Biology subjects

Matsuda, M.

Publications and source records attributed to Matsuda, M..

35 records · Page 2Linked to original sources

bioRxiv2022

Accumulated 3-phosphoglycerate in darkness is a primary starting substrate for stepwise photosynthetic induction process in cyanobacteria

Photosynthesis must maintain stability and robustness throughout fluctuating natural environments. In cyanobacteria, dark-to-light transition leads to drastic metabolic changes from dark respiratory metabolism to CO2 fixation through the Calvin-Benson-Bassham (CBB) cycle using energy and redox equivalents provided by photosynthetic electron transfer. Previous studies showed that catabolic metabolism supports the smooth transition into CBB cycle metabolism. However, metabolic mechanisms for robust initiation of photosynthesis are poorly understood due to lack of dynamic metabolic characterizations of dark-to-light transitions. Here, we show rapid (on a time scale of seconds) dynamic changes in absolute metabolite concentrations and 13C tracer incorporation after strong or weak light irradiation in the cyanobacterium Synechocystis sp. PCC 6803. Integration of this data enables estimation of time-resolved nonstationary metabolic flux underlying CBB cycle activation. This dynamic metabolic analysis indicates that downstream glycolytic intermediates including phosphoglycerate and phosphoenolpyruvate accumulate under dark conditions as major substrates for initial CO2 fixation. Compared with wild-type Synechocystis, significant delays in the initiation of oxygen evolution are observed in 12 h dark preincubated mutants deficient in glycogen degradation or oxidative pentose phosphate pathway ({Delta}zwf, {Delta}gnd, and{Delta} glgP). Accordingly, the degree of delay in the oxygen evolution initiation is proportional to the accumulated pool size of the glycolytic intermediates. These observations indicate that the accumulation of glycolytic intermediates is essential for efficient metabolism switching under fluctuating light environments.

plant biology↗

bioRxiv2022

Targeting A-Kinase Anchoring Protein 12 Phosphorylation in Hepatic Stellate Cells Regulates Liver Injury and Fibrosis in Mouse Models

Trans-differentiation of hepatic stellate cells (HSCs) to activated state potentiates liver fibrosis through release of extracellular matrix (ECM) components, distorting the liver architecture. Since limited antifibrotics are available, pharmacological intervention targeting activated HSCs may be considered for therapy. A-kinase anchoring protein 12 (AKAP12) is a scaffolding protein that directs protein kinases A/C (PKA/PKC) and cyclins to specific locations spatiotemporally controlling their biological effects. It has been shown that AKAP12s scaffolding functions are altered by phosphorylation. In previously published work, observed an association between AKAP12 phosphorylation and HSC activation. In this work we demonstrate that AKAP12s scaffolding activity towards the endoplasmic reticulum (ER)-resident collagen chaperone, heat-shock protein 47 (HSP47) is strongly inhibited by AKAP12s site-specific phosphorylation in activated HSCs. CRISPR-directed gene editing of AKAP12s phospho- sites restores its scaffolding towards HSP47, inhibiting HSP47s collagen maturation functions and HSC activation. AKAP12 phospho-editing dramatically inhibits fibrosis, ER stress response, HSC inflammatory signaling and liver injury in mice. Our overall findings suggest a pro-fibrogenic role of AKAP12 phosphorylation that may be targeted for therapeutic intervention in liver fibrosis.

cell biology↗

bioRxiv2022

Voltage-Sensing Phosphatase (VSP) Regulates Endocytosis-Dependent Nutrient Absorption in Chordate Enterocytes

Voltage-sensing phosphatase (VSP) is a unique membrane protein that translates membrane electrical activities into the changes of phosphoinositide profiles. VSP orthologs from various species have been intensively investigated toward their biophysical properties, primarily using a heterologous expression system. In contrast, the physiological role of VSP in native tissues remains largely unknown. Here we report that zebrafish VSP (Dr-VSP) is functionally expressed on the endomembranes of lysosome-rich enterocytes (LREs) that mediate dietary protein absorption via endocytosis in the zebrafish mid-intestine. Dr-VSP-deficient LREs were remarkably defective in forming endosomal vacuoles after initial uptake of dextran and mCherry. Dr-VSP-deficient zebrafish exhibited growth restriction and higher mortality during the critical period when zebrafish larvae rely primarily on exogenous feeding via intestinal absorption. Furthermore, our comparative study on marine invertebrate Ciona intestinalis VSP (Ci-VSP) revealed co-expression with endocytosis-associated genes in absorptive epithelial cells of the Ciona digestive tract, corresponding to zebrafish LREs. These findings signify a crucial role of VSP in regulating endocytosis-dependent nutrient absorption in specialized enterocytes across animal species. Summary statementVoltage-sensing phosphatase (VSP) is identified in absorptive enterocytes, revealing its crucial role in promoting endocytosis and nutrient absorption during early development.

cell biology↗

bioRxiv2021

ERK-mediated Curvature Feedback Regulates Branching Morphogenesis in Lung Epithelial Tissue

Intricate branching patterns emerge in internal organs because of the repetitive presence of simple deformations in epithelial tissues. During murine lung development, epithelial cells in distal tips of a single tube require fibroblast growth factor (FGF) signals generated by their surrounding mesenchyme to form repetitive tip bifurcations. However, it remains unknown how the cells employ FGF signaling to convert their behaviors to achieve the recursive branching processes. Here we show a self-sustained epithelial regulatory system during the murine lung branching morphogenesis, mediated by extracellular signal-regulated kinase (ERK), which acts as a downstream driver of FGF signaling. We found that tissue-scale curvature regulated ERK activity in the lung epithelium using two-photon live cell imaging and mechanical perturbations. ERK was activated specifically in epithelial tissues with a positive curvature, regardless of whether the change in curvature was attributable to morphogenesis or artificial perturbations. Moreover, ERK activation accelerated actin polymerization specifically at the apical side of cells, and mechanically contributed to the extension of the apical membrane, leading to a decrease in epithelial tissue curvature. These results indicate the existence of negative feedback loop between tissue curvature and ERK activity beyond scale. We confirmed that this regulation was sufficient to generate the recursive branching processes by a mathematical model. Taken together, we propose that ERK mediates the curvature feedback loop underlying the process of branching morphogenesis in developing lungs.

developmental biology↗

bioRxiv2021

Redundant and specific roles of EGFR ligands in the ERK activation waves during collective cell migration of MDCK cells

Epidermal growth factor receptor (EGFR) plays a pivotal role in collective cell migration by mediating cell-to-cell propagation of extracellular signal-regulated kinase (ERK) activation. Here, we aimed to determine which EGFR ligands mediate the ERK activation waves by gene knockout. Four of the seven known EGFR ligands are expressed in MDCK cells. We found that epidermal growth factor (EGF)-deficient cells exhibited lower basal ERK activity than the cells deficient in heparin-binding EGF (HBEGF), transforming growth factor alpha (TGF) or epiregulin (EREG), but all cell lines deficient in a single EGFR ligand retained the ERK activation waves. Therefore, we knocked out the EGFR ligand genes in decreasing order of expression. ERK activation waves were markedly suppressed, albeit incompletely, only when all four EGFR ligands were knocked-out. Re-expression of the EGFR ligands revealed that all but HBEGF could restore the ERK activation waves. Aiming at complete elimination of the ERK activation waves, we further attempted to knockout Nrg1, a ligand for ErbB3 and ErbB4, and found that Nrg1 deficiency induced growth arrest in the absence of all four EGFR ligand genes expressed in MDCK cells. Collectively, these results showed that EGFR ligands exhibit remarkable redundancy in the propagation of ERK activation waves during collective cell migration.

cell biology↗

bioRxiv2021

LMO7-dependent apical constriction requires the binding of the Myosin heavy chain

Apical constriction, or reduction of the apical domain, underlies many morphogenetic events during development, such as furrow or tube formation. Actomyosin complexes play an essential role in apical constriction, however the detailed analysis of molecular mechanisms is still pending. Here we show that Lim domain only protein 7 (Lmo7), a multidomain adaptor at apical junctions, promotes apical constriction in the Xenopus superficial ectoderm, whereas apical domain size increases in Lmo7-depleted cells. Lmo7 is localized to and promotes the formation of the circumferential actomyosin belt adjacent to apical junctions. We find that Lmo7-dependent apical constriction requires the RhoA-ROCK-Non-muscle myosin II (NMII) pathway. Strikingly, Lmo7 binds and recruits NMII heavy chains to apical junctions. Lmo7 overexpression altered the subcellular distribution of Wtip, a sensor of mechanical tension. Our findings suggest that Lmo7 serves as a scaffold organizing the actomyosin network to regulate contractility at apical junctions.

cell biology↗

bioRxiv2021

Metastatic Single Tumor Cells Evade NK Cell-mediated Killing by Thrombin-mediated Loss of the Activating Ligand CD155/PVR/Necl-5

Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is not known. Here we have combined ultra-sensitive bioluminescence whole body imaging with intravital two-photon microscopy involving genetically-encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hrs of arrival, but not thereafter. Intravital dynamic imaging revealed that a disseminated tumor cell in a pulmonary capillary interacts with an NK cell every 2 hrs on average. In the first 4 hrs after tumor cell arrival, 50% of such encounters lead to tumor cell death but after 24 hrs of arrival, nearly 100% of the interactions result in the survival of the tumor cell. This evasion of NK cell surveillance is mediated by thrombin-dependent loss of cell surface CD155/PVR/Necl-5, a ligand for the NK cell activating receptor DNAM-1. This loss prevents the NK cell signaling needed for effective killing of tumor targets. By quantitatively visualizing the evasion of NK cell surveillance, we have uncovered a molecular mechanism for cancer evasion and provided an explanation for the anti-metastatic effect of anticoagulants. SUMMARYIntravital functional two-photon microscopy reveals that metastatic tumor cells lodged in pulmonary capillaries acquire resistance to patrolling NK cells. Protease-mediated loss of the activating ligand CD155/PVR/Necl-5 on tumor cells prevents NK cells from ERK activation and tumor cell killing.

immunology↗

bioRxiv2020

Interplay between medial nuclear stalling and lateral cellular flow underlies cochlear duct morphogenesis

A notable example of spiral architecture in organs is the mammalian cochlear duct, where the duct morphology is critical for hearing function. Molecular genetics has revealed the necessary signaling molecules for the formation of spirals in organs, but it remains unclear how cellular dynamics generate bending and coiling of the cochlear duct during development. Here we show two modes of multicellular dynamics underlying the morphogenetic process by combining deep tissue live-cell imaging, Forster resonance energy transfer (FRET)-based quantitation, and mathematical modeling. First, surgical separation of the cochlear duct revealed that bending forces reside primarily in the medial side of the duct. In the medial pseudostratified epithelium, we found that nuclei stall at the luminal side during interkinetic nuclear migration, which would cause the extension of the luminal side, thereby bending the duct. Second, long-term organ-scale FRET imaging of extracellular signal-regulated kinase (ERK) activity showed that helical ERK activation waves propagate from the duct tip concomitant with the reverse multicellular flow in the lateral side of the duct, resulting in advection-based duct elongation. We propose an interplay of distinct multicellular behaviors underpinning spiral morphogenesis in the developing cochlear duct.

developmental biology↗

bioRxiv2020

Incoherent feedforward regulation via Sox9 and Erk underpins mouse tracheal cartilage development

Tracheal cartilage provides architectural integrity to the respiratory airway, and defects in this structure during embryonic development cause severe congenital anomalies. Previous genetic studies have revealed genes that are critical for the development of tracheal cartilage. However, it is still unclear how crosstalk between these proteins regulates tracheal cartilage formation. Here we show a core regulatory network underlying murine tracheal chondrogenesis from embryonic day (E) 12.5 to E15.5, by combining volumetric imaging of fluorescence reporters, inhibitor assays, and mathematical modeling. We focused on SRY-box transcription factor 9 (Sox9) and extracellular signal-regulated kinase (Erk) in the tracheal mesenchyme, and observed a synchronous, inverted U-shaped temporal change in both Sox9 expression and Erk activity with a peak at E14.5, whereas the expression level of downstream cartilage matrix genes, such as collagen II alpha 1 (Col2a1) and aggrecan (Agc1), monotonically increased. Inhibitor assays revealed that the Erk signaling pathway functions as an inhibitory regulator of tracheal cartilage differentiation during this period. These results suggest that expression of the cartilage matrix genes is controlled by an incoherent feedforward loop via Sox9 and Erk, which is supported by a mathematical model. Furthermore, the modeling analysis suggests that a Sox9-Erk incoherent feedforward regulation augment the robustness against the variation of upstream factors. The present study provides a better understanding of the regulatory network underlying the tracheal development and will be helpful for efficient induction of tracheal organoids.

developmental biology↗

bioRxiv2020

Intravital imaging reveals cell cycle-dependent satellite cell migration during muscle regeneration.

During muscle regeneration, extracellular signal-regulated kinase (ERK) promotes both proliferation and migration. However, the relationship between proliferation and migration is poorly understood in this context. To elucidate this complex relationship on a physiological level, we established an intravital imaging system for measuring ERK activity, migration speed, and cell-cycle phases in mouse muscle satellite cells. We found that in vivo, ERK was maximally activated in satellite cells two days after injury, and this is then followed by increases in cell number and motility. With limited effects of immediate ERK activity on migration, we hypothesized that ERK increases migration speed in the later phase by promoting cell-cycle progression. Our cell-cycle analysis further revealed that in satellite cells, ERK activity is critical for the G1/S transition, and cells migrate more rapidly in the S/G2 phase three days after injury. Finally, migration speed of satellite cells was suppressed after CDK1/2, but not CDK1, inhibitor treatment, demonstrating a critical role of CDK2 in satellite cell migration. Overall, our study demonstrates that in satellite cells, the ERK-CDK2 axis not only promotes the G1/S transition, but also migration speed, which may provide a novel mechanism for efficient muscle regeneration.

cell biology↗

bioRxiv2019

ERK-mediated mechanochemical waves direct collective cell polarization

During collective migration of epithelial cells, the migration direction is aligned over a large, tissue-scale expanse. Although the collective cell migration is known to be directed by mechanical forces transmitted via cell-cell junctions, it remains elusive how the intercellular force transmission is coordinated with intracellular biochemical signaling to achieve collective movements. Here we show that intercellular coupling of extracellular signal-regulated kinase (ERK)-mediated mechanochemical feedback in individual cells yields long-distance transmission of guidance cues. Mechanical stretch activates ERK through epidermal growth factor receptor (EGFR) activation, and the ERK activation triggers cell contraction. In addition, the contraction of the activated cell pulls neighboring cells via cell-cell junctions, evoking another round of ERK activation and contraction in the neighbors. Furthermore, anisotropic contraction based on front-rear cell polarization guarantees unidirectional propagation of ERK activation waves, and in turn, the ERK activation waves direct multicellular alignment of the polarity, leading to long-range ordered migration. Our findings reveal that mechanical forces mediate intercellular signaling underlying sustained transmission of guidance cues for collective cell migration.

cell biology↗

bioRxiv2019

Species-specific oscillation periods of human and mouse segmentation clocks are due to cell autonomous differences in biochemical reaction parameters

While the mechanisms of embryonic development are similar between mouse and human, the tempo is in general slower in human. The cause of interspecies differences in developmental time remains a mystery partly due to lack of an appropriate model system1. Since murine and human embryos differ in their sizes, geometries, and nutrients, we use in vitro differentiation of pluripotent stem cells (PSCs) to compare the same type of cells between the species in similar culture conditions. As an example of well-defined developmental time, we focus on the segmentation clock, oscillatory gene expression that regulates the timing of sequential formation of body segments2-4. In this way we recapitulate the murine and human segmentation clocks in vitro, showing that the species-specific oscillation periods are derived from cell autonomous differences in the speeds of biochemical reactions. Presomitic mesoderm (PSM)-like cells induced from murine and human PSCs displayed the oscillatory expression of HES7, the core gene of the segmentation clock5,6, with oscillation periods of 2-3 hours (mouse PSM) and 5-6 hours (human PSM). Swapping HES7 loci between murine and human genomes did not change the oscillation periods dramatically, denying the possibility that interspecies differences in the sequences of HES7 loci might be the cause of the observed period difference. Instead, we found that the biochemical reactions that determine the oscillation period, such as the degradation of HES7 and delays in its expression, are slower in human PSM compared with those in mouse PSM. With the measured biochemical parameters, our mathematical model successfully accounted for the 2-3-fold period difference between mouse and human. We further demonstrate that the concept of slower biochemical reactions in human cells is generalizable to several other genes, as well as to another cell type. These results collectively indicate that differences in the speeds of biochemical reactions between murine and human cells give rise to the interspecies period difference of the segmentation clock and may contribute to other interspecies differences in developmental time.

developmental biology↗

bioRxiv2019

A parallel cell-cycle entry pathway with inverted G1 signaling and alternate point of no return

Cell-cycle entry relies on an orderly progression of signaling events. To start, cells first activate the kinase cyclin D-CDK4/6, which leads to eventual inactivation of the retinoblastoma protein Rb. Hours later, cells inactivate APC/CCDH1 and cross the final commitment point. However, many cells with genetically deleted cyclin Ds, which activate and confer specificity to CDK4/6, can compensate and proliferate. Despite its importance in cancer, how this alternate pathway operates and whether wild-type cells use this pathway remain unknown. Here, using single-cell microscopy, we demonstrate that cells with acutely inhibited CDK4/6 enter the cell cycle with slowed and fluctuating cyclin E-CDK2 activity. Surprisingly, in this alternate pathway, the order of APC/CCDH1 and Rb inactivation is inverted in both cell lines and wild-type mice. Finally, we show that as a consequence of the signaling inversion, Rb inactivation replaces APC/CCDH1 inactivation as the point of no return. Together, we provide molecular characterization of a parallel cell-cycle entry pathway, and reveal temporal plasticity that underlies the G1 regulatory circuit.

cell biology↗

bioRxiv2019

Morphine analgesia and mu opioid receptor signaling require programed death protein 1 (PD-1)

Opioids such as morphine produce analgesia via mu opioid receptor (MOR), but opioid receptor signaling is not fully understood. Here we report that morphine analgesia and MOR signaling require neuronal Programmed cell death protein-1 (PD-1). We found that morphine-induced antinociception following systemic or intrathecal injection was compromised in Pd1-/- mice. Morphine analgesia was also abrogated in wild-type mice after treatment with Nivolumab, a clinically used anti-PD-1 monoclonal antibody. Morphine produced analgesia by suppressing calcium currents in DRG neurons and excitatory synaptic transmission in spinal cord neurons, but strikingly, both actions were impaired by PD-1 blockade. In a mouse model of bone cancer, the antinociceptive action of systemic morphine was compromised in Pd1-/- mice. Finally, PD-L1 and morphine produce synergistic analgesia. Our findings demonstrate that PD-1 also acts as a neuro checkpoint inhibitor and mediates opioid-induced analgesia and MOR signaling in neurons.

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↗

bioRxiv2017

Single-cell quantification of the concentration and dissociation constant of endogenous proteins

Kinetic simulation is a useful approach for the elucidation of complex cell-signaling systems. To perform the numerical simulations required for kinetic modeling, parameters such as the protein concentration and dissociation constant (Kd) are essential. However, only a limited number of parameters have been measured experimentally in living cells. Here, we describe a method for quantifying the concentration and Kd of endogenous proteins at the single-cell level with CRISPR/Cas9-mediated knock-in and fluorescence cross-correlation spectroscopy (FCCS). First, the mEGFP gene was knocked-in at the end of the MAPK1 gene, which encoded ERK2, through homology-directed repair or microhomology-mediated end joining. Next, the HaloTag gene was further knocked-in at the end of the RSK2 gene. Protein concentrations of endogenous ERK2-mEGFP and RSK2-HaloTag were quantified in living cells by fluorescence correlation spectroscopy (FCS), revealing substantial cellular heterogeneities. Interestingly, the levels of ERK2-mEGFP and RSK2-HaloTag were strongly positively correlated, suggesting a global mechanism underlying their expressions. In addition, FCCS measurement revealed temporal changes in the apparent Kd values of the binding between ERK2-mEGFP and RSK2-HaloTag in response to EGF stimulation. Our method provides a new approach for quantification of the endogenous protein concentration and dissociation constant in living cells.

biochemistry↗

bioRxiv2017

Cross-inhibition of JNK by p38 visualized by multiplexed fluorescence imaging

AbstractThe stress activated protein kinases (SAPKs), c-Jun N-terminal kinase (JNK) and p38, are important players in cell fate decisions in response to environmental stress signals. Crosstalk signaling between JNK and p38 is emerging as an important regulatory mechanism in the inflammatory and stress responses. However, it is still unknown how this crosstalk affects signaling dynamics, cell-to-cell variation, and cellular responses at the single-cell level. To address these questions, we established a multiplexed live-cell imaging system based on kinase translocation reporters to simultaneously monitor JNK and p38 activities with high specificity and sensitivity at single-cell resolution. Various stresses, such as anisomycin, osmotic stress, and UV irradiation, and pro-inflammatory cytokines activated JNK and p38 with various dynamics. In all cases, however, p38 suppressed JNK activity in a cross-inhibitory manner. We demonstrate that p38 antagonizes JNK through both transcriptional and post-translational mechanisms. This cross-inhibition of JNK appears to generate cellular heterogeneity in JNK activity after stress exposure. Our data indicate that this heterogeneity in JNK activity plays a role in fractional killing in response to UV stress. Our highly sensitive multiplexed imaging system enables detailed investigation into the p38-JNK interplay in single cells.\n\nOne Sentence SummaryCross-inhibition by p38 generates cell-to-cell variability in JNK activity.

cell biology↗