Biology subjects

Ohnishi, K.

Publications and source records attributed to Ohnishi, K..

8 recordsLinked to original sources

bioRxiv2026

Dual-color fluorescent reporters resolve Alu and LINE-1 retrotransposition in single cells

LINE-1 (L1) is transcribed by RNA polymerase (Pol) II, whereas the non-coding Short INterspersed Element (SINE) Alu is transcribed by Pol III and exploits L1 ORF2p for mobilization. Conventional L1 retrotransposition reporters rely on spliceosome-dependent introns and cannot directly monitor Alu retrotransposition. Here, we developed EGFPTet, a fluorescent SINE reporter in which EGFP is interrupted by a Tetrahymena thermophila group I self-splicing intron. EGFPTet enabled flow cytometry, fluorescence microscopy, cell sorting, and time-course analysis of Alu mobilization without colony formation. Reporter activation required ORF2p reverse transcriptase activity, and recovered Alu-EGFPTet insertions displayed retrotransposition hallmarks. EGFPTet quantified Alu retrotransposition across cell lines and host-factor perturbations and was repurposed to detect mobilization of the phylogenetically distinct mouse B2 SINE. Defined amino acid substitutions generated an mTurquoise2 derivative without redesigning the self-splicing junctions, allowing dual-color analysis of Alu trans-mobilization and L1 cis retrotransposition in individual cells. In this assay, wild-type L1 supported a higher Alu retrotransposition frequency than the ORF1p-mutant L1. Despite their shared dependence on ORF2p, Alu co-expression did not significantly reduce L1 retrotransposition, and Alu/L1 dual-positive counts exceeded independence-based expectations, indicating preferential co-occurrence of the two reporter signals. EGFPTet provides a fluorescent platform for investigating the shared and distinct cellular conditions that support SINE and L1 mobilization.

molecular biology↗

bioRxiv2026

A hidden thermal mechanism in inhibitory ligand-gated chloride channels

Temperature profoundly shapes neural activity, with temperature-sensitive excitatory cation channels serving as key molecular components of thermosensation and neuronal excitability. Here we show that the glutamate-gated chloride channel AVR-14B, a Cys-loop receptor and principal molecular target of the antiparasitic drug ivermectin in the nematode Brugia malayi, exhibits temperature-dependent gating. Application of glutamate to AVR-14B evoked rapidly desensitizing transient currents; however, above approximately 24 {degrees}C, an additional non-desensitizing sustained current component emerged. Mechanistically, warming altered the gating behavior of the channel, thereby conferring intrinsic temperature sensitivity. Mutational and structural analyses revealed that ions permeate via lateral fenestrations distinct from the central axial pore, forming a noncanonical pathway for temperature-dependent gating. This temperature-dependent gating determines drug efficacy: ivermectin failed to activate AVR-14B below the thermal threshold at which the sustained current emerges. Finally, AVR-14B-null C. elegans showed enhanced heat tolerance, even though wild-type animals generally fail to thrive above 25 {degrees}C, confirming that this molecular mechanism governs organismal physiology. Similar temperature-dependent gating was observed in the human glycine receptor, indicating a conserved principle within this receptor class. Our findings identify inhibitory ligand-gated ion channels as intrinsic thermosensors and uncover a mechanism by which temperature can switch a single receptor between phasic and tonic inhibition, with implications for neural function and temperature-mediated therapeutics.

biophysics↗

bioRxiv2025

Neural mechanisms underlying reward processing and social cognition: a replication study with a Japanese sample

Neural functions underlying reward processing and social cognition play a critical role in everyday decision-making. Given that these processes may be shaped by cultural factors, it is essential to examine their cross-cultural generalizability. In this study, we used functional MRI to scan native Japanese speakers as they performed two well-established experimental paradigms: the Monetary Incentive Delay (MID) task for reward processing and the Theory of Mind (ToM) task for social cognition. We successfully replicated previous findings. Specifically, in the MID task, reward expectation and reward outcome were associated with neural activity in the ventral striatum and ventromedial prefrontal cortex. In the ToM task, social cognition was linked to activation in the temporoparietal junction. Notably, the posterior cingulate cortex was engaged in both tasks, suggesting its integrative role across cognitive domains. Together, these results replicate and extend earlier work, supporting the cross-cultural generalizability of the neural mechanisms underlying reward and social cognition, and further validating our fMRI protocol for future research.

neuroscience↗

bioRxiv2024

Chemotherapy resistance due to epithelial-to-mesenchymal transition is caused by abnormal lipid metabolic balance

Invasive cancer is defined by the loss of epithelial cell traits resulting from the ectopic expression of epithelial-mesenchymal transition (EMT)-related transcription factors such as Snail. Although EMT is known to impart chemoresistance to cancer cells, the precise molecular mechanisms remain elusive. We found that Snail expression confers chemoresistance by upregulating the cholesterol efflux pump ABCA1 as a countermeasure to the excess of cytotoxic free cholesterol relative to its major interaction partner in cellular membranes, sphingomyelin. This imbalance is introduced by the transcriptional repression of enzymes involved in the biosynthesis of sphingomyelin by Snail. Inhibiting esterification of cholesterol, which renders it inert, selectively suppresses growth of a xenograft model of Snail-positive kidney cancer. Our findings offer a new perspective on lipid-targeting strategies for invasive cancer therapy.

cell biology↗

bioRxiv2021

MAP4K3 inhibits Sirtuin-1 to repress LKB1-AMPK to promote amino acid dependent activation of mTORC1

mTORC1 is the key rheostat controlling cellular metabolic state. Of the various inputs to mTORC1, the most potent effector of intracellular nutrient status is amino acid supply. Despite an established role for MAP4K3 in promoting mTORC1 activation in the presence of amino acids, the signaling pathway by which MAP4K3 controls mTORC1 activation remains unknown. Here we examined the process of MAP4K3 activation of mTORC1 and found that MAP4K3 represses the LKB1-AMPK pathway to prevent TSC1/2 complex inactivation of Rheb. When we sought the regulatory link between MAP4K3 and LKB1 inhibition, we discovered that MAP4K3 physically interacts with the master nutrient regulatory factor Sirtuin-1 and phosphorylates Sirtuin-1 to repress LKB1 activation. Our results reveal the existence of a novel signaling pathway linking amino acid satiety with MAP4K3-dependent suppression of SIRT1 to inactivate the repressive LKB1-AMPK pathway and thereby potently activate the mTORC1 complex to dictate the metabolic disposition of the cell.

cell biology↗

bioRxiv2021

All-trans retinoic acid induces GADD34 gene expression via transcriptional regulation by Six1-TLE3 and post-transcriptional regulation by p38-TTP in skeletal muscle

All-trans retinoic acid (ATRA) increases the sensitivity to unfolded protein response (UPR) in differentiating leukemic blasts. The downstream transcriptional factors of PERK, a major arm of UPR, regulates muscle differentiation. However, the role of growth arrest and DNA damage-inducible protein 34 (GADD34), one of the downstream factors of PERK, and the effects of ATRA on GADD34 expression in muscle remain unclear. In this study, we identified ATRA increased the GADD34 expression independent of the PERK signal in the gastrocnemius muscle of mice. ATRA up-regulated GADD34 expression through the transcriptional activation of it via inhibiting the interaction of homeobox Six1 and transcription co-repressor TLE3 with the MEF3-binding site on the GADD34 gene promoter in myoblasts. ATRA also inhibited the interaction of TTP, which induces mRNA degradation, with AU-rich element on GADD34 mRNA via p38 MAPK, resulting in the instability of GADD34 mRNA. Overexpressed GADD34 in myoblasts changes the type of myosin heavy chain in myotubes. These results suggest ATRA increases GADD34 expression via transcriptional and post-transcriptional regulation in myoblasts, which changes muscle fiber type in myotubes.

biochemistry↗

bioRxiv2020

Cellular identity and Ca2+ signaling activity of the non-reproductive GnRH system in the Ciona larva

Tunicate larvae have a non-reproductive gonadotropin-releasing hormone (GnRH) system with multiple ligands and receptor heterodimerization enabling complex regulation. In the Ciona larva, one of the gnrh genes, gnrh2, is conspicuously expressed in the motor ganglion and nerve cord, which are homologous structures to the hindbrain and spinal cord, respectively, of vertebrates. The gnrh2 gene is also expressed in the proto-placodal sensory neurons, which are the proposed homologue of vertebrate olfactory neurons. The tunicate larvae occupy a non-reproductive dispersal stage, yet the role of their GnRH system remains elusive. In this study, we investigated neuronal types of gnrh2-expressing cells in Ciona larvae and visualized the activity of these cells by fluorescence imaging using a calcium sensor protein. Some cholinergic neurons and dopaminergic cells express gnrh2, suggesting that GnRH plays a role in controlling swimming behavior. However, none of the gnrh2-expressing cells overlap with glycinergic or GABAergic neurons. A role in motor control is also suggested by the correlation between the activity of some gnrh2-expressing cells and tail movements. Interestingly, gnrh2-positive ependymal cells in the nerve cord, known as a kind of glia cells, actively produced Ca2+ transients, suggesting that neuroendocrine signaling occurs in the glia cells of the nerve cord.

neuroscience↗

bioRxiv2019

Mechanoreceptor-mediated circuit regulates cold tolerance in Caenorhabditis elegans

C. elegans mechanoreceptors located in the ASG sensoryneuron have been found to sense temperature -- a key trait for animal survival. Experimental loss of xanthine dehydrogenase (XDH-1) function in the AIN and AVJ interneurons resulted in reduced cold tolerance and atypical neuronal response to changes in temperature. These interneurons are synapse with upstream neurons such as the mechanoreceptor-expressing ASG. Ca2+ imaging revealed that ASG responsiveness to temperature change via mechanoreceptor DEG-1, a Degenerin/Epithelial Sodium Channel (DEG/ENaC), affects downstream AIN and AVJ circuits. Ectopic expression of DEG-1 in the ASE gustatory neuron resulted in acquisition of thermosensitivity, while electrophysiological analysis revealed that DEG-1 was involved in temperature sensation. Together, these results suggest that cold tolerance is regulated by mechanoreceptor-mediated circuit calculation.

genetics↗