Knockout of all ErbB-family genes delineates their roles in proliferation, survival and migration.

The ErbB-family receptors play pivotal roles in the proliferation, migration and survival of epithelial cells. Because our knowledge on the ErbB-family receptors has been largely obtained by the exogenous application of their ligands, it remains unknown to what extent each of the ErbB members contributes to these outputs. We here knocked out each ErbB gene, various combinations of ErbB genes or all ErbB genes in Madin-Darby canine kidney cells to delineate the contribution of each gene. ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) activation waves during collective cell migration were mediated primarily by ErbB1 and secondarily by the ErbB2 and ErbB3 heterodimer. Either ErbB1 or the ErbB2 and ErbB3 complex was sufficient for the G1/S progression. The saturation cell density was markedly reduced in cells deficient in all ErbB proteins, but not in cells retaining only ErbB2, which cannot bind to ligands. Thus, a ligand-independent ErbB2 activity is sufficient for preventing apoptosis at high cell density. In short, systematic knockout of ErbB-family genes has delineated the roles of each ErbB receptor.

Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle.

Eukaryotic cells spend most of their life in interphase of the cell cycle. Understanding the rich diversity of metabolic and genomic regulation that occurs in interphase requires the demarcation of precise phase boundaries in situ. Here, we report the properties of two genetically encoded fluorescence sensors, Fucci(CA) and Fucci(SCA), which enable real-time monitoring of interphase and cell-cycle biology. We re-engineered the Cdt1-based sensor from the original Fucci system to respond to S phase-specific CUL4Ddb1-mediated ubiquitylation alone or in combination with SCFSkp2-mediated ubiquitylation. In cultured cells, Fucci(CA) produced a sharp triple color-distinct separation of G1, S, and G2, while Fucci(SCA) permitted a two-color readout of G1 and S/G2. Fucci(CA) applications included tracking the transient G1 phase of rapidly dividing mouse embryonic stem cells and identifying a window for UV-irradiation damage in S phase. These results show that Fucci(CA) is an essential tool for quantitative studies of interphase cell-cycle regulation.

Visual quantification of prostaglandin E2 discharge from a single cell.

Calcium transients drive cells to discharge prostaglandin E2 (PGE2). We visualized PGE2-induced protein kinase A (PKA) activation and quantitated PGE2 secreted from a single cell by combining fluorescence microscopy and a simulation model. For this purpose, we first prepared PGE2-producer cells that express either an optogenetic or a chemogenetic calcium channel stimulator: OptoSTIM1 or Gq-DREADD, respectively. Second, we prepared reporter cells expressing the Gs-coupled PGE2 reporter EP2 and the PKA biosensor Booster-PKA, which is based on the principle of Förster resonance energy transfer (FRET). Upon the stimulation-induced triggering of calcium transients, a single producer cell discharges PGE2 to stimulate PKA in the surrounding reporter cells. Due to the flow of the medium, the PKA-activated area exhibited a comet-like smear when HeLa cells were used. In contrast, radial PKA activation was observed when confluent MDCK cells were used, indicating that PGE2 diffusion was restricted to the basolateral space. By fitting the radius of the PKA-activated area to a simulation model based on simple diffusion, we estimated that a single HeLa cell secretes 0.25 fmol PGE2 upon a single calcium transient to activate PKA in more than 1000 neighboring cells. This model also predicts that the PGE2 discharge rate is comparable to the diffusion rate. Thus, our method quantitatively envisions that a single calcium transient affects more than 1000 neighboring cells via PGE2.Key words: prostaglandin E2, imaging, intercellular communication, biosensor, quantification.

Rhodopsin-mediated light-off-induced protein kinase A activation in mouse rod photoreceptor cells.

Light-induced extrasynaptic dopamine release in the retina reduces adenosine 3',5'-cyclic monophosphate (cAMP) in rod photoreceptor cells, which is thought to mediate light-dependent desensitization. However, the fine time course of the cAMP dynamics in rods remains elusive due to technical difficulty. Here, we visualized the spatiotemporal regulation of cAMP-dependent protein kinase (PKA) in mouse rods by two-photon live imaging of retinal explants of PKAchu mice, which express a fluorescent biosensor for PKA. Unexpectedly, in addition to the light-on-induced suppression, we observed prominent light-off-induced PKA activation. This activation required photopic light intensity and was confined to the illuminated rods. The estimated maximum spectral sensitivity of 489 nm and loss of the light-off-induced PKA activation in rod-transducin-knockout retinas strongly suggest the involvement of rhodopsin. In support of this notion, rhodopsin-deficient retinal explants showed only the light-on-induced PKA suppression. Taken together, these results suggest that, upon photopic light stimulation, rhodopsin and dopamine signals are integrated to shape the light-off-induced cAMP production and following PKA activation. This may support the dark adaptation of rods.

FRET-assisted photoactivation of flavoproteins for in vivo two-photon optogenetics.

Optical dimerizers have been developed to untangle signaling pathways, but they are of limited use in vivo, partly due to their inefficient activation under two-photon (2P) excitation. To overcome this problem, we developed Förster resonance energy transfer (FRET)-assisted photoactivation, or FRAPA. On 2P excitation, mTagBFP2 efficiently absorbs and transfers the energy to the chromophore of CRY2. Based on structure-guided engineering, a chimeric protein with 40% FRET efficiency was developed and named 2P-activatable CRY2, or 2paCRY2. 2paCRY2 was employed to develop a RAF1 activation system named 2paRAF. In three-dimensionally cultured cells expressing 2paRAF, extracellular signal-regulated kinase (ERK) was efficiently activated by 2P excitation at single-cell resolution. Photoactivation of ERK was also accomplished in the epidermal cells of 2paRAF-expressing mice. We further developed an mTFP1-fused LOV domain that exhibits efficient response to 2P excitation. Collectively, FRAPA will pave the way to single-cell optical control of signaling pathways in vivo.

Visualization of Spatially-Controlled Vasospasm by Sympathetic Nerve-Mediated ROCK Activation.

Contraction of vascular smooth muscle is regulated primarily by calcium concentration and secondarily by ROCK activity within the cells. In contrast to the wealth of information regarding regulation of calcium concentration, little is known about the spatiotemporal regulation of ROCK activity in live blood vessels. Here, we report ROCK activation in subcutaneous arterioles in a transgenic mouse line that expresses a genetically encoded ROCK biosensor based on the principle of FÓ§rster resonance energy transfer by two-photon excitation in vivo imaging. Rapid vasospasm was induced upon laser ablation of arterioles, concomitant with a transient increase in calcium concentration in arteriolar smooth muscles. Unlike the increase in calcium concentration, vasoconstriction and ROCK activation continued for several minutes after irradiation. Both the ROCK inhibitor, fasudil, and the ganglionic nicotinic acetylcholine receptor blocker, hexamethonium, inhibited laser-induced ROCK activation and reduced the duration of vasospasm at the segments distant from the irradiated point. These observations suggest that vasoconstriction is initially triggered by a rapid surge of cytoplasmic calcium and then maintained by sympathetic nerve-mediated ROCK activation.

Two-photon AMPK and ATP imaging reveals the bias between rods and cones in glycolysis utility.

In vertebrates, retinal rod and cone photoreceptor cells rely significantly on glycolysis. Lactate released from photoreceptor cells fuels neighboring retinal pigment epithelium cells and Müller glial cells through oxidative phosphorylation. To understand this highly heterogeneous metabolic environment around photoreceptor cells, single-cell analysis is needed. Here, we visualized cellular AMP-activated protein kinase (AMPK) activity and ATP levels in the retina by two-photon microscopy. Transgenic mice expressing a hyBRET-AMPK biosensor were used for measuring the AMPK activity. GO-ATeam2 transgenic mice were used for measuring the ATP level. Temporal metabolic responses were successfully detected in the live retinal explants upon drug perfusion. A glycolysis inhibitor, 2-deoxy-d-glucose (2-DG), activated AMPK and reduced ATP. These effects were clearly stronger in rods than in cones. Notably, rod AMPK and ATP started to recover at 30 min from the onset of 2-DG perfusion. Consistent with these findings, ex vivo electroretinogram recordings showed a transient slowdown in rod dim flash responses during a 60-min 2-DG perfusion, whereas cone responses were not affected. Based on these results, we propose that cones surrounded by highly glycolytic rods become less dependent on glycolysis, and rods also become less dependent on glycolysis within 60 min upon the glycolysis inhibition.

A Dual Promoter System to Monitor IFN-γ Signaling in vivo at Single-cell Resolution.

IFN-γ secreted from immune cells exerts pleiotropic effects on tumor cells, including induction of immune checkpoint and antigen presentation, growth inhibition, and apoptosis induction. We combined a dual promoter system with an IFN-γ signaling responsive promoter to generate a reporter named the interferon sensing probe (ISP), which quantitates the response to IFN-γ by means of fluorescence and bioluminescence. The integration site effect of the transgene is compensated for by the PGK promoter-driven expression of a fluorescent protein. Among five potential IFN-γ-responsive elements, we found that the interferon γ-activated sequence (GAS) exhibited the best performance. When ISP-GAS was introduced into four cell lines and subjected to IFN-γ stimulation, dose-dependency was observed with an EC50 ranging from 0.2 to 0.9 ng/mL, indicating that ISP-GAS can be generally used as a sensitive biosensor of IFN-γ response. In a syngeneic transplantation model, the ISP-GAS-expressing cancer cells exhibited bioluminescence and fluorescence signals in an IFN-γ receptor-dependent manner. Thus, ISP-GAS could be used to quantitatively monitor the IFN-γ response both in vitro and in vivo.Key words: in vivo imaging, tumor microenvironment, interferon-gamma, dual promoter system.

An amphipathic helix of vinexin α is necessary for a substrate stiffness-dependent conformational change in vinculin.

Extracellular matrix (ECM) stiffness regulates various cell behaviors, including cell differentiation, proliferation and migration. Vinculin and vinexin α (an isoform encoded by the SORBS3 gene), both of which localize to focal adhesions, cooperatively function as mechanosensors of ECM stiffness. On a rigid ECM, vinexin α interacts with vinculin and induces a conformational change in vinculin to give an 'open' form, which promotes nuclear localization of Yes-associated protein (YAP, also known as YAP1) and transcriptional coactivator with a PDZ-binding motif (TAZ, also known as WWTR1) (hereafter YAP/TAZ). However, the detailed mechanism by which vinexin α induces the conformational change in vinculin has not been revealed. Here, we identify an amphipathic helix named H2 as a novel vinculin-binding site in vinexin α. The H2 helix interacts with the vinculin D1b subdomain and promotes the formation of a talin-vinculin-vinexin α ternary complex. Mutations in the H2 region not only impair the ability of vinexin α to induce the ECM stiffness-dependent conformational change in vinculin but also to promote nuclear localization of YAP/TAZ on rigid ECM. Taken together, these results demonstrate that the H2 helix in vinexin α plays a critical role in ECM stiffness-dependent regulation of vinculin and cell behaviors.

Stochastic ERK activation induced by noise and cell-to-cell propagation regulates cell density-dependent proliferation.

The extracellular signal-regulated kinase (ERK) plays a central role in the signaling cascades of cell growth. Here, we show that stochastic ERK activity pulses regulate cell proliferation rates in a cell density-dependent manner. A fluorescence resonance energy transfer (FRET) biosensor revealed that stochastic ERK activity pulses fired spontaneously or propagated from adjacent cells. Frequency, but not amplitude, of ERK activity pulses exhibited a bell-shaped response to the cell density and correlated with cell proliferation rates. Consistently, synthetic ERK activity pulses generated by a light-switchable CRaf protein accelerated cell proliferation. A mathematical model clarified that 80% and 20% of ERK activity pulses are generated by the noise and cell-to-cell propagation, respectively. Finally, RNA sequencing analysis of cells subjected to the synthetic ERK activity pulses suggested the involvement of serum responsive factor (SRF) transcription factors in the gene expression driven by the ERK activity pulses.

Spatiotemporal ATP Dynamics during AKI Predict Renal Prognosis.

BACKGROUND: Depletion of ATP in renal tubular cells plays the central role in the pathogenesis of kidney diseases. Nevertheless, inability to visualize spatiotemporal in vivo ATP distribution and dynamics has hindered further analysis. METHODS: A novel mouse line systemically expressing an ATP biosensor (an ATP synthase subunit and two fluorophores) revealed spatiotemporal ATP dynamics at single-cell resolution during warm and cold ischemic reperfusion (IR) with two-photon microscopy. This experimental system enabled quantification of fibrosis 2 weeks after IR and assessment of the relationship between the ATP recovery in acute phase and fibrosis in chronic phase. RESULTS: Upon ischemia induction, the ATP levels of proximal tubule (PT) cells decreased to the nadir within a few minutes, whereas those of distal tubule (DT) cells decreased gradually up to 1 hour. Upon reperfusion, the recovery rate of ATP in PTs was slower with longer ischemia. In stark contrast, ATP in DTs was quickly rebounded irrespective of ischemia duration. Morphologic changes of mitochondria in the acute phase support the observation of different ATP dynamics in the two segments. Furthermore, slow and incomplete ATP recovery of PTs in the acute phase inversely correlated with fibrosis in the chronic phase. Ischemia under conditions of hypothermia resulted in more rapid and complete ATP recovery with less fibrosis, providing a proof of concept for use of hypothermia to protect kidney tissues. CONCLUSIONS: Visualizing spatiotemporal ATP dynamics during IR injury revealed higher sensitivity of PT cells to ischemia compared with DT cells in terms of energy metabolism. The ATP dynamics of PTs in AKI might provide prognostic information.

Biliverdin Reductase-A Deficiency Brighten and Sensitize Biliverdin-binding Chromoproteins.

Tissue absorbance, light scattering, and autofluorescence are significantly lower in the near-infrared (NIR) range than in the visible range. Because of these advantages, NIR fluorescent proteins (FPs) are in high demand for in vivo imaging. Nevertheless, application of NIR FPs such as iRFP is still limited due to their dimness in mammalian cells. In contrast to GFP and its variants, iRFP requires biliverdin (BV) as a chromophore. The dimness of iRFP is at least partly due to rapid reduction of BV by biliverdin reductase-A (BLVRA). Here, we established biliverdin reductase-a knockout (Blvra-/-) mice to increase the intracellular BV concentration and, thereby, to enhance iRFP fluorescence intensity. As anticipated, iRFP fluorescence intensity was significantly increased in all examined tissues of Blvra-/- mice. Similarly, the genetically encoded calcium indicator NIR-GECO1, which is engineered based on another NIR FP, mIFP, exhibited a marked increase in fluorescence intensity in mouse embryonic fibroblasts derived from Blvra-/- mice. We expanded this approach to an NIR light-sensing optogenetic tool, the BphP1-PpsR2 system, which also requires BV as a chromophore. Again, deletion of the Blvra gene markedly enhanced the light response in HeLa cells. These results indicate that the Blvra-/- mouse is a versatile tool for the in vivo application of NIR FPs and NIR light-sensing optogenetic tools.Key words: in vivo imaging, near-infrared fluorescent protein, biliverdin, biliverdin reductase, optogenetic tool.

Single-cell quantification of the concentrations and dissociation constants of endogenous proteins.

Kinetic simulation is a useful approach for elucidating complex cell-signaling systems. The numerical simulations required for kinetic modeling in live cells critically require parameters such as protein concentrations and dissociation constants (Kd ). However, only a limited number of parameters have been measured experimentally in living cells. Here we describe an approach 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. First, the mEGFP gene was knocked in at the end of the mitogen-activated protein kinase 1 (MAPK1) gene, encoding extracellular signal-regulated kinase 2 (ERK2), through homology-directed repair or microhomology-mediated end joining. Next, the HaloTag gene was knocked in at the end of the ribosomal S6 kinase 2 (RSK2) gene. We then used fluorescence correlation spectroscopy to measure the protein concentrations of endogenous ERK2-mEGFP and RSK2-HaloTag fusion constructs in living cells, revealing substantial heterogeneities. Moreover, fluorescence cross-correlation spectroscopy analyses revealed temporal changes in the apparent Kd values of the binding between ERK2-mEGFP and RSK2-HaloTag in response to epidermal growth factor stimulation. Our approach presented here provides a robust and efficient method for quantifying endogenous protein concentrations and dissociation constants in living cells.

PAR-3 controls endothelial planar polarity and vascular inflammation under laminar flow.

Impaired cell polarity is a hallmark of diseased tissue. In the cardiovascular system, laminar blood flow induces endothelial planar cell polarity, represented by elongated cell shape and asymmetric distribution of intracellular organelles along the axis of blood flow. Disrupted endothelial planar polarity is considered to be pro-inflammatory, suggesting that the establishment of endothelial polarity elicits an anti-inflammatory response. However, a causative relationship between polarity and inflammatory responses has not been firmly established. Here, we find that a cell polarity protein, PAR-3, is an essential gatekeeper of GSK3ß activity in response to laminar blood flow. We show that flow-induced spatial distribution of PAR-3/aPKCλ and aPKCλ/GSK3ß complexes controls local GSK3ß activity and thereby regulates endothelial planar polarity. The spatial information for GSK3ß activation is essential for flow-dependent polarity to the flow axis, but is not necessary for flow-induced anti-inflammatory response. Our results shed light on a novel relationship between endothelial polarity and vascular homeostasis highlighting avenues for novel therapeutic strategies.

Experimental pathology by intravital microscopy and genetically encoded fluorescent biosensors.

The invention of two-photon excitation microscopes widens the potential application of intravital microscopy (IVM) to the broad field of experimental pathology. Moreover, the recent development of fluorescent protein-based, genetically encoded biosensors provides an ideal tool to visualize the cell function in live animals. We start from a brief review of IVM with two-photon excitation microscopes and genetically encoded biosensors based on the principle of Förster resonance energy transfer (FRET). Then, we describe how IVM using biosensors has revealed the pathogenesis of several disease models.

Efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control of cell signaling.

Optogenetics is a powerful tool to precisely manipulate cell signaling in space and time. For example, protein activity can be regulated by several light-induced dimerization (LID) systems. Among them, the phytochrome B (PhyB)-phytochrome-interacting factor (PIF) system is the only available LID system controlled by red and far-red lights. However, the PhyB-PIF system requires phycocyanobilin (PCB) or phytochromobilin as a chromophore, which must be artificially added to mammalian cells. Here, we report an expression vector that coexpresses HO1 and PcyA with Ferredoxin and Ferredoxin-NADP+ reductase for the efficient synthesis of PCB in the mitochondria of mammalian cells. An even higher intracellular PCB concentration was achieved by the depletion of biliverdin reductase A, which degrades PCB. The PCB synthesis and PhyB-PIF systems allowed us to optogenetically regulate intracellular signaling without any external supply of chromophores. Thus, we have provided a practical method for developing a fully genetically encoded PhyB-PIF system, which paves the way for its application to a living animal.

Two Decades of Genetically Encoded Biosensors Based on Förster Resonance Energy Transfer.

Two decades have passed since the development of the first calcium indicator based on the green fluorescent protein (GFP) and the principle of Förster resonance energy transfer (FRET). During this period, researchers have advanced many novel ideas for the improvement of such genetically encoded FRET biosensors, which have allowed them to expand their targets from small molecules to signaling proteins and physicochemical properties. Although the merits of "genetically encoded" FRET biosensors became clear once various cell lines were established and several transgenic organisms were generated, the road to these developments was not necessarily a smooth one. Moreover, even today the development of new FRET biosensors remains a very labor-intensive, trial-and-error process. Therefore, at this junction, it may be worthwhile to summarize the progress of the FRET biosensor and discuss the future direction of its development and application.Key words: FRET, biosensor, fluorescent protein.

Gliding Basal Cell Migration of the Urothelium during Wound Healing.

Collective cell migration during wound healing has been extensively studied in the epidermis. However, it remains unknown whether the urothelium repairs wounds in a manner similar to the epidermis. By in vivo two-photon excitation microscopy of transgenic mice that express fluorescent biosensors, we studied the collective cell migration of the urothelium in comparison with that of the epidermis. In vivo time-lapse imaging revealed that, even in the absence of a wound, urothelial cells continuously moved and sometimes glided as a sheet over the underlying lamina propria. On abrasion of the epithelium, the migration speed of each epidermal cell was inversely correlated with the distance to the wound edge. Repetitive activation waves of extracellular signal-regulated kinase (ERK) were generated at and propagated away from the wound edge. In contrast, urothelial cells glided as a sheet over the lamina propria without any ERK activation waves. Accordingly, the mitogen-activated protein kinase/ERK kinase inhibitor PD0325901 decreased the migration velocity of the epidermis but not the urothelium. Interestingly, the tyrosine kinase inhibitor dasatinib inhibited migration of the urothelium as well as the epidermis, suggesting that the gliding migration of the urothelium is an active, not a passive, migration. In conclusion, the urothelium glides over the lamina propria to fill wounds in an ERK-independent manner, whereas the epidermis crawls to cover wounds in an ERK-dependent manner.

SH3BP1, an exocyst-associated RhoGAP, inactivates Rac1 at the front to drive cell motility.

The coordination of the several pathways involved in cell motility is poorly understood. Here, we identify SH3BP1, belonging to the RhoGAP family, as a partner of the exocyst complex and establish a physical and functional link between two motility-driving pathways, the Ral/exocyst and Rac signaling pathways. We show that SH3BP1 localizes together with the exocyst to the leading edge of motile cells and that SH3BP1 regulates cell migration via its GAP activity upon Rac1. SH3BP1 loss of function induces abnormally high Rac1 activity at the front, as visualized by in vivo biosensors, and disorganized and instable protrusions, as revealed by cell morphodynamics analysis. Consistently, constitutively active Rac1 mimics the phenotype of SH3BP1 depletion: slow migration and aberrant cell morphodynamics. Our finding that SH3BP1 downregulates Rac1 at the motile-cell front indicates that Rac1 inactivation in this location, as well as its activation by GEF proteins, is a fundamental requirement for cell motility.

A Novel Morphological Marker for the Analysis of Molecular Activities at the Single-cell Level.

For more than a century, hematoxylin and eosin (H&E) staining has been the de facto standard for histological studies. Consequently, the legacy of histological knowledge is largely based on H&E staining. Due to the recent advent of multi-photon excitation microscopy, the observation of live tissue is increasingly being used in many research fields. Adoption of this technique has been further accelerated by the development of genetically encoded biosensors for ions and signaling molecules. However, H&E-based histology has not yet begun to fully utilize in vivo imaging due to the lack of proper morphological markers. Here, we report a genetically encoded fluorescent marker, NuCyM (Nucleus, Cytosol, and Membrane), which is designed to recapitulate H&E staining patterns in vivo. We generated a transgenic mouse line ubiquitously expressing NuCyM by using a ROSA26 bacterial artificial chromosome (BAC) clone. NuCyM evenly marked the plasma membrane, cytoplasm and nucleus in most tissues, yielding H&E staining-like images. In the NuCyM-expressing cells, cell division of a single cell was clearly observed as five basic phases during M phase by three-dimensional imaging. We next crossed NuCyM mice with transgenic mice expressing an ERK biosensor based on the principle of Förster resonance energy transfer (FRET). Using NuCyM, ERK activity in each cell could be extracted from the FRET images. To further accelerate the image analysis, we employed machine learning-based segmentation methods, and thereby automatically quantitated ERK activity in each cell. In conclusion, NuCyM is a versatile cell morphological marker that enables us to grasp histological information as with H&E staining.Key words: in vivo imaging, histology, machine learning, molecular activity.