Biology subjects

Kawamura, R.

Publications and source records attributed to Kawamura, R..

7 recordsLinked to original sources

bioRxiv2026

Developmental emergence of spatiotemporal coordination in cerebellar Purkinje cell populations

Coordinated activity across neuronal populations is fundamental to brain function, yet how such network-level organization emerges during development remains incompletely understood. Here, we performed whole-cerebellar calcium imaging at cellular scale in zebrafish larvae to examine the developmental maturation of Purkinje cell population dynamics. Visual stimulation recruited large, spatially organized populations of Purkinje cells whose responses depended on inferior olive input and were associated with optokinetic behavior. Notably, in the absence of stimuli, Purkinje cells formed transient assemblies exhibiting distance-dependent coordination. During development, long-range coordination progressively emerged, extending initially local correlations into distributed population-wide coordination. Early enucleation, but not dark rearing, disrupted the developmental refinement of long-range coordination and induced aberrant population clustering, suggesting that early retina-dependent signals contribute to cerebellar network development. Together, these findings reveal key organizational features underlying the developmental emergence of coordinated cerebellar population dynamics and suggest that early retina-dependent signals shape population-level organization.

neuroscience↗

bioRxiv2026

GapR is a nucleoid-associated protein that stiffens and overtwists DNA to promote transcription and replication

GapR is an -proteobacterial nucleoid-associated protein reported to either directly regulate transcription of AT-rich DNA or to regulate transcription indirectly through sensing DNA topology and modulating topoisomerase activity. We use single-DNA micromechanics, biolayer interferometry (BLI), and computational analysis to study GapR transcriptional regulation. Micromechanics experiments show that GapR overtwists DNA and shortens its contour length. GapR binding also substantially increases DNA bending persistence length and twist stiffness, while promoting duplex DNA melting at higher forces. Nonequilibrium binding experiments show GapR-DNA complexes to be extremely stable, with essentially no dissociation on hour-long time scales. Strikingly, our BLI experiments show that GapR has high affinity for AT-rich DNA while our micromechanics show that GapR binding is enhanced by pre-twisting of DNA, validating GapR affinity for both forms. By analyzing published GapR binding data, we differentiate between AT-rich and overtwisted DNA binding, revealing that overtwisted DNA primarily determines GapR localization. Lastly, we show that these two binding modes exert opposite effects on transcription. AT-rich binding stimulates initiation, likely by promoting RNA polymerase progression, while overtwisted DNA binding represses transcription, possibly by stabilizing local overtwist at promoters. We propose that GapR binding promotes a conformation of DNA ideal for efficient replication and transcription progression. 3 Key pointsO_LIGapR is a DNA structuring protein that binds AT-rich and overtwisted, positively supercoiled DNA C_LIO_LIGapR binding stiffens DNA, limits plectoneme formation, and melts undertwisted DNA at higher force C_LIO_LIGapR binding modes exert opposing effects on transcription initiation while promoting elongation C_LI

molecular biology↗

bioRxiv2024

Isolation and manipulation of meiotic spindles from mouse oocytes reveals migration regulated by pulling force during asymmetric division

Spindles are essential for accurate chromosome segregation in all eukaryotic cells. This study presents a novel approach for isolating fresh mammalian spindles from mouse oocytes, establishing it as a valuable in vitro model system for a wide range of possible studies. Our method enables the investigation of the physical properties and migration force of meiotic spindles in oocytes. We found that the spindle length decreases upon isolation from the oocyte. Combining this observation with direct measurements of spindle mechanics, we examined the forces governing spindle migration during oocyte asymmetric division. Our findings suggest that spindle migration is regulated by a pulling force and a net tensile force of approximately 680 pN is applied to the spindle in vivo during the migration process. This method, unveiling insights into spindle dynamics, holds promise as a robust model for future investigations into spindle formation and chromosome separation. We also found that the same approach could not isolate spindles from somatic cells, indicative of mammalian oocytes having a unique spindle organization amenable to isolation. Significance StatementSpindles are essential for accurate chromosome segregation in all eukaryotic cells, yet studying their mechanical properties in vivo remains challenging. Here, we present a novel method for isolating intact, functional spindles from live mouse oocytes, establishing a powerful in vitro model for dissecting spindle mechanics. Using this system, we reveal that spindle length shortens upon isolation and quantify a net tensile force of approximately 680 pN applied to the spindle during migration, implicating a pulling mechanism in asymmetric division. Notably, this isolation approach does not succeed in somatic cells, highlighting a unique organization of the mammalian oocyte spindle. This platform opens new avenues for understanding spindle dynamics, force generation, and chromosome segregation in meiosis.

cell biology↗

bioRxiv2024

Spike protein of the SARS-CoV-2 omicron variant interacts with actin

The omicron variant of SARS-CoV-2 is responsible for the COVID-19 pandemic, serving as a significant origin for the variants still being detected today. It affects the spike protein that most vaccines used to target when the Omicron strain was discovered. Here, we demonstrate that the receptor binding domain (RBD) of the Omicron variant of SARS-CoV-2 exhibits an increased affinity for human angiotensin-converting enzyme type 2 (hACE2) as a viral cell receptor compared to the prototype RBD. We also identified that {beta}- and {gamma}-actin are Omicron-specific binding partners of RBD. Protein complex predictions suggested that many of the Omicron-specific amino acid substitutions might be involved in the affinity of RBD and actin. Accordingly, we highlight the intriguing observation that proteins expected to localize to different cellular compartments exhibit strong binding.

biochemistry↗

bioRxiv2023

Intracellular conformation of amyotrophic lateral sclerosis-causative TDP-43

Transactive response element DNA/RNA-binding protein 43 kDa (TDP-43) is the causative protein of amyotrophic lateral sclerosis (ALS); several ALS-associated mutants of TDP-43 have been identified. TDP-43 has several domains: an N-terminal domain, two RNA/DNA-recognition motifs, and a C-terminal intrinsically disordered region (IDR). Its structures have been partially determined, but the whole structure remains elusive. In this study, we investigate the possible end-to-end distance between the N- and C-termini of TDP-43, its alterations due to ALS-associated mutations in the IDR, and its apparent molecular shape in live cells using Forster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS). Further-more, the interaction between ALS-associated TDP-43 and heteronuclear ribonucleoprotein A1 (hnRNP A1) is slightly stronger than that of wild-type TDP-43. Our findings provide insights into the structure of wild-type and ALS-associated mutants of TDP-43 in a cell.

biophysics↗

bioRxiv2021

Neural activation in the olfactory epithelium of East African cichlid in response to odorant exposure

Fishes use olfaction to gain various information vital for survival and communication. To understand biodiversity in fishes, it is important to identify what receptors individual fish use to detect specific chemical compounds. However, studies of fish olfactory receptors and their ligands are still limited to a few model organisms represented primarily by zebrafish. Here, we tested the neural activation of olfactory sensory neurons (OSNs) in East African cichlids, the most diversified teleost lineage, by in situ hybridization with a c-fos riboprobe. We confirmed that microvillous neurons contributed the most to the detection of amino acids, as in other fishes. Conversely, we found that ciliated neurons contributed the most to detection of conjugated steroids, known as pheromone candidates. We also found that V2Rs, the major expressed receptor in microvillous neurons exhibited differential responsiveness to amino acids, and further suggested that the cichlid-specific duplication of V2R led to ligand differentiation in cichlids by demonstrating a differential response to arginine. Finally, we established a nonlethal method to collect cichlid urine and showed how various OSNs, including V1R+ neurons, respond to male urine. This study provides an experimental basis for understanding how cichlids encode natural odors and illuminates how olfaction has contributed to the diversification of cichlids by combining with phylogenetic studies of olfactory receptors gene evolutions.

neuroscience↗

bioRxiv2021

High-resolution, genome-wide mapping of positive supercoiling in chromosomes

Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently-oriented genes, consistent with the "twin-domain" model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

molecular biology↗