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The article discusses the role of mouse eosinophil-associated ribonuclease-2 (mEAR2) in allergic asthma. The study found that mEAR2 is not necessary for the development and function of eosinophils at rest, but it plays a crucial role in allergic airway inflammation (AAI). The absence of mEAR2 in mice resulted in decreased lung inflammation, reduced eosinophil recruitment, and diminished airway hyperresponsiveness. Additionally, mEAR2 was found to enhance the production of type 2 cytokines and IgE, contributing to the allergic response. The findings suggest that mEAR2 could serve as a potential biomarker for asthma management and a therapeutic target. [Extracted from the article]

Immune responses in humanized mice are generally inefficient without co-transplantation of human thymus or HLA transgenes. Previously, we generated humanized mice via the intra-bone marrow injection of CD133+ cord blood cells into irradiated adult immunodeficient mice (IBMI-huNSG mice), which could mount functional immune responses against HTLV-1, although the underlying mechanisms were still unknown. Here, we investigated thymocyte development in IBMI-huNSG mice, focusing on the roles of human and mouse MHC restriction. IBMI-huNSG mice had normal developmental profiles but aberrant thymic structures. Surprisingly, the thymic medulla-like regions expanded after immunization due to enhanced thymocyte expansion in association with the increase in HLA-DR+ cells, including CD205+ dendritic cells (DCs). The organ culture of thymus from immunized IBMI-huNSG mice with a neutralizing antibody to HLA-DR showed the HLA-DR-dependent expansion of CD4 single positive thymocytes. Mature peripheral T-cells exhibited alloreactive proliferation when co-cultured with human peripheral blood mononuclear cells. Live imaging of the thymus from immunized IBMI-huNSG mice revealed dynamic adhesive contacts of human-derived thymocytes and DCs accompanied by Rap1 activation. These findings demonstrate that an increase in HLA-DR+ cells by immunization promotes HLA-restricted thymocyte expansion in humanized mice, offering a unique opportunity to generate humanized mice with ease. [ABSTRACT FROM AUTHOR]

Myeloid-derived suppressor cells ( MDSCs) cause paraneoplastic leukemoid reactions and facilitate tumor cell metastasis. However, the interaction of MDSCs with tumor cells in live tissue has not been adequately visualized. To accomplish this task, we developed an intravital imaging protocol to observe metastasized tumor cells in mouse lungs. For visualization of the activation of MDSCs, bone marrow cells derived from transgenic mice expressing a Förster resonance energy transfer biosensor for ERK were implanted into host mice. Under a two-photon excitation microscope, numerous polymorphonuclear cells ( PMNs) were found to infiltrate the lungs of tumor-bearing mice in which 4T1 mammary tumor cells were implanted into the footpads. By Förster resonance energy transfer imaging, we found ERK activation in PMNs around the 4T1 tumor emboli in the lungs. Because antibody array analysis implied the involvement of osteopontin ( OPN) in the metastasis of 4T1 cells, we further analyzed the effect of OPN knockdown. The OPN knockdown in 4T1 cells did not affect the cell growth, but markedly suppressed lung metastasis of 4T1 cells and ERK activation in PMNs in the lung. Intravenous injection of recombinant OPN restored the lung metastasis of OPN-deficient 4T1 cells, suggesting that OPN functioned in a paracrine manner. It has been reported that ERK activation of neutrophils causes NETosis and that PMNs promote metastasis of tumor cells by NETosis. In agreement with previous reports, the NETosis inhibitor DNase I inhibited lung metastasis of 4T1 cells. These observations suggest that OPN promotes metastasis of 4T1 cells by activating PMNs and inducing NETosis. [ABSTRACT FROM AUTHOR]

Aging-associated alterations of cellular functions have been implicated in various disorders including cancers. Due to difficulties in identifying aging cells in living tissues, most studies have focused on aging-associated changes in whole tissues or certain cell pools. Thus, it remains unclear what kinds of alterations accumulate in each cell during aging. While analyzing several mouse lines expressing fluorescent proteins (FPs), we found that expression of FPs is gradually silenced in the intestinal epithelium during aging in units of single crypt composed of clonal stem cell progeny. The cells with low FP expression retained the wild-type Apc allele and the tissues composed of them did not exhibit any histological abnormality. Notably, the silencing of FPs was also observed in intestinal adenomas and the surrounding normal mucosae of Apc-mutant mice, and mediated by DNA methylation of the upstream promoter. Our genome-wide analysis then showed that the silencing of FPs reflects specific gene expression alterations during aging, and that these alterations occur in not only mouse adenomas but also human sporadic and hereditary (familial adenomatous polyposis) adenomas. Importantly, pharmacological inhibition of DNA methylation, which suppresses adenoma development in Apc-mutant mice, reverted the aging-associated silencing of FPs and gene expression alterations. These results identify aging-associated gene expression signatures that are heterogeneously induced by DNA methylation and precede intestinal tumorigenesis triggered by Apc inactivation, and suggest that pharmacological inhibition of the signature genes could be a novel strategy for the prevention and treatment of intestinal tumors. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β activated kinase 1 ( TAK1) has been shown to play a crucial role in cell death, differentiation, and inflammation. Here, we live-imaged robust TAK1 activation in Lewis lung carcinoma 3 LL cells implanted into the s.c. tissue of syngeneic C57 BL/6 mice and treated with polyinosinic:polycytidylic acid (PolyI:C). First, we developed and characterized a Förster resonance energy transfer-based biosensor for TAK1 activity. The TAK1 biosensor, named Eevee- TAK1, responded to stress-inducing reagents such as anisomycin, tumor necrosis factor-α, and interleukin1-β. The anisomycin-induced increase in Förster resonance energy transfer was abolished by the TAK1 inhibitor (5z)-7-oxozeaenol. Activity of TAK1 in 3 LL cells was markedly increased by PolyI:C in the presence of macrophages. 3 LL cells expressing Eevee- TAK1 were implanted into mice and observed through imaging window by two-photon excitation microscopy. During the growth of tumor, the 3 LL cells at the periphery of the tumor showed higher TAK1 activity than the 3 LL cells located at the center of the tumor, suggesting that cells at the periphery of the tumor mass were under stronger stress. Injection of PolyI:C, which is known to induce regression of the implanted tumors, induced marked and homogenous TAK1 activation within the tumor tissues. The effect of PolyI:C faded within 4 days. These observations suggest that Eevee- TAK1 is a versatile tool to monitor cellular stress in cancer tissues. [ABSTRACT FROM AUTHOR]

The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers. [ABSTRACT FROM AUTHOR]

Enteric neural crest-derived cells (ENCCs) migrate from the anterior foregut in a rostrocaudal direction to colonize the entire gastroin-testinal tract and to form the enteric nervous system. Genetic approaches have identified many signaling molecules regulating the migration of ENCCs; however, it remains elusive how the activities of the signaling molecules are regulated spatiotemporally during migration. In this study, transgenic mice expressing biosensors based on Forster resonance energy transfer were generated to video the activity changes of the signaling molecules in migrating ENCCs. In an organ culture of embryonic day 11.25 (El 1.25) to E13 guts, ENCCs at the rostral wavefront migrated as a cellular chain faster than the following ENCCs that formed a network. The faster-migrating cells at the wavefront exhibited lower protein kinase A (PKA) activity than did the slower-migrating trailing cells. The activities of Racl and Cdc42 exhibited an inverse correlation with the PKA activity, and PKA activation decreased the Rac 1 activity and migration velocity. PKA activity in ENCCs was correlated positively with the distribution of GDNF and inversely with the distribution of endothelin 3 (ET-3). Accordingly, PKA was activated by GDNF and inhibited by ET-3 in cultured ENCCs. Finally, although the JNK and ERK pathways were previously reported to control the migration of ENCCs, we did not find any correlation of JNK or ERK activity with the migration velocities. These results suggest that external cues regulate the migration of ENCCs by controlling PKA activity, but not ERK or JNK activity, and argue for the importance of live imaging of signaling molecule activities in developing organs. [ABSTRACT FROM AUTHOR]

Abstract: Pippi (phosphatidyl inositol phosphate indicator) is a biosensor based on the principle of FRET (Förster resonance energy transfer), which consists of a pair of fluorescent proteins, CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein), the PH domain sandwiched between them, and K-Ras C-terminal sequence for plasma membrane localization. Due to marked cross-excitation of YFP with the conditions used to excite CFP, initial FRET images obtained by TPE (two-photon excitation) microscopy suffered from low signal-to-noise ratio, hampering the observation of lipids in three-dimensional structures. To solve this problem, YFP and CFP in the original Pippi-PI(3,4)P2 was replaced by sREACh (super resonance energy accepting chromoprotein) and mTFP1 (monomeric teal fluorescent protein), respectively. The biosensor was also fused with an internal control protein, mKeima, where Keima/mTFP1 indicates the FRET efficiency, and indeed epidermal growth factor stimulation increased Keima/mTFP1 in HeLa cells. This biosensor successfully showed PI(3,4)P2 accumulation to the lateral membrane in the MDCK cyst cultured in a three-dimensional environment. Furthermore, other FRET-based biosensors for PIP3 distribution and for tyrosine kinase activity were developed based on this method, suggesting its broad application for visualizing signal transduction events with TPE microscopy. [Copyright &y& Elsevier]

Both TSC2 (tuberin) and forkhead transcription factor FoxO1 are phosphorylated and inhibited by Akt and play important roles in insulin signaling. However, little is known about the relationship between TSC2 and FoxO1. Here we identified TSC2 as a FoxO1-binding protein by using a yeast two-hybrid screening with a murine islet cDNA library. Among FoxOs, only FoxO1 can be associated with TSC2. The physical association between the C terminus of TSC2 (amino acids 1280-1499) and FoxO1 degrades the TSC1-TSC2 complex and inhibits GTPase-activating protein activity of TSC2 toward Rheb. Overexpression of wild type FoxO1 enhances p70 S6K phosphorylation, whereas overexpression of TSC2 can reverse these effects. Knockdown of endogenous FOXO1 in human vascular endothelial cells decreased phosphorylation of p70 S6K. Prolonged overexpression of wild type FoxO1 enhanced phosphorylation of serine 307 of IRS1 and decreased phosphorylation of Akt and FoxO1 itself even in the presence of serum. These data suggest a novel mechanism by which FoxO1 regulates the insulin signaling pathway through negative regulation of TSC2 function. [ABSTRACT FROM AUTHOR]

Dynamin associates with a variety of SH3 domain-containing molecules via a C-terminal praline-rich motif and takes part, with them, in endocytic processes. Here, we have investigated a new dynamin-associating molecule, formin-binding protein 17 (FBP17), involved in deforming the plasma membrane and in endocytosis. FBP17 formed tubular invaginations originating from the plasma membrane. Its N-terminal Fer/CIP4 homology domain, a coiled-coil domain, and a proline-rich motif were required for tubular invagination and self-assembly, by which tubular invagination might be induced. Using anti-FBP17 antibody, we detected positive immunoreactions in the testis that were restricted to the germ cells. We also detected FBP17 in the brain by immunoblotting and in situ hybridization. When COS cells expressing enhanced green fluorescent protein-tagged FBP17 were incubated with fluorescently labeled transferrin, epidermal growth factor, and cholera toxin, these molecules co-localized with FBP17-induced tubular invaginations, suggesting that FBP17 is involved in dynamin-mediated endocytosis in both a clathrin-dependent and -independent manner. These observations therefore indicate that FBP17 interacts with dynamin and regulates endocytosis by forming vesicotubular structures. [ABSTRACT FROM AUTHOR]

Streptomyces virginiae produces γ-butyrolactone autoregulators (virginiae butanolide, VB), which control the biosynthesis of virginiamycin M1 and S. A 6.3-kb region downstream of the virginiamycin S (VS)-resistance operon in S. virginiae was sequenced, and four plausible open reading frames (ORFs) (visA, 1,260 bp; visB, 1,656 bp; visC, 888 bp; visD, 1209 bp) were identified. Homology analysis revealed significant similarities with enzymes involved in the biosynthesis of cyclopeptolide antibiotics: VisA (53% identity, 65% similarity) to l-lysine 2-aminotransferase (NikC) of nikkomycin D biosynthesis, VisB (66% identity, 72% similarity) to 3-hydroxypicolinic acid:AMP ligase of pristinamycin I biosynthesis, VisC (48% identity, 59% similarity) to lysine cyclodeaminase of ascomycin biosynthesis, and VisD (43% identity, 56% similarity) to erythromycin C-22 hydroxylase of erythromycin biosynthesis. Northern blotting as well as high-resolution S1 analysis of the ORFs revealed that they were transcribed as two bicistronic transcripts, namely 3.0-kb visB-visA and another 2.7-kb visC-visD transcript, with promoters locating upstream of visB and visC, respectively. Transcription of the two operons was observed only 1 h after the VB production, which was 2 h before the virginiamycin production. Furthermore, prompt induction of the transcription was observed as a result of external VB addition, suggesting that the expression of the two operons was under the control of VB. [Copyright &y& Elsevier]

The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers. [ABSTRACT FROM AUTHOR]

Shoc2/SUR-8 positively regulates Ras/ERK MAP kinase signaling by serving as a scaffold for Ras and Raf. Here, we examined the role of Shoc2 in the spatio-temporal regulation of Ras by using a fluorescence resonance energy transfer (FRET)-based biosensor, together with computational modeling. In epidermal growth factor-stimulated HeLa cells, RNA-mediated Shoc2 knockdown reduced the phosphorylation of MEK and ERK with half-maximal inhibition, but not the activation of Ras. For the live monitoring of Ras binding to Raf, we utilized a FRET biosensor wherein Ras and the Ras-binding domain of Raf were connected tandemly and sandwiched with acceptor and donor fluorescent proteins for the FRET measurement. With this biosensor, we found that Shoc2 was required for the rapid interaction of Ras with Raf upon epidermal growth factor stimulation. To decipher the molecular mechanisms underlying the kinetics, we developed two computational models that might account for the action of Shoc2 in the Ras-ERK signaling. One of these models, the Shoc2 accelerator model, provided a reasonable explanation of the experimental observations. In this Shoc2 accelerator model, Shoc2 accelerated both the association and dissociation of Ras-Raf interaction. We propose that Shoc2 regulates the spatio-temporal patterns of the Ras-ERK signaling pathway primarily by accelerating the Ras-Raf interaction. [ABSTRACT FROM AUTHOR]

Rap1-GTPase activates integrins and plays an indispensable role in lymphocyte trafficking, but the importance of Rap1 inactivation in this process remains unknown. Here we identified the Rap1-inactivating proteins Rasa3 and Sipa1 as critical regulators of lymphocyte trafficking. The loss of Rasa3 and Sipa1 in T cells induced spontaneous Rap1 activation and adhesion. As a consequence, T cells deficient in Rasa3 and Sipa1 were trapped in the lung due to firm attachment to capillary beds, while administration of LFA1 antibodies or loss of talin1 or Rap1 rescued lung sequestration. Unexpectedly, mutant T cells exhibited normal extravasation into lymph nodes, fast interstitial migration, even greater chemotactic responses to chemokines and sphingosine-1-phosphate, and entrance into lymphatic sinuses but severely delayed exit: mutant T cells retained high motility in lymphatic sinuses and frequently returned to the lymph node parenchyma, resulting in defective egress. These results reveal the critical trafficking processes that require Rap1 inactivation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Horitani, Ueda, Kamioka, Kondo, Ikeda, Naganuma and Kinashi.)

AMP-activated protein kinase (AMPK), a master regulator of cellular metabolism, is a potential target for type 2 diabetes. Although extensive in vitro studies have revealed the complex regulation of AMPK, much remains unknown about the regulation in vivo. We therefore developed transgenic mice expressing a highly sensitive fluorescence resonance energy transfer (FRET)-based biosensor for AMPK, called AMPKAR-EV. AMPKAR-EV allowed us to readily examine the role of LKB1, a canonical stimulator of AMPK, in drug-induced activation and inactivation of AMPK in vitro. In transgenic mice expressing AMPKAR-EV, the AMP analog AICAR activated AMPK in muscle. In contrast, the antidiabetic drug metformin activated AMPK in liver, highlighting the organ-specific action of AMPK stimulators. Moreover, we found that AMPK was activated primarily in fast-twitch muscle fibers after tetanic contraction and exercise. These observations suggest that the AMPKAR-EV mouse will pave a way to understanding the heterogeneous responses of AMPK among cell types in vivo., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

To better understand the roles played by signaling molecules in the bladder, we established a protocol of intravital imaging of the bladder of mice expressing a Förster/fluorescence resonance energy transfer (FRET) biosensor for extracellular signal-regulated kinase (ERK), which plays critical roles not only in cell growth but also stress responses. With an upright two-photon excitation microscope and a vacuum-stabilized imaging window, cellular ERK activity was visualized in the whole bladder wall, from adventitia to urothelium. We found that bladder distention caused by elevated intravesical pressure (IVP) activated ERK in the urothelium, but not in the detrusor smooth muscle. When bladder distension was prevented, high IVP failed to activate ERK, suggesting that mechanical stretch, but not the high IVP, caused ERK activation. To delineate its molecular mechanism, the stretch-induced ERK activation was reproduced in an hTERT-immortalized human urothelial cell line (TRT-HU1) in vitro. We found that uniaxial stretch raised the ATP concentration in the culture medium and that inhibition of ATP signaling by apyrase or suramin suppressed the stretch-induced ERK activation in TRT-HU1 cells. In agreement with this in vitro observation, pretreatment with apyrase or suramin suppressed the high IVP-induced urothelial ERK activation in vivo. Thus, we propose that mechanical stretch induces intravesical secretion of ATP and thereby activates ERK in the urothelium. Our method of intravital imaging of the bladder of FRET biosensor-expressing mice should open a pathway for the future association of physiological stimuli with the activities of intracellular signaling networks., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Vascular hyperpermeability is a pathological hallmark of cancer. Previous in vitro studies have elucidated roles of various signaling molecules in vascular hyperpermeability; however, the activities of such signaling molecules have not been examined in live tumor tissues for technical reasons. Here, by in vivo two-photon excitation microscopy with transgenic mice expressing biosensors based on Förster resonance energy transfer, we examined the activity of protein kinase A (PKA), which maintains endothelial barrier function. The level of PKA activity was significantly lower in the intratumoral endothelial cells than the subcutaneous endothelial cells. PKA activation with a cAMP analogue alleviated the tumor vascular hyperpermeability, suggesting that the low PKA activity in the endothelial cells may be responsible for the tumor-tissue hyperpermeability. Because the vascular endothelial growth factor (VEGF) receptor is a canonical inducer of vascular hyperpermeability and a molecular target of anticancer drugs, we examined the causality between VEGF receptor activity and the PKA activity. Motesanib, a kinase inhibitor for VEGF receptor, activated tumor endothelial PKA and reduced the vascular permeability in the tumor. Conversely, subcutaneous injection of VEGF decreased endothelial PKA activity and induced hyperpermeability of subcutaneous blood vessels. Notably, in cultured human umbilical vascular endothelial cells, VEGF activated PKA rather than decreasing its activity, highlighting the remarkable difference between its actions in vitro and in vivo These data suggested that the VEGF receptor signaling pathway increases vascular permeability, at least in part, by reducing endothelial PKA activity in the live tumor tissue. Cancer Res; 76(18); 5266-76. ©2016 AACR., (©2016 American Association for Cancer Research.)

Transforming growth factor-β activated kinase 1 (TAK1) has been shown to play a crucial role in cell death, differentiation, and inflammation. Here, we live-imaged robust TAK1 activation in Lewis lung carcinoma 3LL cells implanted into the s.c. tissue of syngeneic C57BL/6 mice and treated with polyinosinic:polycytidylic acid (PolyI:C). First, we developed and characterized a Förster resonance energy transfer-based biosensor for TAK1 activity. The TAK1 biosensor, named Eevee-TAK1, responded to stress-inducing reagents such as anisomycin, tumor necrosis factor-α, and interleukin1-β. The anisomycin-induced increase in Förster resonance energy transfer was abolished by the TAK1 inhibitor (5z)-7-oxozeaenol. Activity of TAK1 in 3LL cells was markedly increased by PolyI:C in the presence of macrophages. 3LL cells expressing Eevee-TAK1 were implanted into mice and observed through imaging window by two-photon excitation microscopy. During the growth of tumor, the 3LL cells at the periphery of the tumor showed higher TAK1 activity than the 3LL cells located at the center of the tumor, suggesting that cells at the periphery of the tumor mass were under stronger stress. Injection of PolyI:C, which is known to induce regression of the implanted tumors, induced marked and homogenous TAK1 activation within the tumor tissues. The effect of PolyI:C faded within 4 days. These observations suggest that Eevee-TAK1 is a versatile tool to monitor cellular stress in cancer tissues., (© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

A number of chemical mediators regulate neutrophil recruitment to inflammatory sites either positively or negatively. Although the actions of each chemical mediator on the intracellular signaling networks controlling cell migration have been studied with neutrophils cultured in vitro, how such chemical mediators act cooperatively or counteractively in vivo remains largely unknown. To understand the mechanisms regulating neutrophil recruitment to the inflamed intestine in vivo, we recently generated transgenic mice expressing biosensors based on FRET (Förster resonance energy transfer) and set up two-photon excitation microscopy to observe the gastrointestinal tract in living mice. By measuring FRET in neutrophils, we showed activity changes of protein kinases in the neutrophils recruited to inflamed intestines. In this chapter, we describe the protocol used to visualize the protein kinase activities in neutrophils of the inflamed intestine of transgenic mice expressing the FRET biosensors.

Many chemical mediators regulate neutrophil recruitment to inflammatory sites. Although the actions of each of these chemical mediators have been demonstrated with neutrophils in vitro, how they act cooperatively or counteract each other in vivo remains largely unknown. To understand the behaviors of neutrophils in vivo, the activities of intracellular signaling molecules must be visualized in living tissues. For this purpose, we can use genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET). In this review, we first provide an overview of FRET biosensors and then describe how we can utilize these biosensors to visualize the activity changes of signaling molecules in neutrophils during extravasation. In relation to this topic, we will also describe the development of transgenic mice expressing the FRET biosensors and in vivo two-photon excitation microscopy.

Extracellular signal-regulated kinase (ERK) is a key effector of many growth signalling pathways. In this study, we visualise epidermal ERK activity in living mice using an ERK FRET biosensor. Under steady-state conditions, the epidermis occasionally revealed bursts of ERK activation patterns where ERK activity radially propagated from cell to cell. The frequency of this spatial propagation of radial ERK activity distribution (SPREAD) correlated with the rate of epidermal cell division. SPREADs and proliferation were stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) in a manner dependent on EGF receptors and their cognate ligands. At the wounded skin, ERK activation propagated as trigger wave in parallel to the wound edge, suggesting that ERK activation propagation can be superimposed. Furthermore, by visualising the cell cycle, we found that SPREADs were associated with G2/M cell cycle progression. Our results provide new insights into how cell proliferation and transient ERK activity are synchronised in a living tissue.

Many chemical mediators regulate neutrophil recruitment to inflammatory sites. Although the actions of each chemical mediator have been demonstrated with neutrophils in vitro, how such chemical mediators act cooperatively or counteractively in vivo remains largely unknown. Here, by in vivo two-photon excitation microscopy with transgenic mice expressing biosensors based on Förster resonance energy transfer, we time-lapse-imaged the activities of extracellular signal-regulated kinase (ERK) and protein kinase A (PKA) in neutrophils in inflamed intestinal tissue. ERK activity in neutrophils rapidly increased during spreading on the endothelial cells and showed positive correlation with the migration velocity on endothelial cells or in interstitial tissue. Meanwhile, in the neutrophils migrating in the interstitial tissue, high PKA activity correlated negatively with migration velocity. In contradiction to previous in vitro studies that showed ERK activation by prostaglandin E2 (PGE2) engagement with prostaglandin receptor EP4, intravenous administration of EP4 agonist activated PKA, inhibited ERK, and suppressed migration of neutrophils. The opposite results were obtained using nonsteroidal antiinflammatory drugs (NSAIDs). Therefore, NSAID-induced enteritis may be caused at least partially by the inhibition of EP4 receptor signaling of neutrophils. Our results demonstrate that ERK positively regulates the neutrophil recruitment cascade by promoting adhesion and migration steps., (© 2014 Mizuno et al.)

The mechanism behind the spatiotemporal control of cancer cell dynamics and its possible association with cell proliferation has not been well established. By exploiting the intravital imaging technique, we found that cancer cell motility and invasive properties were closely associated with the cell cycle. In vivo inoculation of human colon cancer cells bearing fluorescence ubiquitination-based cell cycle indicator (Fucci) demonstrated an unexpected phenomenon: S/G2/M cells were more motile and invasive than G1 cells. Microarray analyses showed that Arhgap11a, an uncharacterized Rho GTPase-activating protein (RhoGAP), was expressed in a cell-cycle-dependent fashion. Expression of ARHGAP11A in cancer cells suppressed RhoA-dependent mechanisms, such as stress fiber formation and focal adhesion, which made the cells more prone to migrate. We also demonstrated that RhoA suppression by ARHGAP11A induced augmentation of relative Rac1 activity, leading to an increase in the invasive properties. RNAi-based inhibition of Arhgap11a reduced the invasion and in vivo expansion of cancers. Additionally, analysis of human specimens showed the significant up-regulation of Arhgap11a in colon cancers, which was correlated with clinical invasion status. The present study suggests that ARHGAP11A, a cell cycle-dependent RhoGAP, is a critical regulator of cancer cell mobility and is thus a promising therapeutic target in invasive cancers.

In recent years, fluorescence imaging has received particular attention, due to increasing availabilities of fluorescent proteins and dyes, which had driven the development of novel biosensors. Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to difficulties in stable expression of FRET biosensors. In this study, we report efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were generated by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harboring Tol2 recombination sites. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.

Two-photon excitation microscopy was used to visualized two different modes of invasion at perivascular and intraparenchymal regions of rat C6 glioblastoma cells that were orthotopically implanted into rat brains. Probes based on the principle of Förster resonance energy transfer (FRET) further revealed that glioblastoma cells penetrating the brain parenchyma showed higher Rac1 and Cdc42 activities and lower RhoA activity than those advancing in the perivascular regions. This spatial regulation of Rho-family GTPase activities was recapitulated in three-dimensional spheroid invasion assays with rat and human glioblastoma cells, in which multipod glioblastoma cells that invaded the gels and led the other glioblastoma cells exhibited higher Rac1 and Cdc42 activities than the trailing glioblastoma cells. We also studied the Cdc42-specific guanine nucleotide exchange factor Zizimin1 (also known as DOCK9) as a possible contributor to this spatially controlled activation of Rho-family GTPases, because it is known to play an essential role in the extension of neurites. We found that shRNA-mediated knockdown of Zizimin1 inhibited formation of pseudopodia and concomitant invasion of glioblastoma cells both under a 3D culture condition and in vivo. Our results suggest that the difference in the activity balance of Rac1 and Cdc42 versus RhoA determines the mode of glioblastoma invasion and that Zizimin1 contributes to the invasiveness of glioblastoma cells with high Rac1 and Cdc42 activities.

Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to particular difficulties in the development of transgenic mice that express FRET biosensors. In this study, we report the efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were created by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harbouring Tol2 recombination sites. High expression of the biosensors in a wide range of cell types allowed us to screen newborn mice simply by inspection. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.

Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have shed new light on the spatiotemporal dynamics of signaling molecules. Among them, intramolecular FRET biosensors have been increasingly used due to their high sensitivity and user-friendliness. Time-consuming optimizations by trial and error, however, obstructed the development of intramolecular FRET biosensors. Here we report an optimized backbone for rapid development of highly sensitive intramolecular FRET biosensors. The key concept is to exclude the "orientation-dependent" FRET and to render the biosensors completely "distance-dependent" with a long, flexible linker. We optimized a pair of fluorescent proteins for distance-dependent biosensors, and then developed a long, flexible linker ranging from 116 to 244 amino acids in length, which reduced the basal FRET signal and thereby increased the gain of the FRET biosensors. Computational simulations provided insight into the mechanisms by which this optimized system was the rational strategy for intramolecular FRET biosensors. With this backbone system, we improved previously reported FRET biosensors of PKA, ERK, JNK, EGFR/Abl, Ras, and Rac1. Furthermore, this backbone enabled us to develop novel FRET biosensors for several kinases of RSK, S6K, Akt, and PKC and to perform quantitative evaluation of kinase inhibitors in living cells.

EGF-induced activation of ERK has been extensively studied by both experimental and theoretical approaches. Here, we used a simulation model based mostly on experimentally determined parameters to study the ERK-mediated negative feedback regulation of the Ras guanine nucleotide exchange factor, son of sevenless (SOS). Because SOS1 is phosphorylated at multiple serine residues upon stimulation, we evaluated the role of the multiplicity by building two simulation models, which we termed the decisive and cooperative phosphorylation models. The two models were constrained by the duration of Ras activation and basal phosphorylation level of SOS1. Possible solutions were found only in the decisive model wherein at least three, and probably more than four, phosphorylation sites decisively suppress the SOS activity. Thus, the combination of experimental approaches and the model analysis has suggested an unexpected role of multiple phosphorylations of SOS1 in the negative regulation.

Situated downstream of Ras is a key signaling molecule, Raf1. Increase in Ca(2+) concentration has been shown to modulate the Ras-dependent activation of Raf1; however, the mechanism underlying this effect remains elusive. Here, to characterize the role of Ca(2+) in Ras signaling to Raf1, we used a synthetic guanine nucleotide exchange factor (GEF) for Ras, eGRF. In HeLa cells expressing eGRF, Ras was activated by the cAMP analogue 007 as efficiently as by epidermal growth factor (EGF), whereas the activation of Raf1, MEK, and ERK by 007 was about half of that by EGF. Using a biosensor based on fluorescence resonance energy transfer, it was found that activation of Raf1 at the plasma membrane required not only Ras activation but also an increase in Ca(2+) concentration or inhibition of calmodulin. Furthermore, the Ca(2+)-dependent activation of Raf1 was found to be abrogated by knockdown of Shoc2, a scaffold protein that binds both Ras and Raf1. These observations indicated that the Shoc2 scaffold protein modulates Ras-dependent Raf1 activation in a Ca(2+)- and calmodulin-dependent manner.

Both TSC2 (tuberin) and forkhead transcription factor FoxO1 are phosphorylated and inhibited by Akt and play important roles in insulin signaling. However, little is known about the relationship between TSC2 and FoxO1. Here we identified TSC2 as a FoxO1-binding protein by using a yeast two-hybrid screening with a murine islet cDNA library. Among FoxOs, only FoxO1 can be associated with TSC2. The physical association between the C terminus of TSC2 (amino acids 1280-1499) and FoxO1 degrades the TSC1-TSC2 complex and inhibits GTPase-activating protein activity of TSC2 toward Rheb. Overexpression of wild type FoxO1 enhances p70 S6K phosphorylation, whereas overexpression of TSC2 can reverse these effects. Knockdown of endogenous FOXO1 in human vascular endothelial cells decreased phosphorylation of p70 S6K. Prolonged overexpression of wild type FoxO1 enhanced phosphorylation of serine 307 of IRS1 and decreased phosphorylation of Akt and FoxO1 itself even in the presence of serum. These data suggest a novel mechanism by which FoxO1 regulates the insulin signaling pathway through negative regulation of TSC2 function.

Rap1 is a small GTPase that regulates adherens junction maturation. It remains elusive how Rap1 is activated upon cell-cell contact. We demonstrate for the first time that Rap1 is activated upon homophilic engagement of vascular endothelial cadherin (VE-cadherin) at the cell-cell contacts in living cells and that MAGI-1 is required for VE-cadherin-dependent Rap1 activation. We found that MAGI-1 localized to cell-cell contacts presumably by associating with beta-catenin and that MAGI-1 bound to a guanine nucleotide exchange factor for Rap1, PDZ-GEF1. Depletion of MAGI-1 suppressed the cell-cell contact-induced Rap1 activation and the VE-cadherin-mediated cell-cell adhesion after Ca2+ switch. In addition, relocation of vinculin from cell-extracellular matrix contacts to cell-cell contacts after the Ca2+ switch was inhibited in MAGI-1-depleted cells. Furthermore, inactivation of Rap1 by overexpression of Rap1GAPII impaired the VE-cadherin-dependent cell adhesion. Collectively, MAGI-1 is important for VE-cadherin-dependent Rap1 activation upon cell-cell contact. In addition, once activated, Rap1 upon cell-cell contacts positively regulate the adherens junction formation by relocating vinculin that supports VE-cadherin-based cell adhesion.

Platelet endothelial adhesion molecule-1 (PECAM-1) is a part of intercellular junctions and triggers intracellular signaling cascades upon homophilic binding. The intracellular domain of PECAM-1 is tyrosine phosphorylated upon homophilic engagement. However, it remains unclear which tyrosine kinase phosphorylates PECAM-1. We sought to isolate tyrosine kinases responsible for PECAM-1 phosphorylation and identified Fer as a candidate, based on expression cloning. Fer kinase specifically phosphorylated PECAM-1 at the immunoreceptor tyrosine-based inhibitory motif. Notably, Fer induced tyrosine phosphorylation of SHP-2, which is known to bind to the immunoreceptor tyrosine-based inhibitory motif of PECAM-1, and Fer also induced tyrosine phosphorylation of Gab1 (Grb2-associated binder-1). Engagement-dependent PECAM-1 phosphorylation was inhibited by the overexpression of a kinase-inactive mutant of Fer, suggesting that Fer is responsible for the tyrosine phosphorylation upon PECAM-1 engagement. Furthermore, by using green fluorescent protein-tagged Fer and a time-lapse fluorescent microscope, we found that Fer localized at microtubules in polarized and motile vascular endothelial cells. Fer was dynamically associated with growing microtubules in the direction of cell-cell contacts, where p120catenin, which is known to associate with Fer, colocalized with PECAM-1. These results suggest that Fer localized on microtubules may play an important role in phosphorylation of PECAM-1, possibly through its association with p120catenin at nascent cell-cell contacts.