Biology subjects

Komori, T.

Publications and source records attributed to Komori, T..

9 recordsLinked to original sources

bioRxiv2026

Mesoscopic cortical activities associated with pupil-linked perceptions inferred via explainable machine learning

Pupil dilation reflects arousal-related neural processes and is closely linked to sensory perception, attention, and cognitive state, but the mesoscopic cortical dynamics that accompany stimulus-evoked dilation remain unclear. Here, we combined simultaneous pupillometry and wide-field Ca{superscript 2} imaging in mice with explainable machine learning to identify cortical activity patterns predictive of pupil dilation. Cortical activity was recorded during hindpaw somatosensory stimulation, visual pattern change, and visual contextual stimulation using an visual oddball paradigm; spontaneous pupil dilation was additionally analyzed using a public resting-state dataset. To reduce multicollinearity in wide-field imaging signals, cortical activity was decomposed into independent components, which were used as inputs to recurrent neural network (RNN) decoders. Models reliably predicted sensory-evoked pupil dilation above shuffled-label controls when sessions with pre-existing pupil dilation were excluded, indicating that stimulus-evoked dilation was associated with distinct cortical activity patterns. Feature attribution using SHapley Additive exPlanations (SHAP) and spatial back-mapping revealed condition-dependent cortical signatures. Activity around the retrosplenial cortex and higher-order visual areas contributed to predictions during somatosensory and visual pattern stimulation, whereas secondary motor cortex activity was prominent during visual contextual stimulation. Spontaneous pupil dilation required lower-frequency cortical signals for decoding, suggesting partly distinct dynamics from sensory-evoked dilation. These findings demonstrate that explainable machine learning can extract candidate cortical activity patterns associated with pupil-linked arousal from high-dimensional wide-field Ca{superscript 2} imaging data. Because feature importance does not establish causality, the identified regions provide hypothesis-generating targets for future circuit manipulation studies.

neuroscience↗

bioRxiv2025

Anti-amyloid beta therapy resolves stroke recovery impairment caused by Alzheimer's disease

Stroke and dementia are common comorbidities and a growing concern causing disability in aging societies worldwide. Although anti-amyloid beta (anti-A{beta}) antibodies have recently been anticipated to relieve preclinical Alzheimers disease pathology, we discovered that post-stroke administration of anti-A{beta} antibodies restored neural repair for stroke recovery impeded by cerebral A{beta} accumulation. Neuronal recovery-associated gene expression for stroke recovery was considerably impaired even by slight A{beta} accumulation in murine and human brain. Slight A{beta} accumulation had less impact on neurons without stroke but caused a unique myeloid immunity after an ischemic stroke that enhanced the inflammatory cascades impeding neural repair for stroke recovery. Aducanumab administration after ischemic stroke prevented formation of this malignant myeloid immunity, resolving the impairment of post-stroke neural repair caused by cerebral A{beta} accumulation. Thus, our study has revealed the ability of anti-A{beta} therapies to restore functional recovery after a stroke with cerebral A{beta} accumulation.

neuroscience↗

bioRxiv2024

Discovery of functional NLRs using expression level, high-throughput transformation, and large-scale phenotyping

Protecting crops from pests and diseases is vital for the sustainable agricultural systems needed for food security. Introducing functional resistance genes to enhance the plant immune system is an effective method of disease control, but identifying new immune receptors is time-consuming and resource intensive. We observed that functional immune receptors of the NLR class show a signature of high expression in uninfected plants across both monocot and dicot species. Here we show that this signature, combined with high throughput crop transformation, can be used to rapidly identify candidate NLRs from diverse plant species and validate pathogen resistance directly in crop plants. As a proof of concept, we generated a wheat transgenic library carrying 995 NLRs from 18 grass species. Screening the collection with the stem rust pathogen Puccinia graminis, which is a major threat to wheat production, we confirm 19 new resistance genes. This pipeline facilitates resistance gene discovery, unlocking a large gene pool of diverse and non-domesticated plant species and providing in-planta gene validation of disease resistance directly in crops.

plant biology↗

bioRxiv2022

Novel phosphorylation of Rab29 that regulates its localization and lysosomal stress response in concert with LRRK2

Rab proteins are small GTPases that regulate a myriad of intracellular membrane trafficking events. Rab29 is one of the Rab proteins phosphorylated by leucine-rich repeat kinase 2 (LRRK2), a Parkinsons disease-associated kinase. Recent studies suggest that Rab29 regulates LRRK2, whereas the mechanism by which Rab29 is regulated remained unclear. Here we report a novel phosphorylation in Rab29 that is not mediated by LRRK2 and occurs under lysosomal overload stress. Mass spectrometry analysis identified the phosphorylation site of Rab29 as Ser185, and cellular expression studies of phosphomimetic mutants of Rab29 at Ser185 unveiled the involvement of this phosphorylation in counteracting lysosomal enlargement. PKC was deemed to be responsible for this phosphorylation and control the lysosomal localization of Rab29 in concert with LRRK2. These results implicate PKC in the lysosomal stress response pathway comprised of Rab29 and LRRK2, and further underscore the importance of this pathway in the mechanisms underlying lysosomal homeostasis.

molecular biology↗

bioRxiv2022

ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid cells

Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, for insertion of long transgenes such as fluorescent reporters in human cells, the optimal type of HDR donors remains unclear. In this study, we established a simple and efficient CRISPR-mediated knock-in method for long transgenes using linear dsDNA and ssDNA donors, and systematically compared the performance of these two donors for endogenous gene tagging in human non-transformed diploid cells. Quantification using flow cytometry revealed higher efficiency of fluorescent tagging with dsDNA donors than with ssDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was higher with dsDNA donors than with ssDNA. Moreover, in off-target integration analyses, dsDNA and ssDNA were comparably prone to non-homologous integration. These results indicate that ssDNA is not superior to dsDNA as long HDR donors for gene knock-in in human cells.

cell biology↗

bioRxiv2021

Quantitative analysis of CRISPR-del for complete gene knockout in human diploid cells

The advance of CRISPR/Cas9 technology has enabled us easily to generate gene knockout cell lines by introducing insertion/deletion mutations (indels) at the target site via the error-prone non-homologous end joining repair system. Frameshift-promoting indels can disrupt gene functions by generation of a premature stop codon. However, there is growing evidence that targeted genes are not always knocked-out by the indel-based gene disruption. In this study, we established a pipeline of CRISPR-del, which induces a large chromosomal deletion by cutting two different target sites, to perform "complete" gene knockout efficiently in non-transformed human diploid RPE1 cells. By optimizing several procedures, the CRISPR-del pipeline allowed us to generate knockout cell lines harboring bi-allelic large chromosomal deletions in a high-throughput manner. Quantitative analyses show that the frequency of gene deletion with this approach is much higher than that of conventional CRISPR-del methods. The lengths of the deleted genomic regions demonstrated in this study are longer than those of 95% of the human protein-coding genes. Furthermore, the pipeline enables the generation of a model cell line having a bi-allelic cancer-associated chromosomal deletion. Overall, these data lead us to propose that the CRISPR-del pipeline is a high-throughput approach for performing "complete" gene knockout in RPE1 cells.

cell biology↗

bioRxiv2021

The oncogenic Runx3-Myc axis defines p53-deficient osteosarcomagenesis

Osteosarcoma (OS) in human patients is characterized by genetic alteration of TP53. Osteoprogenitor-specific p53-deleted mice (OS mice) have been widely used to study the process of osteosarcomagenesis. However, the molecular mechanisms responsible for the development of OS upon p53 inactivation remain largely unknown. In this study, we detected prominent RUNX3/Runx3 expression in human and mouse p53-deficient OS. Myc was aberrantly upregulated by Runx3 via mR1, a consensus Runx site in the Myc promoter, in a manner dependent on p53 deficiency. Reduction of the Myc level by disruption of mR1 or Runx3 knockdown decreased the tumorigenicity of p53-deficient OS cells and effectively suppressed OS development in OS mice. Furthermore, Runx inhibitors exerted therapeutic effects on OS mice. Together, these results show that p53 deficiency promotes osteosarcomagenesis in human and mouse by allowing Runx3 to induce oncogenic Myc expression.

cancer biology↗

bioRxiv2020

Roles of lysosomotropic agents on LRRK2 activation and Rab10 phosphorylation

Leucine-rich repeat kinase 2 (LRRK2), the major causative gene product of autosomal-dominant Parkinsons disease, is a protein kinase that phosphorylates a subset of Rab GTPases. Since pathogenic LRRK2 mutations increase its ability to phosphorylate Rab GTPases, elucidating the mechanisms of how Rab phosphorylation is regulated by LRRK2 is of great importance. We have previously reported that chloroquine-induced lysosomal stress facilitates LRRK2 phosphorylation of Rab10 to maintain lysosomal homeostasis. Here we reveal that Rab10 phosphorylation by LRRK2 is potently stimulated by treatment of cells with a set of lysosome stressors and clinically used lysosomotropic drugs. These agents commonly promoted the formation of LRRK2-coated enlarged lysosomes and extracellular release of lysosomal enzyme cathepsin B, the latter being dependent on LRRK2 kinase activity. In contrast to the increase in Rab10 phosphorylation, treatment with lysosomotropic drugs did not increase the enzymatic activity of LRRK2, as monitored by its autophosphorylation at Ser1292 residue, but rather enhanced the molecular proximity between LRRK2 and its substrate Rab GTPases on the cytosolic surface of lysosomes. Lysosomotropic drug-induced upregulation of Rab10 phosphorylation was likely a downstream event of Rab29 (Rab7L1)-mediated enzymatic activation of LRRK2. These results suggest a regulated process of Rab10 phosphorylation by LRRK2 that is associated with lysosomal overload stress, and provide insights into the novel strategies to halt the aberrant upregulation of LRRK2 kinase activity.

molecular biology↗

bioRxiv2017

Real-time observation of flexible domain movements in Cas9

The CRISPR-associated protein Cas9 is a widely used genome editing tool that recognizes and cleaves target DNA through the assistance of a single-guide RNA (sgRNA). Structural studies have demonstrated the multi-domain architecture of Cas9 and sequential domain movements upon binding to the sgRNA and the target DNA. These studies also hinted at the flexibility between domains, but whether these flexible movements occur in solution is unclear. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single-molecule FRET. The flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain in Cas9 only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo-Cas9, which may play a role in the assembly with the sgRNA. Collectively, our results highlight the potential role of domain fluctuations in driving Cas9-catalyzed DNA cleavage.

biophysics↗