The critical role of Rap1-GAPs Rasa3 and Sipa1 in T cells for pulmonary transit and egress from the lymph nodes.

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.

Thymocyte Development of Humanized Mice Is Promoted by Interactions with Human-Derived Antigen Presenting Cells upon Immunization.

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.

Rap1 organizes lymphocyte front-back polarity via RhoA signaling and talin1.

Lymphocyte trafficking requires fine-tuning of chemokine-mediated cell migration. This process depends on cytoskeletal dynamics and polarity, but its regulation remains elusive. We quantitatively measured cell polarity and revealed critical roles performed by integrin activator Rap1 in this process, independent of substrate adhesion. Rap1-deficient naive T cells exhibited impaired abilities to reorganize the actin cytoskeleton into pseudopods and actomyosin-rich uropods. Rap1-GTPase activating proteins (GAPs), Rasa3 and Sipa1, maintained an unpolarized shape; deletion of these GAPs spontaneously induced cell polarization, indicative of the polarizing effect of Rap1. Rap1 activation required F-actin scaffolds, and stimulated RhoA activation and actomyosin contractility at the rear. Furthermore, talin1 acted on Rap1 downstream effectors to promote actomyosin contractility in the uropod, which occurred independently of substrate adhesion and talin1 binding to integrins. These findings indicate that Rap1 signaling to RhoA and talin1 regulates chemokine-stimulated lymphocyte polarization and chemotaxis in a manner independent of adhesion.

Distinct bidirectional regulation of LFA1 and α4ß7 by Rap1 and integrin adaptors in T cells under shear flow.

Bidirectional control of integrin activation plays crucial roles in cell adhesive behaviors, but how integrins are specifically regulated by inside-out and outside-in signaling has not been fully understood. Here, we report distinct bidirectional regulation of major lymphocyte homing receptors LFA1 and α4ß7 in primary T cells. A small increase of Rap1 activation in L-selectin-mediated tether/rolling was boosted by the outside-in signaling from ICAM1-interacting LFA1 through subsecond, simultaneous activation of Rap1 GTPase and talin1, but not kindlin-3, resulting in increased capture and slowing. In contrast, none of them were required for tether/rolling by α4ß7 on MAdCAM1. High Rap1 activation with chemokines or the loss of Rap1-inactivating proteins Rasa3 and Sipa1 increased talin1/kindlin-3-dependent arrest with high-affinity binding of LFA1 to membrane-anchored ICAM1. However, despite increased affinity of α4ß7, activated Rap1 severely suppressed adhesion on MAdCAM1 under shear flow, indicating the critical importance of a sequential outside-in/inside-out signaling for α4ß7.

A platform of BRET-FRET hybrid biosensors for optogenetics, chemical screening, and in vivo imaging.

Genetically encoded biosensors based on the principle of Förster resonance energy transfer comprise two major classes: biosensors based on fluorescence resonance energy transfer (FRET) and those based on bioluminescence energy transfer (BRET). The FRET biosensors visualize signaling-molecule activity in cells or tissues with high resolution. Meanwhile, due to the low background signal, the BRET biosensors are primarily used in drug screening. Here, we report a protocol to transform intramolecular FRET biosensors to BRET-FRET hybrid biosensors called hyBRET biosensors. The hyBRET biosensors retain all properties of the prototype FRET biosensors and also work as BRET biosensors with dynamic ranges comparable to the prototype FRET biosensors. The hyBRET biosensors are compatible with optogenetics, luminescence microplate reader assays, and non-invasive whole-body imaging of xenograft and transgenic mice. This simple protocol will expand the use of FRET biosensors and enable visualization of the multiscale dynamics of cell signaling in live animals.

A Highly Sensitive FRET Biosensor for AMPK Exhibits Heterogeneous AMPK Responses among Cells and Organs.

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.

Intravital Förster resonance energy transfer imaging reveals osteopontin-mediated polymorphonuclear leukocyte activation by tumor cell emboli.

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.

Intravital imaging of mouse urothelium reveals activation of extracellular signal-regulated kinase by stretch-induced intravesical release of ATP.

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.

Identification of Aging-Associated Gene Expression Signatures That Precede Intestinal Tumorigenesis.

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.

In Vivo FRET Imaging of Tumor Endothelial Cells Highlights a Role of Low PKA Activity in Vascular Hyperpermeability.

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.

Interphase adhesion geometry is transmitted to an internal regulator for spindle orientation via caveolin-1.

Despite theoretical and physical studies implying that cell-extracellular matrix adhesion geometry governs the orientation of the cell division axis, the molecular mechanisms that translate interphase adhesion geometry to the mitotic spindle orientation remain elusive. Here, we show that the cellular edge retraction during mitotic cell rounding correlates with the spindle axis. At the onset of mitotic cell rounding, caveolin-1 is targeted to the retracting cortical region at the proximal end of retraction fibres, where ganglioside GM1-enriched membrane domains with clusters of caveola-like structures are formed in an integrin and RhoA-dependent manner. Furthermore, Gαi1-LGN-NuMA, a well-known regulatory complex of spindle orientation, is targeted to the caveolin-1-enriched cortical region to guide the spindle axis towards the cellular edge retraction. We propose that retraction-induced cortical heterogeneity of caveolin-1 during mitotic cell rounding sets the spindle orientation in the context of adhesion geometry.

Visualization of Signaling Molecules During Neutrophil Recruitment in Transgenic Mice Expressing FRET Biosensors.

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.

Live imaging of transforming growth factor-ß activated kinase 1 activation in Lewis lung carcinoma 3LL cells implanted into syngeneic mice and treated with polyinosinic:polycytidylic acid.

Transforming growth factor-ß 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-ß. 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.

[In vivo imaging of intracellular signaling molecules: Come/Stop signals of neutrophils during acute inflammation].

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.

Circuit-dependent striatal PKA and ERK signaling underlies rapid behavioral shift in mating reaction of male mice.

The selection of reward-seeking and aversive behaviors is controlled by two distinct D1 and D2 receptor-expressing striatal medium spiny neurons, namely the direct pathway MSNs (dMSNs) and the indirect pathway MSNs (iMSNs), but the dynamic modulation of signaling cascades of dMSNs and iMSNs in behaving animals remains largely elusive. We developed an in vivo methodology to monitor Förster resonance energy transfer (FRET) of the activities of PKA and ERK in either dMSNs or iMSNs by microendoscopy in freely moving mice. PKA and ERK were coordinately but oppositely regulated between dMSNs and iMSNs by rewarding cocaine administration and aversive electric shocks. Notably, the activities of PKA and ERK rapidly shifted when male mice became active or indifferent toward female mice during mating behavior. Importantly, manipulation of PKA cascades by the Designer Receptor recapitulated active and indifferent mating behaviors, indicating a causal linkage of a dynamic activity shift of PKA and ERK between dMSNs and iMSNs in action selection.

Intercellular propagation of extracellular signal-regulated kinase activation revealed by in vivo imaging of mouse skin.

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.

In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines.

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.