Biology subjects

Shibata, T.

Publications and source records attributed to Shibata, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2021

Tracheal motile cilia require CAMSAP3 for formation of central microtubule pair and coordinated beating

Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body, which are linked by a transition zone. The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that CAMSAP3, a protein that can stabilize the minus end of a microtubule, concentrates at multiple sites of the cilium-basal body complex, including the upper region of the transition zone or the axonemal basal plate where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the basal plate, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme, and thereby supports the coordinated motion of multicilia in airway epithelial cells.

cell biology↗

bioRxiv2021

Autonomous epithelial folding induced by an intracellular mechano-polarity feedback loop

Epithelial tissues form folded structures during embryonic development and organogenesis. Whereas substantial efforts have been devoted to identifying mechanical and biochemical mechanisms that induce folding, how they interact remains poorly understood. Here we propose a mechano-biochemical model for dorsal fold formation in the early Drosophila embryo, an epithelial folding event induced by shifts of cell polarity. Based on experimentally observed apical domain homeostasis, we couple cell mechanics to polarity and find that mechanical changes following the initial polarity shifts alter cell geometry, which in turn influences the reaction-diffusion of polarity proteins, thus forming a feedback loop between mechanics and polarity. This model can induce spontaneous fold formation in silico, recapitulate polarity and shape changes observed in vivo, and confer robustness to tissue shape change against small fluctuations in mechanics and polarity. These findings reveal emergent properties of a developing epithelium under control of intracellular mechano-polarity coupling.

biophysics↗

bioRxiv2021

Multiple Monkey Pose Estimation Using OpenPose

This paper proposes a system for pose estimation on monkeys, in diverse and challenging scenarios, and under complex social interactions by using OpenPose. In comparison to most animals used for research, Monkeys present additional difficulties for pose estimation. Multiple degrees of freedom, unique complex postures, intricated social interactions, among others. Our monkey OpenPose trained model is robust against these difficulties. It achieves similar performance as in human pose estimation models, and it can run in Realtime.

neuroscience↗

bioRxiv2020

Expansion of Human Papillomavirus-Specific T Cells in Periphery and Cervix in a Therapeutic Vaccine Recipient Whose Cervical High-Grade Squamous Intraepithelial Lesion Regressed

Advances in high-throughput sequencing have revolutionized the manner with which we can study T cell responses. We describe a woman who received a human papillomavirus (HPV) therapeutic vaccine called PepCan, and experienced complete resolution of her cervical high-grade squamous intraepithelial lesion. By performing bulk T cell receptor (TCR) {beta} deep sequencing of peripheral blood mononuclear cells before and after 4 vaccinations, 70 putatively vaccine-specific clonotypes were identified for being significantly increased using a beta-binomial model. Interferon-{gamma} enzyme-linked immunospot assay identified significantly increased HPV-specific T cell response in the HPV 16 E6 91-115 region after 4 vaccinations (p=0.023). T cells with specificity to this region were sorted and analyzed using single-cell RNA-seq and TCR sequencing, HPV specificity in 60 of the 70 clonotypes identified to be vaccine-specific was demonstrated. TCR {beta} bulk sequencing of the cervical liquid-based cytology samples and cervical formalin-fixed paraffin-embedded samples before and after 4 vaccinations demonstrated the presence of these HPV-specific T cells in the cervix. Combining traditional and cutting-edge immunomonitoring techniques enabled us to demonstrate expansion of HPV-antigen specific T cells in the periphery and cervix.

immunology↗

bioRxiv2020

Small molecule targeting r(UGGAA)n disrupts RNA foci and alleviates disease phenotype in Drosophila model

Synthetic small molecules modulating RNA structure and function have therapeutic potential for RNA diseases. Here we report our discovery that naphthyridine carbamate dimer (NCD) targets disease-causing r(UGGAA)n repeat RNAs in spinocerebellar ataxia type 31 (SCA31). Structural analysis of the NCD-UGGAA/UGGAA complex by nuclear magnetic resonance (NMR) spectroscopy clarified the mode of binding that recognizes four guanines in UGGAA/UGGAA pentad by hydrogen bonding with four naphthyridine moieties of two NCD molecules. Biological studies show that NCD disrupts naturally occurring RNA foci built on r(UGGAA)n repeat RNA known as nuclear stress bodies (nSBs) by interfering with RNA-protein interactions resulting in the suppression of nSBs-mediated splicing event. Feeding NCD to larvae of the Drosophila model of SCA31 alleviates disease phenotype induced by toxic r(UGGAA)n repeat RNA. These studies demonstrated that small molecules targeting toxic repeat RNAs are a promising chemical tool for studies on repeat expansion diseases.

biochemistry↗

bioRxiv2020

Binary decision between asymmetric and symmetric cell division is defined by the balance of PAR proteins in C. elegans embryos

Coordination between cell differentiation and proliferation during development requires the balance between asymmetric and symmetric modes of cell division. However, the cellular intrinsic cue underlying the binary choice between these two division modes remains elusive. Here we show evidence in Caenorhabditis elegans that the invariable lineage of the division modes is programmed by the balance between antagonizing complexes of partitioning-defective (PAR) proteins. By uncoupling unequal inheritance of PAR proteins from that of fate determinants during zygote division, we demonstrated that changes in the balance between PAR-2 and PAR-6 are sufficient to re-program the division modes from symmetric to asymmetric and vice versa in two-cell stage embryos. The division mode adopted occurs independently of asymmetry in cytoplasmic fate determinants, cell-size asymmetry, and cell-cycle asynchrony between the sister cells. We propose that the balance between antagonizing PAR proteins represents an intrinsic self-organizing cue for binary specification of the division modes during development.

cell biology↗

bioRxiv2020

MacaquePose: A novel 'in the wild' macaque monkey pose dataset for markerless motion capture

Video-based markerless motion capture permits quantification of an animals pose and motion, with a high spatiotemporal resolution in a naturalistic context, and is a powerful tool for analyzing the relationship between the animals behaviors and its brain functions. Macaque monkeys are excellent non-human primate models, especially for studying neuroscience. Due to the lack of a dataset allowing training of a deep neural network for the macaques markerless motion capture in the naturalistic context, it has been challenging to apply this technology for macaques-based studies. In this study, we created MacaquePose, a novel open dataset with manually labeled body part positions for macaques in naturalistic scenes, consisting of >13,000 images, refined by researchers. We show that the pose estimation performance of an artificial neural network trained with the dataset is close to that of a human-level. The MacaquePose will provide a platform for innovative behavior analysis for non-human primate.

neuroscience↗

bioRxiv2020

CD47 prevents the elimination of diseased fibroblasts in scleroderma

Scleroderma is a devastating fibrotic autoimmune disease. Current treatments are partly effective in preventing disease progression, but do not remove fibrotic tissue. Here, we evaluated whether scleroderma fibroblasts take advantage of the "dont-eat-me-signal" CD47 and whether blocking CD47 enables the bodys immune system to get rid of diseased fibroblasts. To test this approach, we used a Jun-inducible scleroderma model. We first demonstrated in patient samples that scleroderma upregulated JUN and increased promotor accessibilities of both JUN and the CD47. Next, we established our scleroderma model demonstrating that Jun mediated skin fibrosis through the hedgehog-dependent expansion of CD26+Sca1-fibroblasts in mice. In a niche-independent adaptive transfer model, JUN steered graft survival and conferred increased self-renewal to fibroblasts. In vivo, JUN enhanced the expression of CD47, and inhibiting CD47 eliminated an ectopic fibroblast graft and increased in vitro phagocytosis. In the syngeneic mouse, depleting macrophages ameliorated skin fibrosis. Therapeutically, combined CD47 and IL6 blockade reversed skin fibrosis in mice and led to the rapid elimination of ectopically transplanted scleroderma cells. Altogether, our study is the first to demonstrate the efficiency of combining different immunotherapies in treating scleroderma and provide a rationale for combining CD47 and IL6 inhibition in clinical trials.

immunology↗

bioRxiv2020

Identification of differential gene expression pattern in lens epithelial cells derived from cataractous and non-cataractous lenses of Shumiya cataract rat

The Shumiya cataract rat (SCR) is a model for hereditary cataract. Two-third of these rats develop lens opacity within 10-11-weeks. Onset of cataract is attributed to the synergetic effect of lanosterol synthase (Lss) and farnesyl-diphosphate farnesyltransferase 1 (Fdft1) mutant alleles that lead to cholesterol deficiency in the lenses, which in turn adversely affects lens biology including the growth and differentiation of lens epithelial cells (LECs). Nevertheless, the molecular events and changes in gene expression associated with the onset of lens opacity in SCR is poorly understood. In the present study, a microarray-based approach was employed to analyze comparative gene expression changes in LECs isolated from the pre-cataractous and cataractous stages of lenses of 5-, 8- and 10-week-old SCRs. The changes in gene expression observed in microarray results in the LECs were further validated using real-time PCR and western blot analyses. Lens opacity was not observed in 5-week-old rats. However, histological analysis revealed mild dysplasia in the anterior suture and poorly differentiated fiber cells at the bow region. Expression of approximately 100 genes, including major intrinsic protein of lens fiber (MIP and Aquaporin0), deoxyribonuclease II beta (Dnase2b), heat shock protein B1 (Hspb1), and crystallin {gamma} ({gamma}Cry) B, C, and F were found to be significantly downregulated (0.07-0.5 fold) in rat LECs derived from cataract lenses compared to that in noncataractous lenses (control). Thus, our study aimed to identify the gene expression patterns during cataract formation in SCRs, which may be responsible for cataractogenesis in SCR. We proposed that mutation in lanosterol synthase was responsible for the downregulation of genes associated with lens fiber differentiation, which in turn leads to the formation of cataract in these rats. Our findings may have wider implications in understanding the effect of cholesterol deficiency and the role of cholesterol-lowering therapeutics on cataractogenesis.

cell biology↗

bioRxiv2020

Adherens junction serves to generate cryptic lamellipodia required for collective migration of epithelial cells

Collective migration of epithelial cells plays crucial roles in various biological processes such as cancer invasion. In migrating epithelial sheets, leader cells form lamellipodia to advance, and follower cells also form similar motile apparatus at cell-cell boundaries, which are called cryptic lamellipodia (c-lamellipodia). Using adenocarcinoma-derived epithelial cells, we investigated how c-lamellipodia are generated, and found that they sporadically grew from Ecadherin-based adherens junctions (AJs). WAVE and Arp2/3 complexes were localized along the AJs, and silencing them not only interfered with c-lamellipodia formation but also prevented follower cells from trailing the leaders. Disruption of AJs by removing E-catenin resulted in uncontrolled c-lamellipodia growth, and this was brought about by myosin II activation and the resultant contraction of AJ-associated actomyosin cables. Additional observations indicated that c-lamellipodia tended to grow at mechanically weak sites of the junction. We conclude that AJs not only tie cells together but also generate c-lamellipodia by recruiting actin regulators, enabling epithelial cells to undergo ordered collective migration.

cell biology↗

bioRxiv2020

Performance Evaluation of Markerless 3D Skeleton Pose Estimates with Pop Dance Motion Sequence

The evaluation of markerless pose estimation performed by OpenPose has been getting much attention from researchers of human movement studies. This work aims to evaluate and compare the output joint positions estimated by the OpenPose with a marker-based motion-capture data recorded on a pop dance motion. Although the marker-based motion capture can accurately measure and record the human joint positions, this particular set-up is expensive. The framework to compare the outputs of the markerless method to the ground truth marker-based joint remains unknown, especially for complex body motion. Synchronization, camera calibration, and 3D reconstruction by fusing the outputs of the markerless method (OpenPose) are discussed. In this case study, the comparison results illustrate that even if the markerless method expects to fail when the subjects body parts are self-occluded, the average magnitude errors for each key points are less than 700 mm.

neuroscience↗

bioRxiv2020

Evaluation of DNA extraction protocols from liquid-based cytology specimens for studying cervical microbiota

Cervical microbiota (CM) are considered an important factor affecting the progression of cervical intraepithelial neoplasia (CIN) and are implicated in the persistence of human papillomavirus (HPV). Collection of liquid-based cytology (LBC) samples is routine for cervical cancer screening and HPV genotyping and can be used for long-term cytological biobanking. We sought to determine whether it is possible to access microbial DNA from LBC specimens, and compared the performance of four different extraction protocols: (ZymoBIOMICS DNA Miniprep Kit; QIAamp PowerFecal Pro DNA Kit; QIAamp DNA Mini Kit; and IndiSpin Pathogen Kit) and their ability to capture the diversity of CM from LBC specimens. LBC specimens from 20 patients (stored for 716 {+/-} 105 days) with CIN values of 2 or 3 were each aliquoted for each of the four kits. Loss of microbial diversity due to long-term LBC storage could not be assessed due to lack of fresh LBC samples. Comparisons with other types of cervical sampling were not performed. We observed that all DNA extraction kits provided equivalent accessibility to the cervical microbial DNA within stored LBC samples. Approximately 80% microbial genera were shared among all DNA extraction protocols. Potential kit contaminants were observed as well. Variation between individuals was a significantly greater influence on the observed microbial composition than was the method of DNA extraction. We also observed that HPV16 was significantly associated with community types that were not dominated by Lactobacillus iners.

microbiology↗

bioRxiv2019

Polar pattern formation induced by contact following locomotion in a multicellular system

Biophysical mechanisms underlying collective cell migration of eukaryotic cells have been studied extensively in recent years. One paradigm that induces cells to correlate their motions is contact inhibition of locomotion, by which cells migrating away from the contact site. Here, we report that tail-following behavior at the contact site, termed contact following locomotion (CFL), can induce a non-trivial collective behavior in migrating cells. We show the emergence of a traveling band showing polar order in a mutant Dictyostelium cell that lacks chemotactic activity. The traveling band is dynamic in the sense that it continuously assembled at the front of the band and disassembled at the back. A mutant cell lacking cell adhesion molecule TgrB1 did not show both the traveling band formation and CFL. We thus conclude that CFL is the cell-cell interaction underlying the traveling band formation. We then develop an agent-based simulation with CFL, which shows the role of CFL in the formation of traveling band. We further show that the polar order phase consists of subpopulations that exhibit characteristic transversal motions with respect to the direction of band propagation. These findings describe a novel mechanism of collective cell migration involving cell-cell interactions capable of inducing traveling band with polar order.

biophysics↗

bioRxiv2019

ppGpp functions as an alarmone in metazoa

Guanosine 3,5-bis(pyrophosphate) (ppGpp) functions as a second messenger in bacteria to adjust their physiology in response to environmental changes. In recent years, the ppGpp-specific hydrolase, metazoan SpoT homolog-1 (Mesh1), was shown to have important roles for growth under nutrient deficiency in Drosophila melanogaster. Curiously, however, ppGpp has never been detected in animal cells, and therefore the physiological relevance of this molecule, if any, in metazoans has not been established. Here, we report the detection of ppGpp in Drosophila and human cells and demonstrate that ppGpp accumulation induces metabolic changes, cell death, and eventually lethality in Drosophila. Our results provide the first evidence of the existence and function of the ppGpp-dependent stringent response in animals.

molecular biology↗

bioRxiv2019

Nucleosides drive histiocytosis in SLC29A3 disorders by activating TLR7

A lysosomal transmembrane protein SLC29A3 transports nucleosides from lysosomes to the cytoplasm. Loss-of-function mutations of the SLC29A3 gene cause lysosomal nucleoside storage in monocyte/macrophages, leading to their accumulation called histiocytosis in humans and mice. Little is known, however, about a mechanism behind nucleoside-dependent histiocytosis. TLR7, an innate immune sensors for single stranded RNA, bind and respond to nucleosides. We here show that they drive nucleoside-mediated histiocytosis. Patrolling monocyte/macrophages accumulate in the spleen of Slc29a3-/- mice but not Slc29a3-/- Tlr7-/- mice. Accumulated patrolling monocyte/macrophages stored nucleosides derived from cell corpse. TLR7 was recruited to phagosomes and activated as evidenced by TLR7-dependent phagosomal maturation. TLR7 induced hyper-responsiveness to M-CSF in Slc29a3-/- monocyte/macrophages. These results suggest that TLR7 drives histiocytosis in SLC29A3 disorders. One Sentence SummarySLC29A3 disorders are caused by activation of TLR7 with accumulated nucleosides in lysosomes.

immunology↗

bioRxiv2019

A unified simulation model for understanding the diversity of cancer evolution

Because cancer evolution underlies the therapeutic difficulties of cancer, it is clinically important to understand the evolutionary dynamics of cancer. Thus far, a number of evolutionary processes have been proposed to be working in cancer evolution. However, there exists no simulation model that can describe the different evolutionary processes in a unified manner. In this study, we constructed a unified simulation model for describing the different evolutionary processes and performed sensitivity analysis on the model to determine the conditions in which cancer growth is driven by each of the different evolutionary processes. Our sensitivity analysis has successfully provided a series of novel insights into the evolutionary dynamics of cancer. For example, we found that, while a high neutral mutation rate shapes neutral intratumor heterogeneity (ITH) characterized by a fractal-like pattern, a stem cell hierarchy can also contribute to shaping neutral ITH by apparently increasing the mutation rate. Although It has been reported that the evolutionary principle shaping ITH shifts from selection to accumulation of neutral mutations during colorectal tumorigenesis, our simulation revealed the possibility that this evolutionary shift is triggered by drastic evolutionary events that occur in a a short time and confer a marked fitness increase on one or a few cells. This result helps us understand that each process works not separately but simultaneously and continuously as a series of phases of cancer evolution. Collectively, this study serves as a basis to understand in greater depth the diversity of cancer evolution.

cancer biology↗

bioRxiv2019

Mesenchymal-Epithelial Transition Regulates Initiation of Pluripotency Exit before Gastrulation

The pluripotent epiblast gives rise to all tissues and organs in an adult body. Its differentiation starts at gastrulation when the epiblast generates mesoderm and endoderm germ layers through a process called epithelial-mesenchymal transition (EMT). Although gastrulation EMT coincides with loss of epiblast pluripotency, pluripotent cells in development and in vitro can adopt either mesenchymal or epithelial morphology. The relationship between epiblasts cellular morphology and its pluripotency is not well understood. In this work, using chicken epiblast and mammalian pluripotency stem cell (PSC) models, we show that PSCs undergo a mesenchymal-epithelial transition (MET) prior to EMT-associated pluripotency loss. Epiblast MET and its subsequent EMT are two distinct processes. The former, a partial MET, is associated with reversible initiation of pluripotency exit; whereas the latter, a full EMT, is associated with complete and irreversible pluripotency loss. We provide evidence that integrin-mediated cell-matrix interaction is a key player in pluripotency exit regulation. We propose that epiblast partial MET is an evolutionarily conserved process among all amniotic vertebrates and its developmental function is to mediate planar symmetry-breaking within an epithelialized epiblast, taking place after epiblast MET but before gastrulation EMT.

developmental biology↗

bioRxiv2019

Single-cell DNA and RNA sequencing reveals the dynamics of intra-tumor heterogeneity in a colorectal cancer model

Intra-tumor heterogeneity (ITH) encompasses cellular differences in tumors and is related to clinical outcomes, such as drug resistance. However, little is known about the dynamics of ITH, owing to the lack of time-series analysis at the single-cell level. We performed single-cell exome and transcriptome sequencing of 200 cells and investigated how ITH is generated from one single cell in a mouse colorectal cancer model. The ITH of the transcriptome increased after transplantation from cultured organoids, while that of the exome decreased. Mutations generated in the culture did not greatly change at the transplantation at the bulk-cell level. The RNA ITH increase was due to the emergence of new transcriptional subpopulations. In contrast to the initial cells expressing mesenchymal-marker genes, new subpopulations repressed these genes at transplantation. Analyses of colorectal cancer data from The Cancer Genome Atlas revealed a high proportion of metastatic cases in human subjects with expression patterns similar to the new cell subpopulations in mouse. These results suggest that the birth of transcriptional subpopulations may be a key for adaptation to drastic micro-environmental changes when cancer cells have sufficient genetic alterations at later tumor stages. This study revealed an evolutionary dynamics of single-cell RNA and DNA changes in tumor progression, giving insights into the mesenchymal-epithelial transformation of tumor cells at metastasis in colorectal cancer.

genomics↗