Biology subjects

Nakamura, M.

Publications and source records attributed to Nakamura, M..

At least 109 records · Page 6Linked to original sources

bioRxiv2020

Autocrine insulin pathway signaling regulates actin dynamics in cell wound repair

Cells are exposed to frequent mechanical and/or chemical stressors that can compromise the integrity of the plasma membrane and underlying cortical cytoskeleton. The molecular mechanisms driving the immediate repair response launched to restore the cell cortex and circumvent cell death are largely unknown. Using microarrays and drug-inhibition studies to assess gene expression, we find that initiation of cell wound repair in the Drosophila model is dependent on translation, whereas transcription is required for subsequent steps. We identified 253 genes whose expression is up-regulated (80) or down-regulated (173) in response to laser wounding. A subset of these genes were validated using RNAi knockdowns and exhibit aberrant actomyosin ring assembly and/or actin remodeling defects. Strikingly, we find that the canonical insulin signaling pathway controls actin dynamics through the actin regulators Girdin and Chickadee (profilin), and its disruption leads to abnormal wound repair. Our results provide new insight for understanding how cell wound repair proceeds in healthy individuals and those with diseases involving wound healing deficiencies.

developmental biology↗

bioRxiv2020

β-catenin Signaling Regulates Cell Fate Decisions at the Transition Zone of the Chondro-Osseous Junction During Fracture Healing

Chondrocytes within the fracture callus transform into osteoblasts during bone regeneration, but the molecular mechanisms regulating this process are unknown. Wnt ligands are expressed within the fracture callus, and hypertrophic chondrocytes undergoing transformation to osteoblasts exhibit nuclear localization of {beta}-catenin, indicating active Wnt signaling in these cells. Here, we show that conditional knock out (cKO) of {beta}-catenin in chondrocytes inhibits the transformation of chondrocytes to osteoblasts, while stabilization of {beta}-catenin in chondrocytes accelerates this process. After cKO, chondrocyte-derived cells were located in the bone marrow cavity and upon re-fracture formed cartilage. Lineage tracing in wild type mice revealed that in addition to osteoblasts, chondrocytes give rise to stem cells that contribute to repair of subsequent fractures. These data indicate that Wnt signaling directs cell fate choices of chondrocytes during fracture healing by stimulating transformation of chondrocytes to osteoblasts, and provide a framework for developing Wnt-therapies to stimulate repair.

developmental biology↗

bioRxiv2020

Self-labeling of proteins with chemical fluorescent dyes in BY-2 cells and Arabidopsis seedlings

AO_SCPLOWBSTRACTC_SCPLOWSynthetic chemical fluorescent dyes are promising tools for many applications in biology. SNAP tagging provides a unique opportunity for labeling of specific proteins in vivo with synthetic dyes for studying for example endocytosis, or super-resolution microscopy. However, despite the potential, chemical dye tagging has not been used effectively in plants. A major drawback was the limited knowledge regarding cell wall and membrane permeability of synthetic dyes. Twenty-six out of 31 synthetic dyes were taken up into BY-2 cells, eight were not taken up and can thus serve for measuring endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine (TMR) and silicon-rhodamine (SiR) 647 were used to SNAP tag -tubulin. Successful tagging was verified by live cell imaging and visualization of microtubules arrays in interphase and during mitosis. Fluorescence activation-coupled protein labeling (FAPL) with DRBG-488 was used to observe PIN2 endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the newly forming cell plate during mitosis. Together the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety to cell biological processes.

cell biology↗

bioRxiv2020

Intravenous edaravone plus therapeutic hypothermia offers limited neuroprotection in the hypoxic-ischaemic newborn piglet

Therapeutic hypothermia is a standard therapy for neonatal hypoxic-ischaemic encephalopathy. One potential additional therapy is the free radical scavenger edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one). To compare the neuroprotective effects of edaravone plus therapeutic hypothermia with those of therapeutic hypothermia alone after a hypoxic-ischaemic insult in the newborn piglet, anaesthetized piglets were subjected to 40 min of hypoxia (3-5% inspired oxygen) and cerebral ischaemia was assessed by cerebral blood volume. Body temperature was maintained at 38.5 {degrees}C in the normothermia group (NT, n = 8) and at 34 {degrees}C (24 h after the insult) in the therapeutic hypothermia (TH, n = 7) and therapeutic hypothermia plus edaravone (3 mg intravenous every 12 h for 3 days after the insult; TH+EV, n = 6) groups under mechanical ventilation. Five days after the insult, the mean (standard deviation) neurological scores were 10.9 (5.7) in the NT group, 17.0 (0.4) in the TH group (p = 0.025 vs. NT) and 15.0 (3.9) in the TH+EV group. The histopathological score of the TH+EV group showed no significant improvement compared with that of the other groups. In conclusion, edaravone plus therapeutic hypothermia had no additive neuroprotective effects after hypoxia-ischaemia in neurological and histopathological assessments.

neuroscience↗

bioRxiv2020

Immunological role of primary cilia of dendritic cells in human skin disease

Primary cilia are a unique organelle, known to provide a signaling hub for variety of cell activities. Their potential role(s) in human immune homeostasis and diseases, however, have yet to be explored. Here, we show that human dendritic cells (DCs) express primary cilia-like structure. The primary cilia growth during DC proliferation by GM-CSF was shut off by DC maturation agents, suggesting the role of primary cilia to transduce proliferation signaling. PDGFR pathway, one of proliferation signal in primary cilia, promoted DC proliferation in a dependent manner of intra-flagellar transport system. In epidermis with atopic dermatitis patients, aberrant ciliated langerhans cells and keratinocytes with showing immature state were observed that may play a potential role in inflammation and skin barrier disorder.

cell biology↗

bioRxiv2019

Wash and the WASH Regulatory Complex function in Nuclear Envelope budding

Nuclear envelope budding is a recently described phenomenon wherein large macromolecular complexes can be packaged inside the nucleus and be extruded through the nuclear membranes, completely bypassing nuclear pores. While factors have been identified both as cargos or actively involved in this process, much remains unknown about the molecules that generate the forces and membrane deformations which appear inherent. Using fluorescence and electron microscopy, biochemical and cell biological assays, and genetic perturbations in the Drosophila model, we identify Wash, its regulatory complex, and Arp2/3 as novel players in NE-budding. Surprisingly, Washs role in this process is bipotent and, independent of SHRC/Arp2/3, its perturbation disrupts the normal homotypic Lamin A/B meshworks that are necessary for NE-budding to occur. In addition to NE-budding emerging as important in additional cellular processes and organisms, its incredible similarity to herpesvirus egress suggests new avenues for exploration in both normal and disease biology.

cell biology↗

bioRxiv2019

Transcriptomic profiling of plaque psoriasis and cutaneous T cell subsets during treatment with secukinumab

The IL17A inhibitor secukinumab is efficacious for the treatment of psoriasis. In order to define its mechanism of action, it is important to understand its impact on psoriatic whole skin tissue as well as specific skin-resident immune cell populations such as T lymphocytes. In this study, we treated 15 moderate-to-severe plaque psoriasis patients with secukinumab and characterized the longitudinal transcriptomic changes of whole lesional skin tissue and cutaneous CD4+ T effector cells (Teffs), CD4+ T regulatory cells (Tregs), and CD8+ T effector cells during 12 weeks of treatment. Secukinumab was clinically effective, with 100%, 47%, and 27% of patients in the study achieving PASI75, PASI90, and PASI100 by week 12, respectively. At baseline prior to treatment, we observed that IL17A overexpression predominates in psoriatic CD8+ T cells rather than Teffs, supporting the importance of IL-17-secreting CD8+ T cells (Tc17) compared to IL-17-secreting CD4+ T cells (Th17) cells in the pathogenesis of psoriasis. Although secukinumab targets only IL17A, we observed rapid reduction of IL17A, IL17F, IL23A, IL23R, and IFNG expression in lesional skin as soon as 2 weeks after initiation of treatment and normalization of expression by week 12. Secukinumab treatment resulted in resolution of 89-97% of psoriasis-associated expression differences in both bulk tissue and T cell subsets by week 12 of treatment. Overall, secukinumab appears to rapidly reverse many of the molecular hallmarks of psoriasis.

genomics↗

bioRxiv2019

Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells

BackgroundEndothelial cells (ECs) make up the innermost layer throughout the entire vasculature. Their phenotypes and physiological functions are initially regulated by developmental signals and extracellular stimuli. The underlying molecular mechanisms responsible for the diverse phenotypes of ECs from different organs are not well understood.\n\nResultsTo characterize the transcriptomic and epigenomic landscape in the vascular system, we cataloged gene expression and active histone marks in nine types of human ECs (generating 148 genome-wide datasets) and carried out a comprehensive analysis with chromatin interaction data. We identified 3,765 EC-specific enhancers, some of which were associated with disease-associated genetic variations. We also identified various candidate marker genes for each EC type. Notably, reflecting the developmental origins of ECs and their roles in angiogenesis, vasculogenesis and wound healing.\n\nConclusionsWhile the importance of several HOX genes for early vascular development and adult angiogenesis in pathological conditions has been reported, a systematic analysis of the regulation and roles of HOX genes in mature tissue cells has been lacking. These datasets provide a valuable resource for understanding the vascular system and associated diseases.

genomics↗

bioRxiv2019

Temporal-Spatial Visualization of Endogenous Chromosome Rearrangements in Living Cells

Visualizing the real-time dynamics of genome rearrangement in single living cells is core to studying genomics and diagnostics. Here, we report a robust, versatile approach named CRISPR Live-cell fluorescent in situ hybridization (LiveFISH) for multi-locus genome tracking and cytogenetic detection in a broad variety of cell types including primary cells. LiveFISH utilizes an intrinsic stability switch of CRISPR guide RNAs, which enables efficient and accurate detection of chromosomal disorders such as Patau Syndrome in prenatal amniotic fluid cells and allows multi-locus tracking in human T lymphocytes. Using LiveFISH, we are able to detect and track real-time spatiotemporal dynamics of non-homologous endogenous chromosome translocations induced by gene editing. This new approach enables FISH imaging in living primary cells, which can provide useful insights into the spatiotemporal changes of genome organization and rearrangements in normal and diseased primary cells and will enable fast cytogenetic visualization of various gene-editing associated chromosomal translocations.

bioengineering↗

bioRxiv2019

Age-related changes to macrophages are detrimental to fracture healing

The elderly population suffers from higher rates of complications during fracture healing that result in increased morbidity and mortality. Inflammatory dysregulation is associated with increased age and is a contributing factor to the myriad of age-related diseases. Therefore, we investigated age-related changes to an important cellular regulator of inflammation, the macrophage, and the impact on fracture healing outcomes. We demonstrated that old mice (24 months) have delayed fracture healing with significantly less bone and more cartilage compared to young mice (3 months). The quantity of infiltrating macrophages into the fracture callus was similar in old and young mice. However, RNA-seq analysis demonstrated distinct differences in the transcriptomes of macrophages derived from the fracture callus of old and young mice, with an upregulation of M1/pro-inflammatory genes in macrophages from old mice as well as dysregulation of other immune-related genes. Preventing infiltration of the fracture site by macrophages in old mice improved healing outcomes, with significantly more bone in the calluses of treated mice compared to age-matched controls. After preventing infiltration by macrophages, the macrophages within the fracture callus were collected and examined via RNA-seq analysis, and their transcriptome resembled macrophages from young calluses. Taken together, infiltrating macrophages from old mice demonstrate detrimental age-related changes, and depleting infiltrating macrophages can improve fracture healing in old mice.

immunology↗

bioRxiv2019

White matter alterations in autism spectrum disorder and attention-deficit/hyperactivity disorder in relation to sensory profile

BackgroundAutism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD) have high rates of co-occurrence and share atypical behavioral characteristics, including sensory problems. The present diffusion tensor imaging (DTI) study was conducted to examine whether and how white matter abnormalities are observed in adult populations with developmental disabilities (DD) and to determine how brain-sensory relationships are either shared between or distinct to ASD and ADHD.\n\nMethodsWe collected DTI data from adult developmental disorder (DD) populations (a primary diagnosis of ASD: n=105, ADHD: n=55) as well as age and sex matched typically developed (TD) participants (n=58). Voxel-wise fractional anisotropy (FA), mean diffusivity, axial diffusivity, and radial diffusivity (RD) were analyzed using tract-based spatial statistics. The severities of sensory problems were assessed using the Adolescent/Adult Sensory Profile (AASP).\n\nResultsCategorical analyses identified voxel clusters showing significant effects of DD on FA and RD in the posterior portion of the corpus callosum and its extension in the right hemisphere. Furthermore, regression analyses using the AASP scores revealed that slopes in relationships of FA or RD with the degree of sensory problems were parallel between the two DDs in large parts of the affected corpus callosum regions, although a small but significant cluster did exist showing interaction between the diagnosis of DD and an AASP subscale score on RD.\n\nConclusionsThese results indicate that white matter abnormalities and their relationships to sensory problems are largely shared between ASD and ADHD, with localized abnormalities showing significant between-diagnosis differences within DD. (247 words)

neuroscience↗

bioRxiv2019

Cortical surface architecture endophenotype and correlates of clinical diagnosis of autism spectrum disorder

AimPrior structural MRI studies demonstrated atypical gray matter characteristics in siblings of individuals with autism spectrum disorder (ASD). However, they did not clarify which aspect of gray matter presents the endophenotype. Further, because they did not enroll siblings of TD people, they underestimated the difference between individuals with ASD and their unaffected siblings. The current study aimed to solve these questions.\n\nMethodsWe recruited 30 pairs of adult male siblings (15 of them have an ASD endophenotype, other 15 pairs not) and focused on four gray matter parameters: cortical volume and three surface-based parameters (cortical thickness, fractal dimension, and sulcal depth [SD]). First, we sought to identify a pattern of an ASD endophenotype, comparing the four parameters. Then, we compared individuals with ASD and their unaffected siblings in the cortical parameters to identify neural correlates for the clinical diagnosis accounting for the difference between TD siblings.\n\nResultsA sparse logistic regression with a leave-one-pair-out cross-validation showed the highest accuracy for the identification of an ASD endophenotype (73.3%) with the SD compared with the other three parameters. A bootstrapping analysis accounting for the difference in the SD between TD siblings showed a significantly large difference between individuals with ASD and their unaffected siblings in six out of 68 regions-of-interest accounting for multiple comparisons.\n\nConclusionsThis proof-of-concept study suggests that an ASD endophenotype emerges in SD and that neural correlates for the clinical diagnosis can be dissociated from the endophenotype when we accounted for the difference between TD siblings. (248/250 words)

neuroscience↗

bioRxiv2019

Modeling the Human Segmentation Clock with Pluripotent Stem Cells

Pluripotent stem cells (PSCs) have increasingly been used to model different aspects of embryogenesis and organ formation1. Despite recent advances in the in vitro induction of major mesodermal lineages and mesoderm-derived cell types2,3, experimental model systems that can recapitulate more complex biological features of human mesoderm development and patterning are largely missing. Here, we utilized induced pluripotent stem cells (iPSCs) for the stepwise in vitro induction of presomitic mesoderm (PSM) and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modeling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We succeeded to observe oscillatory expression of core segmentation clock genes, including HES7 and DKK1, and identified novel oscillatory genes in human iPSC-derived PSM. We furthermore determined the period of the human segmentation clock to be around five hours and showed the presence of dynamic traveling wave-like gene expression within in vitro induced human PSM. Utilizing CRISPR/Cas9-based genome editing technology, we then targeted genes, for which mutations in patients with abnormal axial skeletal development such as spondylocostal dysostosis (SCD) (HES7, LFNG and DLL3) or spondylothoracic dysostosis (STD) (MESP2) have been reported. Subsequent analysis of patient-like iPSC knock-out lines as well as patient-derived iPSCs together with their genetically corrected isogenic controls revealed gene-specific alterations in oscillation, synchronization or differentiation properties, validating the overall utility of our model system, to recapitulate not only key features of human somitogenesis but also to provide novel insights into diseases associated with the formation and patterning of the human axial skeleton.

developmental biology↗

bioRxiv2019

The kinesin-like protein Pavarotti functions non-canonically to regulate actin dynamics

Pavarotti, the Drosophila MKLP1 ortholog, is a kinesin-like protein that with Tumbleweed (MgcRacGAP) works together as the centralspindlin complex. This complex is essential for cytokinesis where it helps to organize the contractile actomyosin ring at the equator of dividing cells by activating the RhoGEF Pebble. Actomyosin rings also function as the driving force during cell wound repair. We previously showed that Tumbleweed and Pebble are required for the cell wound repair process. Here, we show that Pavarotti also functions during wound repair and confirm that while Pavarotti, Tumbleweed, and Pebble are utilized during this cellular repair, it is not as the conserved centralspindlin complex. Surprisingly, in vitro and in vivo work show that the classically microtubule-associated Pavarotti binds directly to actin and has a non-canonical role directly regulating actin dynamics. We show that Pavarotti also works independently from Tumbleweed in several actin-related processes during the normal developmental process of oogenesis.

cell biology↗

bioRxiv2019

A NMF-based approach to discover overlooked differentially expressed gene regions from single-cell RNA-seq data

Single-cell RNA sequencing has enabled researchers to quantify the transcriptomes of individual cells, infer cell types, and investigate differential expression among cell types, which will lead to a better understanding of the regulatory mechanisms of cell states. Transcript diversity caused by phenomena such as aberrant splicing events have been revealed, and differential expression of previously unannotated transcripts might be overlooked by annotation-based analyses. Accordingly, we have developed an approach to discover overlooked differentially expressed (DE) gene regions that complements annotation-based methods. We applied our algorithm to two datasets and discovered several intriguing DE transcripts, including a transcript related to the modulation of neural stem/progenitor cell differentiation.

bioinformatics↗

bioRxiv2018

Machine learning approach to identify resting-state functional connectivity pattern serving as an endophenotype of autism spectrum disorder

Endophenotype refers to a measurable and heritable component between genetics and diagnosis and exists in both individuals with a diagnosis and their unaffected siblings. We aimed to identify a pattern of endophenotype consisted of multiple connections. We enrolled adult male individuals with autism spectrum disorder (ASD) endophenotype (i.e., individuals with ASD and their unaffected siblings) and individuals without ASD endophenotype (i.e., pairs of typical development (TD) siblings) and utilized a machine learning approach to classify people with and without endophenotypes, based on resting-state functional connections (FCs). A sparse logistic regression successfully classified people as to the endophenotype (area under the curve=0.78, classification accuracy=75%), suggesting the existence of endophenotype pattern. A binomial test identified that nine FCs were consistently selected as inputs for the classifier. The least absolute shrinkage and selection operator with these nine FCs predicted severity of communication impairment among individuals with ASD (r=0.68, p=0.021). In addition, two of the nine FCs were statistically significantly correlated with the severity of communication impairment (r=0.81, p=0.0026 and r=-0.60, p=0.049). The current findings suggest that an ASD endophenotype pattern exists in FCs with a multivariate manner and is associated with clinical ASD phenotype.

neuroscience↗

bioRxiv2018

GnasR201C Induces Murine Pancreatic Cystic Neoplasms through Suppression of YAP1 Signaling and Transcriptional Reprogramming

Background & AimsSomatic \"hotspot\" mutations of GNAS, which encodes for the alpha subunit of stimulatory G-protein, are present in ~60% of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. There are currently no cognate animal models that recapitulate the biology of mutant Gnas-induced IPMNs, and the underlying mechanisms that lead to the cystic pathway of neoplasia in the pancreas remain unknown.\n\nMethodsWe generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras; Gnas mice) where pancreas-specific GnasR201C expression was induced by doxycycline administration. In this model, mutant Kras is constitutively expressed, and control mice were produced through absence of doxycycline. Separate cohorts of mice were utilized for timed necropsies and for Kaplan-Meier survival analysis. Isogenic cell lines (with doxycycline inducible mutant Gnas expression) were propagated from the resulting pancreatic ductal adenocarcinoma (PDAC).\n\nResultsCo-expression of KrasG12D and GnasR201C resulted in the development of pancreatic cystic lesions resembling human IPMNs in 100% of mice, with higher grades of epithelial dysplasia observed over time. Approximately one-third of Kras; Gnas mice developed PDAC at a median of 38 weeks post doxycycline induction. GnasR201C did not accelerate oncogenic transformation with KrasG12D, but rather, reprogrammed Ras-induced neoplasms towards a well-differentiated phenotype. GnasR201C induction led to activation of the inhibitory Hippo kinase cascade and cytoplasmic sequestration of phosphorylated YAP1 protein, a phenomenon that was also observed in human IPMN with GNAS mutations.\n\nConclusionsGNASR201C functions not as a traditional oncogene, but rather as an \"oncomodulator\" of KRAS-mediated pancreatic neoplasia, through suppression of YAP1 and transcriptional reprogramming towards a differentiated (large ductal) phenotype.

cancer biology↗

bioRxiv2017

CLASP stabilizes microtubule plus ends after severing to facilitate light-induced microtubule reorientation

Central to building and reorganizing cytoskeletal arrays is the creation of new polymers. While nucleation has been the major focus of study for new microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. As severing produces new plus ends behind the stabilizing GTP-cap, an important and unanswered question is how these are stabilized in vivo to promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing and find that CLASP is required for rapid cortical array reorientation. In clasp mutants both rescue of shrinking plus ends and the regrowth of plus ends immediately after severing are reduced, computational modeling reveals that it is the specific stabilization of severed ends that explains CLASPs function in promoting microtubule amplification by severing and cortical array reorientation.

cell biology↗