The neocortex is essential for higher-order brain functions such as cognition, perception, language, and motor control. Although extracellular signal-regulated kinase (ERK) activity in neurons has been well studied for its role in memory formation, its activity patterns in the awake mice brain remain unclear. This study aimed to investigate the spatiotemporal dynamics of ERK activity in neocortical neurons of awake mice using in vivo imaging. We used Förster resonance energy transfer (FRET)-based biosensor and two-photon microscopy to examine ERK activity in vivo through a cranial window. Visual cortical neurons exhibited NMDA receptor-dependent ERK activation in response to visual stimuli. Beyond stimulus-induced activation, we discovered spontaneous and stochastic ERK activation patterns categorized as single, multi-pulsatile, and sustained activations, which were consistent across various cortical regions, including the visual and somatosensory cortices. Furthermore, synchronized ERK activation among neurons was observed, suggesting a coordinated ERK activation mechanism within neuronal networks that may contribute to sensory information processing. These findings reveal a highly dynamic ERK activation in neocortical cells and provide new insights regarding the functional role of ERK in the awake brain.

ABSTRACT Duration of nightly rest is a trait that varies between individuals, influenced by a complex interplay between multiple genetic and environmental factors. The central circadian clock that orchestrates daily rhythm in behavior/sleep resides in the suprachiasmatic nucleus (SCN). Yet, how the SCN encodes the “length” of the circadian rest phase (ρ) remains an open question. Here we demonstrate that the unique G-protein-subtype Gz contributes to this process by sculpting the waveform of the circadian cAMP-PKA activity rhythm within the SCN. Genetic deletion and subsequent rescue of Gz expression reversibly altered the ρ duration, shifting it from ∼10 h in Gz +/+ mice to ∼7.5 h in Gz -/-; mice, accompanied by proportional changes in peak cAMP-PKA activity duration and transcriptome remodeling in the SCN. Notably, intra-SCN cAMP activation led to behavioral rest, with Gz specifically shaping this response without affecting 24-h rhythmicity. These findings suggest that the SCN is not merely a rhythm-generator, but actively allocates the ρ-rest period via Gz signaling.

The discovery of the senescence-associated secretory phenotype (SASP) has reshaped our understanding of cellular senescence, shifting its role from a solely tumor-suppressive mechanism to a potential driver of chronic inflammation and age-related diseases. Accordingly, senolytic drugs, which selectively eliminate senescent cells, have garnered considerable interest due to promising preclinical studies. However, concerns remain regarding the reproducibility and generalizability of these findings. In this cross-laboratory study, we rigorously tested the senolytic efficacy of a GLS1 inhibitor and an anti-PD-1 antibody—agents previously reported to reduce the burden of p16 INK4a -positive senescent cells and improve health outcomes in aged mice. Contrary to earlier reports, our study demonstrates that neither GLS1 inhibition nor PD-1 blockade significantly reduced p16 INK4a -positive cell burden or improved aging-related health parameters. Although we do not seek to discredit prior work, our results underscore the need for rigorous design, standardized protocols, and independent validation to ensure reliable senolytics before clinical translation.

Current fluorescent protein-based multiplexed cell labeling techniques suffer from limited discrimination power due to stochastic color selection and large gene sizes from tandem repeats of multiple fluorescent proteins. We developed Caterpie, a rationally designed system using engineered split fluorescent proteins that enables deterministic identification of 20 distinct cell populations with 97% accuracy and reduced gene sizes. Through computational structure-guided design, we engineered enhanced split mNeonGreen3A and split sfCherry3C variants that achieve performance comparable to split CFP2, the best-performing split fluorescent protein. Our systematic library of trichromatic 11th β-strand tags with up to 12 tandem repeats enables predictable, high-fidelity labeling for precise cell targeting. This technology addresses critical limitations in simultaneous identification of multiple defined cell populations.

Collective cell migration is coordinated by the front-to-rear intercellular propagation of EGFR-Ras-ERK pathway activation. However, the molecular mechanisms integrating front-to-rear information into this intercellular signaling cascade, particularly the determinants of cellular front-side specification, remain elusive. We visualized the activity of EGFR, Ras, Rac1 and Rab5A (hereafter Rab5) by using FRET biosensors and chemogenetic tools. Whereas EGFR activation was uniformly observed within cells, Ras activation was biased to the front side within cells. The polarized Ras activation depended on Merlin and Rac1, which also showed front-biased activation. Furthermore, Rab5, a crucial regulator of cell migration, demonstrated similar front-biased activation and was found to function downstream of Ras while being necessary for Rac1 activation. Thus, the positive feedback loop consisting of Ras, Rab5 and Rac1 is activated primarily at the front of collectively migrating cells. These findings offer new spatio-temporal insight into processing front-rear information during collective cell migration.

The behavior of cells is governed by signals originating from their local environment, including mechanical forces exerted on the cells. Forces are transduced by mechanosensitive proteins, which can impinge on signaling cascades that are also activated by growth factors. We investigated the cross-talk between mechanical and biochemical signals in the regulation of intracellular signaling networks in epithelial monolayers. Phosphoproteomic and transcriptomic analyses on epithelial monolayers subjected to mechanical strain revealed the activation of extracellular signal-regulated kinase (ERK) downstream of the epidermal growth factor receptor (EGFR) as a predominant strain-induced signaling event. Strain-induced EGFR-ERK signaling depended on mechanosensitive E-cadherin adhesions. Proximity labeling showed that the metalloproteinase ADAM17, an enzyme that mediates shedding of soluble EGFR ligands, was closely associated with E-cadherin. A probe that we developed to monitor ADAM-mediated shedding demonstrated that mechanical strain induced ADAM activation. Mechanically induced ADAM activation was essential for mechanosensitive, E-cadherin-dependent EGFR-ERK signaling. Together, our data demonstrate that mechanical strain transduced by E-cadherin adhesion triggers the shedding of EGFR ligands that stimulate downstream ERK activity. Our findings illustrate how mechanical signals and biochemical ligands can operate within a linear signaling cascade.

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

Collective cell migration (CCM) is characterized by the coordinated movement of cell groups while maintaining cell-to-cell cohesion. Despite extensive research on CCM, the collective migration of mature epithelial cells over the extracellular matrix in response to external stimuli has not been reported. Using intravital imaging in mice, we identified urothelial CCM (UCCM) triggered by immunogenic substances, including bladder cancer cells (MB49) and uropathogenic Escherichia coli (UPEC). Integrin signaling inhibitors suppress UCCM, significantly enhancing MB49 tumor growth and UPEC bladder infection. UCCM initiation involves Toll-like receptor 4 (TLR4), we designated this TLR4-associated UCCM as the urothelial collective-gliding response (UCGR). Downstream of integrin signaling, urothelial matrix metalloproteinases (MMP)-8 and MMP-9 mediate UCGR. Intravesical instillation of these factors accelerates UCCM and inhibits tumor growth and infection. UCGR may represent a TLR4-associated defense mechanism, offering potential therapeutic strategies for bladder disorders such as refractory cystitis and recurrent non-muscle invasive bladder cancer after endoscopic resection.

Intricate branching patterns emerge in internal organs due to the recurrent occurrence of simple deformations in epithelial tissues. During murine lung development, epithelial cells in distal tips of the single tube require fibroblast growth factor (FGF) signals emanating from their surrounding mesenchyme to form repetitive tip bifurcations. However, it remains unknown how the cells employ FGF signaling to convert their behaviors to achieve the recursive branching processes. Here, we show a mechano-chemical regulatory system underlying lung branching morphogenesis, orchestrated by extracellular signal-regulated kinase (ERK) as a downstream driver of FGF signaling. We found that tissue-scale curvature regulated ERK activity in the lung epithelium using two-photon live cell imaging and mechanical perturbations. ERK activation occurs specifically in epithelial tissues exhibiting positive curvature, regardless of whether the change in curvature was attributable to morphogenesis or perturbations. Moreover, ERK activation accelerates actin polymerization preferentially at the apical side of cells, mechanically contributing to the extension of the apical membrane, culminating in a reduction of epithelial tissue curvature. These results indicate the existence of a negative feedback loop between tissue curvature and ERK activity that transcends spatial scales. Our mathematical model confirms that this regulatory mechanism is sufficient to generate the recursive branching processes. Taken together, we propose that ERK orchestrates a curvature feedback loop pivotal to the self-organized patterning of tissues.

Canonical epidermal growth factor (EGF) receptor (EGFR) activation involves the binding of seven EGFR ligands (EGFRLs); however, their extracellular dynamics remain elusive. Here, employing fluorescent probes and a tool for triggering ectodomain shedding, we show that epiregulin (EREG), a low-affinity EGFRL, rapidly and efficiently activates EGFR in Madin-Darby canine kidney (MDCK) epithelial cells and mouse epidermis. During collective cell migration, EGFR and extracellular signal-regulated kinase (ERK) activation waves propagate in an a disintegrin and metalloprotease 17 (ADAM17) sheddase- and EGFRL-dependent manner. Upon induced EGFRL shedding, low-affinity ligands EREG and amphiregulin (AREG) mediate faster and broader ERK waves than high-affinity ligands. Tight/adherens junction integrity is essential for ERK activation propagation, suggesting that tight intercellular spaces prefer the low-affinity EGFRLs for efficient signal transmission. In EREG-deficient mice, ERK wave propagation and cell migration were impaired during skin wound repair. We additionally show that heparin-binding EGF-like growth factor (HBEGF) primarily promotes surrounding cell motility. 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.

Patient-derived organoids represent a novel platform to recapitulate the cancer cells in the patient tissue. While cancer heterogeneity has been extensively studied by a number of omics approaches, little is known about the spatiotemporal kinase activity dynamics. Here we applied a live imaging approach to organoids derived from 10 pancreatic ductal adenocarcinoma (PDAC) patients to comprehensively understand their heterogeneous growth potential and drug responses. By automated wide-area image acquisitions and analyses, the PDAC cells were non-selectively observed to evaluate their heterogeneous growth patterns. We monitored single-cell ERK and AMPK activities to relate cellular dynamics to molecular dynamics. Furthermore, we evaluated two anti-cancer drugs, a MEK inhibitor, PD0325901, and an autophagy inhibitor, hydroxychloroquine (HCQ), by our analysis platform. Our analyses revealed a phase-dependent regulation of PDAC organoid growth, where ERK activity is necessary for the early phase and AMPK activity is necessary for the late stage of organoid growth. Consistently, we found PD0325901 and HCQ target distinct organoid populations, revealing their combination is widely effective to the heterogeneous cancer cell population in a range of PDAC patient-derived organoid lines. Together, our live imaging quantitatively characterized the growth and drug sensitivity of human PDAC organoids at multiple levels: in single cells, single organoids, and individual patients. This study will pave the way for understanding the cancer heterogeneity and promote the development of new drugs that eradicate intractable cancer.

Acute kidney injury (AKI), typically caused by ischemia, is a common clinical complication with a poor prognosis. Although proteinuria is an important prognostic indicator of AKI, the underlying causal mechanism remains unclear. In vitro studies suggest that podocytes have high ATP demands to maintain their structure and function, however, analyzing their ATP dynamics in living kidneys has been technically challenging. Here, using intravital imaging to visualize a FRET-based ATP biosensor expressed systemically in female mice due to their suitability for glomerular imaging, we monitor the in vivo ATP dynamics in podocytes during ischemia reperfusion injury. ATP levels decrease during ischemia, but recover after reperfusion in podocytes, exhibiting better recovery than in glomerular endothelial cells. However, prolonged ischemia results in insufficient ATP recovery in podocytes, which is inversely correlated with mitochondrial fragmentation and foot process effacement during the chronic phase. Furthermore, preventing mitochondrial fission via pharmacological inhibition ameliorates podocyte injury in vitro, ex vivo, and in vivo. Thus, these findings provide several insights into how ATP depletion and mitochondrial fragmentation contribute to podocyte injury after ischemic AKI and could potentially be therapeutic targets. The involvement of glomerular injury in acute kidney injury (AKI) has not been fully elucidated. Here, authors visualize impaired ATP recovery in podocytes in a murine model of ischemic AKI using intravital ATP imaging and propose accelerated mitochondrial fission as a key factor of ischemic podocyte injury.

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology (PD12)1 May 2024PD12-05 IMMUNOLOGICAL SIGNIFICANCE OF NEWLY DISCOVERED UROTHELIAL COLLECTIVE MIGRATION Takeshi Sano, Zhang Ning, Ryosuke Ikeuchi, Hideaki Takada, Kenji Nakamura, Toru Sakatani, Akihiro Hamada, Yuki Kita, Michiyuki Matsuda, and Takashi Kobayashi Takeshi SanoTakeshi Sano , Zhang NingZhang Ning , Ryosuke IkeuchiRyosuke Ikeuchi , Hideaki TakadaHideaki Takada , Kenji NakamuraKenji Nakamura , Toru SakataniToru Sakatani , Akihiro HamadaAkihiro Hamada , Yuki KitaYuki Kita , Michiyuki MatsudaMichiyuki Matsuda , and Takashi KobayashiTakashi Kobayashi View All Author Informationhttps://doi.org/10.1097/01.JU.0001008772.30001.48.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Intravital imaging of the mouse bladder identified a process of urothelial collective cell migration (uCCM) caused by intravesical administration of various immunogenic substances. This study aimed to investigate the mechanism and immunological significance of this newly discovered urothelial migration. METHODS: J96 (uropathogenic E. coli strain), lipopolysaccharide (LPS), and Bacillus Calmette-Guérin (BCG, Tokyo strain) were individually injected into the bladder of C57BL/6 mice expressing a cyan fluorescent protein in their cell nuclei. While mice were under general anesthesia induced by isoflurane, we used two-photon excitation microscopy, which enables deep tissue imaging, to visualize the urothelium. Dasatinib, a Src inhibitor, and PF-573228, a focal adhesion kinase (FAK) inhibitor, were individually administered intravenously or orally. TAK-242, a Toll-like receptor 4 (TLR4) inhibitor, was administered intravenously, followed by intravesical administration. To induce bladder infection, J96 was injected into the bladder and allowed to incubate for 30 minutes. Bladder extraction took place after six hours for the analysis of J96 infection. Trajectories and velocities of uCCM were analyzed using MetaMorph software. RESULTS: uCCM started 4 to 6 hours after intravesical injection of all the immunogenic substances, including J96, LPS, and BCG (Figure 1). The mean velocity of uCCM induced by these substances was 39.6 μm/h (range 34.3-45.2) while uCCM did not occur without treatment. Remarkably, both intravenous and oral administration of dasatinib and PF-573228 effectively delayed J96-induced uCCM. Intriguingly, the inhibition of uCCM through oral administration of dasatinib and PF-573228 significantly heightened J96 infection. Furthermore, TAK-242 treatment almost completely abrogated LPS-induced uCCM. CONCLUSIONS: uCCM induced by immunogenic substances appeared to depend on the integrin-FAK signaling pathway. Inhibition of this migration enhanced the infection caused by uropathogenic E. coli, suggesting that it may play an immunological role against bladder infection. This immunological function may be associated with the innate immune response, possibly mediated by pattern recognition receptors, such as TLR4. Thus, enhancement of this migration may be a novel strategy against various infectious diseases of the bladder. Download PPT Source of Funding: None © 2024 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 211Issue 5SMay 2024Page: e257 Advertisement Copyright & Permissions© 2024 by American Urological Association Education and Research, Inc.Metrics Author Information Takeshi Sano More articles by this author Zhang Ning More articles by this author Ryosuke Ikeuchi More articles by this author Hideaki Takada More articles by this author Kenji Nakamura More articles by this author Toru Sakatani More articles by this author Akihiro Hamada More articles by this author Yuki Kita More articles by this author Michiyuki Matsuda More articles by this author Takashi Kobayashi More articles by this author Expand All Advertisement PDF downloadLoading ...