Biology subjects

Terai, K.

Publications and source records attributed to Terai, K..

10 recordsLinked to original sources

bioRxiv2026

Hmgb1 release kinetics shape its extracellular functions during regulated cell death

Danger-associated molecular patterns (DAMPs), such as high mobility group protein B1 (HMGB1), are released from dying cells, yet the kinetics and functional consequences of their release remain unclear. Using Hmgb1-mCherry transgenic mice and live-cell imaging, we visualize Hmgb1 and interleukin-1{beta} (IL-1{beta}) secretion at single-cell resolution. Hmgb1 exhibits two distinct release kinetics: short-duration completed within 1 min and long duration spanning several minutes. Mathematical modeling demonstrates that short-duration release generates substantially higher local Hmgb1 concentrations and steeper gradients near the dying cell than long-duration release. Such rapid release may enable one or a few dying cells to produce sufficient Hmgb1 to stimulate neighboring cells, consistent with its role as an alarmin. In vivo imaging of cisplatin-induced kidney injury reveals an inverse correlation between intracellular Hmgb1 levels and monocyte infiltration. Together, these findings suggest that Hmgb1 release kinetics shape its extracellular functions during regulated cell death.

cell biology↗

bioRxiv2025

Intensity Modulation of Trichromatic Split Fluorescent Proteins for Live Cell Mapping

Multiplexed live cell-labeling technologies are essential tools for investigating complex biological systems, yet existing approaches face limitations in achieving reliable discrimination of multiple cell populations. Here, we present color and intensity modulation with split fluorescent proteins for cell labeling (Caterpie), a live cell labeling system that enables high-fidelity identification of multiple cell populations through rational design of split fluorescent proteins. Drawing inspiration from human trichromatic vision, we engineered optimized split fluorescent protein variants--split CFP2, split mNG3A, and split sfCherry3C--and developed a comprehensive library of the 11th {beta}-strand (FP11) tags comprising variable numbers of tandem repeats. Structure-guided protein engineering enhanced the performance of split mNG3A and split sfCherry3C through targeted modifications of their C-terminal sequences and binding interfaces, respectively. By systematically varying the composition and arrangement of FP11 repeats, we selected 20 distinct FP11 tags that enable robust cell population discrimination with 97% accuracy using conventional fluorescence microscopy. In these FP11 tag-expressing cells, we expressed multiple EGF ligands and their receptors and cultured them in bulk to examine the effects of EGF signaling on chemotaxis within the cell population. The results showed that chemotaxis changes according to the intensity of EGFR signaling. The Caterpie system holds significant potential for applications requiring precise identification and tracking of multiple cell populations in complex biological systems.

cell biology↗

bioRxiv2025

E-cadherin mechanotransduction activates EGFR-ERK signaling in epithelial cells by inducing ADAM-mediated ligand shedding

The behavior of cells is governed by signals originating from their local environment, including mechanical forces that cells experience. Forces are transduced by mechanosensitive proteins, which can impinge on signaling cascades that are also activated by growth factor receptors upon ligand binding. We investigated the crosstalk between these mechanical and biochemical signals in the regulation of intracellular signaling networks in epithelial monolayers. Phosphoproteomic and transcriptomic analyses on epithelial monolayers subjected to mechanical strain revealed ERK signaling as a predominant strain-activated hub, initiated at the level of the upstream epidermal growth factor receptor (EGFR). Strain-induced EGFR-ERK signaling depends on mechanosensitive E-cadherin adhesions. Proximity labeling identified the metalloproteinase ADAM17, an enzyme that mediates shedding of soluble EGFR ligands, to be closely associated with E-cadherin. We developed a novel probe for monitoring ADAM-mediated shedding, which demonstrated that mechanical strain induced ADAM activation. Mechanically-induced ADAM activation was essential for mechanosensitive signaling from E-cadherin adhesions towards EGFR-ERK. Collectively, our data demonstrate that mechanical strain transduced by E-cadherin adhesion triggers the shedding of EGFR ligands that stimulate downstream downstream ERK activity. Our findings illustrate how mechanical signals and biochemical ligands can operate within a single, linear signaling cascade. Significance statementCells integrate different types of information that they receive from their local environment to regulate their behavior. This includes biochemical signals, such as growth factors binding to their dedicated receptor. Similarly, cells respond to mechanical forces that they are subjected to. Although biochemical and mechanical signals can elicit similar signaling responses in cells, the interplay between these types of signals is not well understood. Here we unveil that mechanical strain of epithelia modulates the activity of the EGFR-ERK signaling pathway by controlling the availability of growth factors that bind and activate EGFR. This finding demonstrates that biochemical and mechanical signals do not act in a segregated fashion, but rather can function in a linear cascade, shedding light on fundamental principles governing cellular regulation.

cell biology↗

bioRxiv2024

Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters during collective cell migration of epithelial cells

Epidermal growth factor receptor ligands (EGFRLs) consist of seven proteins. In stark contrast to the amassed knowledge concerning the epidermal growth factor receptors themselves, the extracellular dynamics of individual EGFRLs remain elusive. Here, employing fluorescent probes and a tool for triggering ectodomain shedding of EGFRLs, we show that EREG, a low-affinity EGFRL, exhibits the most rapid and efficient activation of EGFR in confluent epithelial cells and mouse epidermis. In Madin-Darby canine kidney (MDCK) renal epithelial cells, EGFR- and ERK-activation waves propagate during collective cell migration in an ADAM17 sheddase- and EGFRL-dependent manner. Upon induction of EGFRL shedding, radial ERK activation waves were observed in the surrounding receiver cells. Notably, the low-affinity ligands EREG and AREG mediated faster and broader ERK waves than the high-affinity ligands. The integrity of tight/adherens junctions was essential for the propagation of ERK activation, implying that the tight intercellular spaces prefer the low-affinity EGFRL to the high-affinity ligands for efficient signal transmission. To validate this observation in vivo, we generated EREG-deficient mice expressing the ERK biosensor and found that ERK wave propagation and cell migration were impaired during skin wound repair. In conclusion, we have quantitatively demonstrated the distinctions among EGFRLs in shedding, diffusion, and target cell activation in physiological contexts. Our findings underscore the pivotal role of low-affinity EGFRLs in rapid intercellular signal transmission.

cell biology↗

bioRxiv2023

Calcium transients trigger switch-like discharge of prostaglandin E2 (PGE2) in an ERK-dependent manner

Prostaglandin E2 (PGE2) is a key player in a plethora of physiological and pathological events. Nevertheless, little is known about the dynamics of PGE2 secretion from a single cell and its effect on the neighboring cells. Here, by observing confluent Madin-Darby canine kidney (MDCK) epithelial cells expressing fluorescent biosensors we demonstrate that calcium transients in a single cell cause PGE2-mediated radial spread of PKA activation (RSPA) in neighboring cells. By in vivo imaging, RSPA was also observed in the basal layer of the mouse epidermis. Experiments with an optogenetic tool revealed a switch-like PGE2 discharge in response to the increasing cytoplasmic Ca2+ concentrations. The cell density of MDCK cells correlated with the frequencies of calcium transients and the following RSPA. The extracellular signal-regulated kinase (ERK) activation also enhanced the frequency of RSPA in MDCK and in vivo. Thus, the PGE2 discharge is regulated temporally by calcium transients and ERK activity.

cell biology↗

bioRxiv2022

A FRET biosensor, SMART, monitors necroptosis in renal tubular epithelial cells in a cisplatin-induced kidney injury model

Necroptosis is a regulated form of cell death involved in various pathological conditions, including ischemic reperfusion injuries, virus infections, and drug-induced tissue injuries. However, it is not fully understood when and where necroptosis occurs in vivo. We previously generated a Forster resonance energy transfer (FRET) biosensor, termed SMART (the sensor for MLKL activation based on FRET), which specifically monitored necroptosis in human and murine cell lines in vitro. Here, we generated transgenic (Tg) mice that expressed the SMART biosensor in various tissues. SMART monitored necroptosis, but not apoptosis or pyroptosis, in primary cells, including peritoneal macrophages and embryonic fibroblasts. Moreover, the FRET signal was elevated in renal tubular cells of cisplatin-treated SMART Tg mice compared to untreated SMART Tg mice. Together, SMART Tg mice may provide a valuable tool for monitoring necroptosis in different types of cells in vitro and in vivo.

cell 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

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

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

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↗