MAP2 expressed in cultured hippocampal neurons, we discovered a crucial protein region responsible for the localization of MAP2, the serine/proline-rich (S/P) region. Our pulse-chase live-cell imaging revealed the slow but steady migration of MAP2 toward distal dendrites, which was not observed in a MAP2 mutant lacking the S/P region, indicating that S/P-dependent transport is vital for the proper localization of MAP2. Furthermore, our experiments using an inhibitor of cytoplasmic Dynein, ciliobrevin D, as well as Dynein knockdown, showed that cytoplasmic Dynein is involved in the transport of MAP2 in dendrites. We also found that Dynein complex binds to MAP2 through the S/P region in heterologous cells. Using mathematical modeling based on experimental data, we confirmed that an intermittent active transport mechanism is essential. Thus, we propose that the cytoplasmic Dynein recruits and transports free MAP2 toward distal dendrites, thereby maintaining the precise dendritic localization of MAP2 in neurons. Our findings shed light on the previously unknown mechanism behind MAP2 localization and provide a new direction for soluble protein trafficking research in the field of cell biology of neurons.

neuroscience↗