Explore the vast range of titles available.

Multiplex live imaging enables simultaneous visualization of multiple signaling pathways in living cells, offering real-time insights into complex cellular networks. This methodology is essential in research fields such as cancer biology, where signaling activities exhibit heterogeneity, feedback regulation, crosstalk, and dynamic changes during pathological progression and the acquisition of therapeutic resistance. While conventional biochemical assays advanced our understanding of signaling signatures through static or population-level analyses, they lack the temporal resolution required to capture dynamic events at single-cell resolution. Recent methodological innovations have expanded multiplex live imaging through several strategies. Spectral multiplexing exploits broadened fluorescent protein palettes and optimized biosensor combinations, sometimes coupled with intracellular multiplexing methods that distinguish signals by targeting fluorescence to subcellular compartments. Intercellular multiplexing distributes reporters across cell populations, and temporal multiplexing leverages optical switching to separate signals over time. Additional modalities such as fluorescence anisotropy, fluorescence lifetime, and Raman imaging provide orthogonal readouts. Furthermore, computational approaches reinforce multiplex strategies by improved spectral unmixing, often complemented by deep learning-based algorithms. Collectively, these advances enable simultaneous tracking of multiple signaling pathways within single cells, revealing how diverse inputs are integrated into cellular responses. Here we review current strategies for multiplex live imaging, especially highlighting its applications to cancer signaling networks. Progress in fluorescent biosensor development, imaging technologies, and computational analysis will further promote the exploration of dynamic cellular regulations in basic research and translational medicine.Key words: multiplex live imaging, fluorescent biosensors, signal dynamics, image analysis, cancer heterogeneity

Fluctuation in signal transduction pathways is frequently observed during mammalian development. However, its role in regulating stem cells has not been explored. Here we tracked spatiotemporal ERK MAPK dynamics in human epidermal stem cells. While stem cells and differentiated cells were distinguished by high and low stable basal ERK activity, respectively, we also found cells with pulsatile ERK activity. Transitions from Basalhi-Pulselo (stem) to Basalhi-Pulsehi, Basalmid-Pulsehi, and Basallo-Pulselo (differentiated) cells occurred in expanding keratinocyte colonies and in response to differentiation stimuli. Pharmacological inhibition of ERK induced differentiation only when cells were in the Basalmid-Pulsehi state. Basal ERK activity and pulses were differentially regulated by DUSP10 and DUSP6, leading us to speculate that DUSP6- mediated ERK pulse down-regulation promotes initiation of differentiation, whereas DUSP10-mediated down-regulation of mean ERK activity promotes and stabilizes postcommitment differentiation. Levels of MAPK1/MAPK3 transcripts correlated with DUSP6 and DUSP10 transcripts in individual cells, suggesting that ERK activity is negatively regulated by transcriptional and posttranslational mechanisms. When cells were cultured on a topography that mimics the epidermal−dermal interface, spatial segregation of mean ERK activity and pulses was observed. In vivo imaging of mouse epidermis revealed a patterned distribution of basal cells with pulsatile ERK activity, and down-regulation was linked to the onset of differentiation. Our findings demonstrate that ERK MAPK signal fluctuations link kinase activity to stem cell dynamics.

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. Our skin is our largest organ; it provides a barrier that protects the underlying tissues and internal organs from the external environment and acts as one of our first lines of defense against infection. Both of these roles subject the skin to wear and tear and so it must constantly create new skin cells to replace those lost or damaged. However, if this renewal process goes awry it can lead to excessive cell growth or skin cancer. To avoid this, cells tightly regulate the pathways that stimulate skin renewal. Skin renewal involves growth signals activating an enzyme called ERK. When and where the ERK enzyme is activated is normally tightly regulated, and many kinds of cancer have been linked to ERK becoming active at the wrong time or in the wrong place. Despite the importance of ERK in skin cells, a number of technical challenges have made it difficult to study how these signals are passed from cell to cell. Hiratsuka et al. have now examined genetically altered mice that produce a fluorescent sensor molecule that makes it possible to see ERK activity in living skin cells. The skin of anesthetized mice was observed under a microscope, and time-lapse videos revealed occasional ‘firework-like’ bursts of ERK activity. At first the ERK enzyme was active in a small cluster of skin cells, then ERK activity was seen in the surrounding cells—appearing to spread outwards over the course of several minutes—before the activity stopped. Hiratsuka et al. named this pattern of activity a ‘Spatial Propagation of Radial ERK Activity Distribution’, or SPREAD for short. By studying SPREADs in the skin on the ears and the back of these mice, Hiratsuka et al. learned that these bursts of ERK activity coincided with skin cell growth; the bursts happened more frequently in the areas where the skin cells were dividing. Applying a chemical that stimulates cell division to the skin of the mice triggered more bursts of ERK activity; whereas fewer bursts were observed if Hiratsuka et al. used other chemicals to block the activity of some of the signaling proteins that work upstream of ERK. Further experiments suggested that SPREADs encourage cells to progress through the cycle of events that leads a cell to divide; blocking these bursts caused the cell to pause at the stage just before it would normally divide. Hiratsuka et al. also observed similar patterns of ERK activity moving out like waves from the edges of skin wounds. Further research using similar methods will reveal how growth signals are triggered and propagated in healthy and diseased tissues, not only in the skin but also other organs such as the liver, intestine, and muscles.

Murine dermis contains functionally and spatially distinct fibroblast lineages that cease to proliferate in early postnatal life. Here, we propose a model in which a negative feedback loop between extracellular matrix (ECM) deposition and fibroblast proliferation determines dermal architecture. Virtual‐tissue simulations of our model faithfully recapitulate dermal maturation, predicting a loss of spatial segregation of fibroblast lineages and dictating that fibroblast migration is only required for wound healing. To test this, we performed in vivo live imaging of dermal fibroblasts, which revealed that homeostatic tissue architecture is achieved without active cell migration. In contrast, both fibroblast proliferation and migration are key determinants of tissue repair following wounding. The results show that tissue‐scale coordination is driven by the interdependence of cell proliferation and ECM deposition, paving the way for identifying new therapeutic strategies to enhance skin regeneration. Synopsis In vivo live imaging of dermal fibroblasts combined with mathematical modeling shows that fibroblast behaviour switching between two distinct states—proliferating and depositing ECM—defines dermal architecture. These findings are relevant for identifying new therapeutic strategies for skin regeneration. Tissue‐scale coordination in murine dermis is driven by the interdependence of cell proliferation and ECM deposition. The tissue architecture is set by a negative feedback loop between ECM deposition/remodelling and proliferation. Fibroblast lineages lose segregation with age. Fibroblast migration is the critical discriminator between dermal development and wound healing. Graphical Abstract In vivo live imaging of dermal fibroblasts combined with mathematical modeling shows that fibroblast behaviour switching between two distinct states—proliferating and depositing ECM—defines dermal architecture. These findings are relevant for identifying new therapeutic strategies for skin regeneration.

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. We applied a comprehensive live imaging approach to organoids derived from 10 pancreatic ductal adenocarcinoma (PDAC) patients. By automated image acquisitions and analyses, we found ERK and AMPK activity are important in the early and late phases of organoid growth, respectively.

Extracellular signal-regulated kinase (ERK) regulates multiple cellular functions through distinct activation patterns. Genetically encoded fluorescent probes are instrumental in dissecting the ERK activity dynamics in living cells. Here we modified a previously reported Förster resonance energy transfer (FRET) probe for ERK, EKAREN5 by replacing its mTurquoise2 and YPet sequences with mTurquoise-GL and a synonymous codon variant of YPet, respectively. The modified biosensor, EKAREN5-gl, showed an increased sensitivity to EGF-induced ERK activation responding to a very low dose (20 pg/ml) of EGF stimulation. We quantitatively characterized two FRET-based ERK probes, EKAREN5 and EKAREN5-gl, and a subcellular kinase translocation-based probe, ERK-KTR. We found the three biosensors differently respond to EGF stimulations with different intensity, duration, and latency. Furthermore, we investigated how the minimal EGF-induced ERK activation affects the downstream transcription in HeLa cells by comprehensive transcriptional analysis. We found the minimal ERK activation leads to a distinct transcriptional pattern from those induced by higher ERK activations. Our study highlights the significance of sensitive fluorescent probes to understand cellular signal dynamics and the role of minimal ERK activation in regulating transcription.Key words: fluorescent probe, ERK, FRET, KTR

The epidermis is maintained by multiple stem cell populations whose progeny differentiate along diverse, and spatially distinct, lineages. Here we show that the transcription factor Gata6 controls the identity of the previously uncharacterized sebaceous duct (SD) lineage and identify the Gata6 downstream transcription factor network that specifies a lineage switch between sebocytes and SD cells. During wound healing differentiated Gata6 + cells migrate from the SD into the interfollicular epidermis and dedifferentiate, acquiring the ability to undergo long-term self-renewal and differentiate into a much wider range of epidermal lineages than in undamaged tissue. Our data not only demonstrate that the structural and functional complexity of the junctional zone is regulated by Gata6, but also reveal that dedifferentiation is a previously unrecognized property of post-mitotic, terminally differentiated cells that have lost contact with the basement membrane. This resolves the long-standing debate about the contribution of terminally differentiated cells to epidermal wound repair. Donati et al. show that following skin wounding a differentiated Gata6 + cell population resident in the sebaceous duct migrates to the interfollicular epidermis and reattaches to the basal membrane, dedifferentiating into stem cells.

The 6.6 M w Iburi–Tobu earthquake struck southern Hokkaido, Japan on 6 September 2018. The earthquake triggered widespread slope collapses in the hills near the epicenter, resulting in destructive landslides that killed 36 people. Volcanic deposits covering the region slid downhill in a flow-like manner suggestive of fluidized landslides. Here, we report a distinctive example of liquefaction in the field, which could be a prerequisite for the generation of fluidized landslides triggered by large earthquakes. In the scarp of a typical landslide, an altered halloysite-bearing volcanic layer is observed at a level almost coincident with the sliding surface. The layer is intensely undulating and can be divided into an upper clay-rich layer and a lower pumice-rich layer, suggesting that the altered layer had liquefied as a result of the strong coseismic ground motion. The layer had been soaked by heavy rainfall just one day before the earthquake and could have liquefied, producing a weak and slippery plane, resulting in the catastrophic landslides in this area.

Solar ultraviolet radiation (UVR) is a major source of skin damage, resulting in inflammation, premature ageing, and cancer. While several UVR-induced changes, including extracellular matrix reorganisation and epidermal DNA damage, have been documented, the role of different fibroblast lineages and their communication with immune cells has not been explored. We show that acute and chronic UVR exposure led to selective loss of fibroblasts from the upper dermis in human and mouse skin. Lineage tracing and in vivo live imaging revealed that repair following acute UVR is predominantly mediated by papillary fibroblast proliferation and fibroblast reorganisation occurs with minimal migration. In contrast, chronic UVR exposure led to a permanent loss of papillary fibroblasts, with expansion of fibroblast membrane protrusions partially compensating for the reduction in cell number. Although UVR strongly activated Wnt signalling in skin, stimulation of fibroblast proliferation by epidermal β-catenin stabilisation did not enhance papillary dermis repair. Acute UVR triggered an infiltrate of neutrophils and T cell subpopulations and increased pro-inflammatory prostaglandin signalling in skin. Depletion of CD4- and CD8-positive cells resulted in increased papillary fibroblast depletion, which correlated with an increase in DNA damage, pro-inflammatory prostaglandins, and reduction in fibroblast proliferation. Conversely, topical COX-2 inhibition prevented fibroblast depletion and neutrophil infiltration after UVR. We conclude that loss of papillary fibroblasts is primarily induced by a deregulated inflammatory response, with infiltrating T cells supporting fibroblast survival upon UVR-induced environmental stress.

Mannose has anticancer activity that inhibits cell proliferation and enhances the efficacy of chemotherapy. How mannose exerts its anticancer activity, however, remains poorly understood. Here, using genetically engineered human cancer cells that permit the precise control of mannose metabolic flux, we demonstrate that the large influx of mannose exceeding its metabolic capacity induced metabolic remodeling, leading to the generation of slow-cycling cells with limited deoxyribonucleoside triphosphates (dNTPs). This metabolic remodeling impaired dormant origin firing required to rescue stalled forks by cisplatin, thus exacerbating replication stress. Importantly, pharmacological inhibition of de novo dNTP biosynthesis was sufficient to retard cell cycle progression, sensitize cells to cisplatin, and inhibit dormant origin firing, suggesting dNTP loss-induced genomic instability as a central mechanism for the anticancer activity of mannose. In order to grow and divide, cells require a variety of sugars. Breaking down sugars provides energy for cells to proliferate and allows them to make more complex molecules, such as DNA. Although this principle also applies to cancer cells, a specific sugar called mannose not only inhibits cancer cell division but also makes them more sensitive to chemotherapy. These anticancer effects of mannose are particularly strong in cells lacking a protein known as MPI, which breaks down mannose. Evidence from honeybees suggests that a combination of mannose and low levels of MPI leads to a build-up of a modified form of mannose, called mannose-6-phosphate, within cells. As a result, pathways required to release energy from glucose become disrupted, proving lethal to these insects. However, it was not clear whether the same processes were responsible for the anticancer effects of mannose. To investigate, Harada et al. removed the gene that encodes the MPI protein in two types of human cancer cells. The experiments showed that mannose treatment was not lethal to these cells but overall slowed the cell cycle – a fundamental process for cell growth and division. More detailed biochemical experiments showed that cancer cells with excess mannose-6-phosphate could not produce the molecules required to make DNA. This prevented them from doubling their DNA – a necessary step for cell division – and responding to stress caused by chemotherapy. Harada et al. also noticed that cancer cells lacking MPI did not all react to mannose treatment in exactly the same way. Therefore, future work will address these diverse reactions, potentially providing an opportunity to use the mannose pathway to search for new cancer treatments.