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The ubiquitylation and degradation of the CDK inhibitor p21 in cells irradiated with low dose of ultraviolet (UV) promoted by the DNA polymerase delta processivity factor Proliferating Cell Nuclear Antigen (PCNA) is described.

Toll-like receptors (TLRs) are involved in the recognition of microbial pathogens. A subset of TLRs, TLR7, TLR8 and TLR9, induces antiviral responses by producing interferon-a (lFN-α). Production of lFN-α is dependent on the Toll-interleukin-1 receptor domain-containing adaptor MyD88. Here we show that MyD88 formed a complex with the transcription factor IRF7 but not with IRF3. The death domain of MyD88 interacted with an inhibitory domain of IRF7, and this interaction resulted in activation of the lFN-α-dependent promoters. Furthermore, the adaptor molecule TRAF6 also bound and activated IRF7. Ubiquitin ligase activity of TRAF6 was required for IRF7 activation. These results indicate that TLR-mediated IFN-α induction requires the formation of a complex consisting of MyD88, TRAF6 and IRF7 as well as TRAF6-dependent ubiquitination. [ABSTRACT FROM AUTHOR]

Live imaging techniques have revolutionized our understanding of paracrine signaling, a crucial form of cell-to-cell communication in biological processes. This review examines recent advances in visualizing and tracking paracrine factors through four key stages: secretion from producing cells, diffusion through extracellular space, binding to target cells, and activation of intracellular signaling within target cells. Paracrine factor secretion can be directly visualized by fluorescent protein tagging to ligand, or indirectly by visualizing the cleavage of the transmembrane pro-ligands or plasma membrane fusion of endosomes comprising the paracrine factors. Diffusion of paracrine factors has been studied using techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence decay after photoactivation (FDAP), and single-molecule tracking. Binding of paracrine factors to target cells has been visualized through various biosensors, including GPCR-activation-based (GRAB) sensors and Förster resonance energy transfer (FRET) probes for receptor tyrosine kinases. Finally, activation of intracellular signaling is monitored within the target cells by biosensors for second messengers, transcription factors, and so on. In addition to the imaging tools, the review also highlights emerging optogenetic and chemogenetic tools for triggering the release of paracrine factors, which is essential for associating the paracrine factor secretion to biological outcomes during the bioimaging of paracrine factor signaling.Key words: paracrine signaling, live imaging, biosensors, optogenetics, chemogenetics.

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

Prostaglandin E 2 (PGE 2 ) is a key player in a plethora of physiological and pathological events. Nevertheless, little is known about the dynamics of PGE 2 secretion from a single cell and its effect on the neighboring cells. Here, by observing confluent Madin-Darby canine kidney (MDCK) epithelial cells expressing fluorescent biosensors, we demonstrate that calcium transients in a single cell cause PGE 2 -mediated radial spread of PKA activation (RSPA) in neighboring cells. By in vivo imaging, RSPA was also observed in the basal layer of the mouse epidermis. Experiments with an optogenetic tool revealed a switch-like PGE 2 discharge in response to the increasing cytoplasmic Ca 2+ concentrations. The cell density of MDCK cells correlated with the frequencies of calcium transients and the following RSPA. The extracellular signal-regulated kinase (ERK) activation also enhanced the frequency of RSPA in MDCK and in vivo. Thus, the PGE 2 discharge is regulated temporally by calcium transients and ERK activity., Competing Interests: TW, SY, KT, DT, SN, MM, KT No competing interests declared, (© 2023, Watabe et al.)

Calcium transients drive cells to discharge prostaglandin E 2 (PGE 2 ). We visualized PGE 2 -induced protein kinase A (PKA) activation and quantitated PGE 2 secreted from a single cell by combining fluorescence microscopy and a simulation model. For this purpose, we first prepared PGE 2 -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 PGE 2 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 PGE 2 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 PGE 2 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 PGE 2 upon a single calcium transient to activate PKA in more than 1000 neighboring cells. This model also predicts that the PGE 2 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 PGE 2 .Key words: prostaglandin E 2 , imaging, intercellular communication, biosensor, quantification.

Prostaglandin E 2 (PGE 2 ) promotes tumor progression through evasion of antitumor immunity. In stark contrast to cyclooxygenase-dependent production of PGE 2 , little is known whether PGE 2 secretion is regulated within tumor tissues. Here, we show that VEGF-dependent release of thromboxane A 2 (TXA 2 ) triggers Ca 2+ transients in tumor cells, culminating in PGE 2 secretion and subsequent immune evasion in the early stages of tumorigenesis. Ca 2+ transients caused cPLA2 activation and triggered the arachidonic acid cascade. Ca 2+ transients were monitored as the surrogate marker of PGE 2 secretion. Intravital imaging of Braf V600E mouse melanoma cells revealed that the proportion of cells exhibiting Ca 2+ transients is markedly higher in vivo than in vitro . The TXA 2 receptor was indispensable for the Ca 2+ transients in vivo , high intratumoral PGE 2 concentration, and evasion of antitumor immunity. Notably, treatment with a VEGF receptor antagonist and an anti-VEGF antibody rapidly suppressed Ca 2+ transients and reduced TXA 2 and PGE 2 concentrations in tumor tissues. These results identify the VEGF-TXA 2 axis as a critical promoter of PGE 2 -dependent tumor immune evasion, providing a molecular basis underlying the immunomodulatory effect of anti-VEGF therapies. SIGNIFICANCE: This study identifies the VEGF-TXA 2 axis as a potentially targetable regulator of PGE 2 secretion, which provides novel strategies for prevention and treatment of multiple types of malignancies., (©2021 American Association for Cancer Research.)

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.

The heart is an endocrine organ, as cardiomyocytes (CMs) secrete natriuretic peptide (NP) hormones. Since the discovery of NPs, no other peptide hormones that affect remote organs have been identified from the heart. We identified osteocrin (Ostn) as an osteogenesis/chondrogenesis regulatory hormone secreted from CMs in zebrafish. ostn mutant larvae exhibit impaired membranous and chondral bone formation. The impaired bones were recovered by CM-specific overexpression of OSTN. We analyzed the parasphenoid (ps) as a representative of membranous bones. In the shortened ps of ostn morphants, nuclear Yap1/Wwtr1-dependent transcription was increased, suggesting that Ostn might induce the nuclear export of Yap1/Wwtr1 in osteoblasts. Although OSTN is proposed to bind to NPR3 (clearance receptor for NPs) to enhance the binding of NPs to NPR1 or NPR2, OSTN enhanced C-type NP (CNP)-dependent nuclear export of YAP1/WWTR1 of cultured mouse osteoblasts stimulated with saturable CNP. OSTN might therefore activate unidentified receptors that augment protein kinase G signaling mediated by a CNP-NPR2 signaling axis. These data demonstrate that Ostn secreted from the heart contributes to bone formation as an endocrine hormone., (© 2017. Published by The Company of Biologists Ltd.)

Toll-like receptors (TLRs) are involved in the recognition of microbial pathogens. A subset of TLRs, TLR7, TLR8 and TLR9, induces antiviral responses by producing interferon-alpha (IFN-alpha). Production of IFN-alpha is dependent on the Toll-interleukin-1 receptor domain-containing adaptor MyD88. Here we show that MyD88 formed a complex with the transcription factor IRF7 but not with IRF3. The death domain of MyD88 interacted with an inhibitory domain of IRF7, and this interaction resulted in activation of the IFN-alpha-dependent promoters. Furthermore, the adaptor molecule TRAF6 also bound and activated IRF7. Ubiquitin ligase activity of TRAF6 was required for IRF7 activation. These results indicate that TLR-mediated IFN-alpha induction requires the formation of a complex consisting of MyD88, TRAF6 and IRF7 as well as TRAF6-dependent ubiquitination.