Artykuły w czasopismach: „Live Cell Imaging Biosensors”

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Yoon, Sangpil, Yijia Pan, Kirk Shung i Yingxiao Wang. "FRET-Based Ca2+ Biosensor Single Cell Imaging Interrogated by High-Frequency Ultrasound". Sensors 20, nr 17 (3.09.2020): 4998. http://dx.doi.org/10.3390/s20174998.

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Fluorescence resonance energy transfer (FRET)-based biosensors have advanced live cell imaging by dynamically visualizing molecular events with high temporal resolution. FRET-based biosensors with spectrally distinct fluorophore pairs provide clear contrast between cells during dual FRET live cell imaging. Here, we have developed a new FRET-based Ca2+ biosensor using EGFP and FusionRed fluorophores (FRET-GFPRed). Using different filter settings, the developed biosensor can be differentiated from a typical FRET-based Ca2+ biosensor with ECFP and YPet (YC3.6 FRET Ca2+ biosensor, FRET-CFPYPet). A high-frequency ultrasound (HFU) with a carrier frequency of 150 MHz can target a subcellular region due to its tight focus smaller than 10 µm. Therefore, HFU offers a new single cell stimulations approach for FRET live cell imaging with precise spatial resolution and repeated stimulation for longitudinal studies. Furthermore, the single cell level intracellular delivery of a desired FRET-based biosensor into target cells using HFU enables us to perform dual FRET imaging of a cell pair. We show that a cell pair is defined by sequential intracellular delivery of the developed FRET-GFPRed and FRET-CFPYPet into two target cells using HFU. We demonstrate that a FRET-GFPRed exhibits consistent 10–15% FRET response under typical ionomycin stimulation as well as under a new stimulation strategy with HFU.

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Snell, Nicole, Vishnu Rao, Kendra Seckinger, Junyi Liang, Jenna Leser, Allison Mancini i M. Rizzo. "Homotransfer FRET Reporters for Live Cell Imaging". Biosensors 8, nr 4 (11.10.2018): 89. http://dx.doi.org/10.3390/bios8040089.

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Förster resonance energy transfer (FRET) between fluorophores of the same species was recognized in the early to mid-1900s, well before modern heterotransfer applications. Recently, homotransfer FRET principles have re-emerged in biosensors that incorporate genetically encoded fluorescent proteins. Homotransfer offers distinct advantages over the standard heterotransfer FRET method, some of which are related to the use of fluorescence polarization microscopy to quantify FRET between two fluorophores of identical color. These include enhanced signal-to-noise, greater compatibility with other optical sensors and modulators, and new design strategies based upon the clustering or dimerization of singly-labeled sensors. Here, we discuss the theoretical basis for measuring homotransfer using polarization microscopy, procedures for data collection and processing, and we review the existing genetically-encoded homotransfer biosensors.

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Secilmis, Melike, Hamza Yusuf Altun, Johannes Pilic, Yusuf Ceyhun Erdogan, Zeynep Cokluk, Busra Nur Ata, Gulsah Sevimli i in. "A Co-Culture-Based Multiparametric Imaging Technique to Dissect Local H2O2 Signals with Targeted HyPer7". Biosensors 11, nr 9 (14.09.2021): 338. http://dx.doi.org/10.3390/bios11090338.

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Multispectral live-cell imaging is an informative approach that permits detecting biological processes simultaneously in the spatial and temporal domain by exploiting spectrally distinct biosensors. However, the combination of fluorescent biosensors with distinct spectral properties such as different sensitivities, and dynamic ranges can undermine accurate co-imaging of the same analyte in different subcellular locales. We advanced a single-color multiparametric imaging method, which allows simultaneous detection of hydrogen peroxide (H2O2) in multiple cell locales (nucleus, cytosol, mitochondria) using the H2O2 biosensor HyPer7. Co-culturing of endothelial cells stably expressing differentially targeted HyPer7 biosensors paved the way for co-imaging compartmentalized H2O2 signals simultaneously in neighboring cells in a single experimental setup. We termed this approach COMPARE IT, which is an acronym for co-culture-based multiparametric imaging technique. Employing this approach, we detected lower H2O2 levels in mitochondria of endothelial cells compared to the cell nucleus and cytosol under basal conditions. Upon administering exogenous H2O2, the cytosolic and nuclear-targeted probes displayed similarly slow and moderate HyPer7 responses, whereas the mitochondria-targeted HyPer7 signal plateaued faster and reached higher amplitudes. Our results indicate striking differences in mitochondrial H2O2 accumulation of endothelial cells. Here, we present the method’s potential as a practicable and informative multiparametric live-cell imaging technique.

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Tiruthani, Karthik, Adam Mischler, Shoeb Ahmed, Jessica Mahinthakumar, Jason M. Haugh i Balaji M. Rao. "Design and evaluation of engineered protein biosensors for live-cell imaging of EGFR phosphorylation". Science Signaling 12, nr 584 (4.06.2019): eaap7584. http://dx.doi.org/10.1126/scisignal.aap7584.

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Live-cell fluorescence microscopy is broadly applied to study the dynamics of receptor-mediated cell signaling, but the availability of intracellular biosensors is limited. A biosensor based on the tandem SH2 domains from phospholipase C–γ1 (PLCγ1), tSH2-WT, has been used to measure phosphorylation of the epidermal growth factor receptor (EGFR). Here, we found that tSH2-WT lacked specificity for phosphorylated EGFR, consistent with the known promiscuity of SH2 domains. Further, EGF-stimulated membrane recruitment of tSH2-WT differed qualitatively from the expected kinetics of EGFR phosphorylation. Analysis of a mathematical model suggested, and experiments confirmed, that the high avidity of tSH2-WT resulted in saturation of its target and interference with EGFR endocytosis. To overcome the apparent target specificity and saturation issues, we implemented two protein engineering strategies. In the first approach, we screened a combinatorial library generated by random mutagenesis of the C-terminal SH2 domain (cSH2) of PLCγ1 and isolated a mutant form (mSH2) with enhanced specificity for phosphorylated Tyr992 (pTyr992) of EGFR. A biosensor based on mSH2 closely reported the kinetics of EGFR phosphorylation but retained cross-reactivity similar to tSH2-WT. In the second approach, we isolated a pTyr992-binding protein (SPY992) from a combinatorial library generated by mutagenesis of the Sso7d protein scaffold. Compared to tSH2-WT and mSH2, SPY992 exhibited superior performance as a specific, moderate-affinity biosensor. We extended this approach to isolate a biosensor for EGFR pTyr1148 (SPY1148). This approach of integrating theoretical considerations with protein engineering strategies can be generalized to design and evaluate suitable biosensors for various phospho-specific targets.

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Houser, Mei CQ, Steven S. Hou, Florian Perrin, Yuliia Turchyna, Brian J. Bacskai, Oksana Berezovska i Masato Maesako. "A Novel NIR-FRET Biosensor for Reporting PS/γ-Secretase Activity in Live Cells". Sensors 20, nr 21 (22.10.2020): 5980. http://dx.doi.org/10.3390/s20215980.

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Presenilin (PS)/γ-secretase plays a pivotal role in essential cellular events via proteolytic processing of transmembrane proteins that include APP and Notch receptors. However, how PS/γ-secretase activity is spatiotemporally regulated by other molecular and cellular factors and how the changes in PS/γ-secretase activity influence signaling pathways in live cells are poorly understood. These questions could be addressed by engineering a new tool that enables multiplexed imaging of PS/γ-secretase activity and additional cellular events in real-time. Here, we report the development of a near-infrared (NIR) FRET-based PS/γ-secretase biosensor, C99 720-670 probe, which incorporates an immediate PS/γ-secretase substrate APP C99 with miRFP670 and miRFP720 as the donor and acceptor fluorescent proteins, respectively. Extensive validation demonstrates that the C99 720-670 biosensor enables quantitative monitoring of endogenous PS/γ-secretase activity on a cell-by-cell basis in live cells (720/670 ratio: 2.47 ± 0.66 (vehicle) vs. 3.02 ± 1.17 (DAPT), ** p < 0.01). Importantly, the C99 720-670 and the previously developed APP C99 YPet-Turquoise-GL (C99 Y-T) biosensors simultaneously report PS/γ-secretase activity. This evidences the compatibility of the C99 720-670 biosensor with cyan (CFP)-yellow fluorescent protein (YFP)-based FRET biosensors for reporting other essential cellular events. Multiplexed imaging using the novel NIR biosensor C99 720-670 would open a new avenue to better understand the regulation and consequences of changes in PS/γ-secretase activity.

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Woehler, Andrew. "Simultaneous Quantitative Live Cell Imaging of Multiple FRET-Based Biosensors". PLoS ONE 8, nr 4 (16.04.2013): e61096. http://dx.doi.org/10.1371/journal.pone.0061096.

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Vilchez Mercedes, Samuel A., Ian Eder, Mona Ahmed, Ninghao Zhu i Pak Kin Wong. "Optimizing locked nucleic acid modification in double-stranded biosensors for live single cell analysis". Analyst 147, nr 4 (2022): 722–33. http://dx.doi.org/10.1039/d1an01802g.

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Banerjee, Swayoma, Luis Rene Garcia i Wayne K. Versaw. "Quantitative Imaging of FRET-Based Biosensors for Cell- and Organelle-Specific Analyses in Plants". Microscopy and Microanalysis 22, nr 2 (16.02.2016): 300–310. http://dx.doi.org/10.1017/s143192761600012x.

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AbstractGenetically encoded Förster resonance energy transfer (FRET)-based biosensors have been used to report relative concentrations of ions and small molecules, as well as changes in protein conformation, posttranslational modifications, and protein–protein interactions. Changes in FRET are typically quantified through ratiometric analysis of fluorescence intensities. Here we describe methods to evaluate ratiometric imaging data acquired through confocal microscopy of a FRET-based inorganic phosphate biosensor in different cells and subcellular compartments of Arabidopsis thaliana. Linear regression was applied to donor, acceptor, and FRET-derived acceptor fluorescence intensities obtained from images of multiple plants to estimate FRET ratios and associated location-specific spectral correction factors with high precision. FRET/donor ratios provided a combination of high dynamic range and precision for this biosensor when applied to the cytosol of both root and leaf cells, but lower precision when this ratiometric method was applied to chloroplasts. We attribute this effect to quenching of donor fluorescence because high precision was achieved with FRET/acceptor ratios and thus is the preferred ratiometric method for this organelle. A ligand-insensitive biosensor was also used to distinguish nonspecific changes in FRET ratios. These studies provide a useful guide for conducting quantitative ratiometric studies in live plants that is applicable to any FRET-based biosensor.

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Dobrzyński, Maciej, Marc-Antoine Jacques i Olivier Pertz. "Mining single-cell time-series datasets with Time Course Inspector". Bioinformatics 36, nr 6 (14.11.2019): 1968–69. http://dx.doi.org/10.1093/bioinformatics/btz846.

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Abstract Summary Thanks to recent advances in live cell imaging of biosensors, microscopy experiments can generate thousands of single-cell time-series. To identify sub-populations with distinct temporal behaviours that correspond to different cell fates, we developed Time Course Inspector (TCI)—a unique tool written in R/Shiny to combine time-series analysis with clustering. With TCI it is convenient to inspect time-series, plot different data views and remove outliers. TCI facilitates interactive exploration of various hierarchical clustering and cluster validation methods. We showcase TCI by analysing a single-cell signalling time-series dataset acquired using a fluorescent biosensor. Availability and implementation https://github.com/pertzlab/shiny-timecourse-inspector. Supplementary information Supplementary data are available at Bioinformatics online.

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Valetdinova, Kamila R., Tuyana B. Malankhanova, Suren M. Zakian i Sergey P. Medvedev. "The Cutting Edge of Disease Modeling: Synergy of Induced Pluripotent Stem Cell Technology and Genetically Encoded Biosensors". Biomedicines 9, nr 8 (5.08.2021): 960. http://dx.doi.org/10.3390/biomedicines9080960.

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The development of cell models of human diseases based on induced pluripotent stem cells (iPSCs) and a cell therapy approach based on differentiated iPSC derivatives has provided a powerful stimulus in modern biomedical research development. Moreover, it led to the creation of personalized regenerative medicine. Due to this, in the last decade, the pathological mechanisms of many monogenic diseases at the cell level have been revealed, and clinical trials of various cell products derived from iPSCs have begun. However, it is necessary to reach a qualitatively new level of research with cell models of diseases based on iPSCs for more efficient searching and testing of drugs. Biosensor technology has a great application prospect together with iPSCs. Biosensors enable researchers to monitor ions, molecules, enzyme activities, and channel conformation in live cells and use them in live imaging and drug screening. These probes facilitate the measurement of steady-state concentrations or activity levels and the observation and quantification of in vivo flux and kinetics. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of the false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the benefits of using biosensors in drug screening. Here, we discuss the possibilities of using biosensor technology in combination with cell models based on human iPSCs and gene editing systems. Furthermore, we focus on the current achievements and problems of using these methods.

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Tany, Ryosuke, Yuhei Goto, Yohei Kondo i Kazuhiro Aoki. "Quantitative live-cell imaging of GPCR downstream signaling dynamics". Biochemical Journal 479, nr 8 (21.04.2022): 883–900. http://dx.doi.org/10.1042/bcj20220021.

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G-protein-coupled receptors (GPCRs) play an important role in sensing various extracellular stimuli, such as neurotransmitters, hormones, and tastants, and transducing the input information into the cell. While the human genome encodes more than 800 GPCR genes, only four Gα-proteins (Gαs, Gαi/o, Gαq/11, and Gα12/13) are known to couple with GPCRs. It remains unclear how such divergent GPCR information is translated into the downstream G-protein signaling dynamics. To answer this question, we report a live-cell fluorescence imaging system for monitoring GPCR downstream signaling dynamics. Genetically encoded biosensors for cAMP, Ca2+, RhoA, and ERK were selected as markers for GPCR downstream signaling, and were stably expressed in HeLa cells. GPCR was further transiently overexpressed in the cells. As a proof-of-concept, we visualized GPCR signaling dynamics of five dopamine receptors and 12 serotonin receptors, and found heterogeneity between GPCRs and between cells. Even when the same Gα proteins were known to be coupled, the patterns of dynamics in GPCR downstream signaling, including the signal strength and duration, were substantially distinct among GPCRs. These results suggest the importance of dynamical encoding in GPCR signaling.

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Su, Yichi, i Ming C. Hammond. "RNA-based fluorescent biosensors for live cell imaging of small molecules and RNAs". Current Opinion in Biotechnology 63 (czerwiec 2020): 157–66. http://dx.doi.org/10.1016/j.copbio.2020.01.001.

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Terutsuki, Daigo, Hidefumi Mitsuno i Ryohei Kanzaki. "3D-Printed Bubble-Free Perfusion Cartridge System for Live-Cell Imaging". Sensors 20, nr 20 (12.10.2020): 5779. http://dx.doi.org/10.3390/s20205779.

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The advent of 3D-printing technologies has had a significant effect on the development of medical and biological devices. Perfusion chambers are widely used for live-cell imaging in cell biology research; however, air-bubble invasion is a pervasive problem in perfusion systems. Although 3D printing allows the rapid fabrication of millifluidic and microfluidic devices with high resolution, little has been reported on 3D-printed fluidic devices with bubble trapping systems. Herein, we present a 3D-printed millifluidic cartridge system with bent and flat tapered flow channels for preventing air-bubble invasion, irrespective of bubble volume and without the need for additional bubble-removing devices. This system realizes bubble-free perfusion with a user-friendly interface and no-time-penalty manufacturing processes. We demonstrated the bubble removal capability of the cartridge by continually introducing air bubbles with different volumes during the calcium imaging of Sf21 cells expressing insect odorant receptors. Calcium imaging was conducted using a low-magnification objective lens to show the versatility of the cartridge for wide-area observation. We verified that the cartridge could be used as a chemical reaction chamber by conducting protein staining experiments. Our cartridge system is advantageous for a wide range of cell-based bioassays and bioanalytical studies, and can be easily integrated into portable biosensors.

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Kellenberger, Colleen A., Chen Chen, Aaron T. Whiteley, Daniel A. Portnoy i Ming C. Hammond. "RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messenger Cyclic di-AMP". Journal of the American Chemical Society 137, nr 20 (15.05.2015): 6432–35. http://dx.doi.org/10.1021/jacs.5b00275.

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TSE, Ho-Yin, Chi Shun Yeung, Chun Yin Lau, Man Yee Cheung, Jianyu Guan, Md Khairul Islam, Paul T. Anastas i Shao-Yuan Leu. "One-pot synthesis to prepare lignin/photoacid nanohybrids for multifunctional biosensors and photo-triggered singlet oxygen generation". Green Chemistry 24, nr 7 (2022): 2904–18. http://dx.doi.org/10.1039/d2gc00196a.

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This study presents a one-pot synthesis approach for a sustainable lignin/photoacid nanohybrid multifunctional biosensor (AL-Por-PP) for fluorescent live cell imaging, bisulfite detection and photo-trigger singlet oxygen generation.

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Kim, Namdoo, Seunghan Shin i Se Won Bae. "cAMP Biosensors Based on Genetically Encoded Fluorescent/Luminescent Proteins". Biosensors 11, nr 2 (31.01.2021): 39. http://dx.doi.org/10.3390/bios11020039.

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Cyclic adenosine monophosphate (cAMP) plays a key role in signal transduction pathways as a second messenger. Studies on the cAMP dynamics provided useful scientific insights for drug development and treatment of cAMP-related diseases such as some cancers and prefrontal cortex disorders. For example, modulation of cAMP-mediated intracellular signaling pathways by anti-tumor drugs could reduce tumor growth. However, most early stage tools used for measuring the cAMP level in living organisms require cell disruption, which is not appropriate for live cell imaging or animal imaging. Thus, in the last decades, tools were developed for real-time monitoring of cAMP distribution or signaling dynamics in a non-invasive manner. Genetically-encoded sensors based on fluorescent proteins and luciferases could be powerful tools to overcome these drawbacks. In this review, we discuss the recent genetically-encoded cAMP sensors advances, based on single fluorescent protein (FP), Föster resonance energy transfer (FRET), single luciferase, and bioluminescence resonance energy transfer (BRET) for real-time non-invasive imaging.

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Biran, Israel, David M. Rissin, Eliora Z. Ron i David R. Walt. "Optical imaging fiber-based live bacterial cell array biosensor". Analytical Biochemistry 315, nr 1 (kwiecień 2003): 106–13. http://dx.doi.org/10.1016/s0003-2697(02)00700-5.

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Bischof, Helmut, Sandra Burgstaller, Markus Waldeck-Weiermair, Thomas Rauter, Maximilian Schinagl, Jeta Ramadani-Muja, Wolfgang F. Graier i Roland Malli. "Live-Cell Imaging of Physiologically Relevant Metal Ions Using Genetically Encoded FRET-Based Probes". Cells 8, nr 5 (22.05.2019): 492. http://dx.doi.org/10.3390/cells8050492.

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Essential biochemical reactions and processes within living organisms are coupled to subcellular fluctuations of metal ions. Disturbances in cellular metal ion homeostasis are frequently associated with pathological alterations, including neurotoxicity causing neurodegeneration, as well as metabolic disorders or cancer. Considering these important aspects of the cellular metal ion homeostasis in health and disease, measurements of subcellular ion signals are of broad scientific interest. The investigation of the cellular ion homeostasis using classical biochemical methods is quite difficult, often even not feasible or requires large cell numbers. Here, we report of genetically encoded fluorescent probes that enable the visualization of metal ion dynamics within individual living cells and their organelles with high temporal and spatial resolution. Generally, these probes consist of specific ion binding domains fused to fluorescent protein(s), altering their fluorescent properties upon ion binding. This review focuses on the functionality and potential of these genetically encoded fluorescent tools which enable monitoring (sub)cellular concentrations of alkali metals such as K+, alkaline earth metals including Mg2+ and Ca2+, and transition metals including Cu+/Cu2+ and Zn2+. Moreover, we discuss possible approaches for the development and application of novel metal ion biosensors for Fe2+/Fe3+, Mn2+ and Na+.

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Hirashima, Tsuyoshi. "Live imaging approach of dynamic multicellular responses in ERK signaling during vertebrate tissue development". Biochemical Journal 479, nr 2 (20.01.2022): 129–43. http://dx.doi.org/10.1042/bcj20210557.

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The chemical and mechanical responses of cells via the exchange of information during growth and development result in the formation of biological tissues. Information processing within the cells through the signaling pathways and networks inherent to the constituent cells has been well-studied. However, the cell signaling mechanisms responsible for generating dynamic multicellular responses in developing tissues remain unclear. Here, I review the dynamic multicellular response systems during the development and growth of vertebrate tissues based on the extracellular signal-regulated kinase (ERK) pathway. First, an overview of the function of the ERK signaling network in cells is provided, followed by descriptions of biosensors essential for live imaging of the quantification of ERK activity in tissues. Then adducing four examples, I highlight the contribution of live imaging techniques for studying the involvement of spatio-temporal patterns of ERK activity change in tissue development and growth. In addition, theoretical implications of ERK signaling are also discussed from the viewpoint of dynamic systems. This review might help in understanding ERK-mediated dynamic multicellular responses and tissue morphogenesis.

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Frey, Wesley D., Ashlyn Y. Anderson, Hyemin Lee, Julie B. Nguyen, Emma L. Cowles, Hua Lu i James G. Jackson. "Phosphoinositide species and filamentous actin formation mediate engulfment by senescent tumor cells". PLOS Biology 20, nr 10 (24.10.2022): e3001858. http://dx.doi.org/10.1371/journal.pbio.3001858.

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Cancer cells survive chemotherapy and cause lethal relapse by entering a senescent state that facilitates expression of many phagocytosis/macrophage-related genes that engender a novel cannibalism phenotype. We used biosensors and live-cell imaging to reveal the basic steps and mechanisms of engulfment by senescent human and mouse tumor cells. We show filamentous actin in predator cells was localized to the prey cell throughout the process of engulfment. Biosensors to various phosphoinositide (PI) species revealed increased concentration and distinct localization of predator PI(4) P and PI(4,5)P2 at the prey cell during early stages of engulfment, followed by a transient burst of PI(3) P before and following internalization. PIK3C2B, the kinase responsible for generating PI(3)P, was required for complete engulfment. Inhibition or knockdown of Clathrin, known to associate with PIK3C2B and PI(4,5)P2, severely impaired engulfment. In sum, our data reveal the most fundamental cellular processes of senescent cell engulfment, including the precise localizations and dynamics of actin and PI species throughout the entire process.

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Ciobanu, Madalina, Dale E. Taylor, Jeremy P. Wilburn i David E. Cliffel. "Glucose and Lactate Biosensors for Scanning Electrochemical Microscopy Imaging of Single Live Cells". Analytical Chemistry 80, nr 8 (kwiecień 2008): 2717–27. http://dx.doi.org/10.1021/ac7021184.

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Houser, Mei C. Q., Shane P. C. Mitchell, Priyanka Sinha, Brianna Lundin, Oksana Berezovska i Masato Maesako. "Endosome and Lysosome Membrane Properties Functionally Link to γ-Secretase in Live/Intact Cells". Sensors 23, nr 5 (28.02.2023): 2651. http://dx.doi.org/10.3390/s23052651.

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Our unique multiplexed imaging assays employing FRET biosensors have previously detected that γ-secretase processes APP C99 primarily in late endosomes and lysosomes in live/intact neurons. Moreover we have shown that Aβ peptides are enriched in the same subcellular loci. Given that γ-secretase is integrated into the membrane bilayer and functionally links to lipid membrane properties in vitro, it is presumable that γ-secretase function correlates with endosome and lysosome membrane properties in live/intact cells. In the present study, we show using unique live-cell imaging and biochemical assays that the endo-lysosomal membrane in primary neurons is more disordered and, as a result, more permeable than in CHO cells. Interestingly, γ-secretase processivity is decreased in primary neurons, resulting in the predominant production of long Aβ42 instead of short Aβ38. In contrast, CHO cells favor Aβ38 over the Aβ42 generation. Our findings are consistent with the previous in vitro studies, demonstrating the functional interaction between lipid membrane properties and γ-secretase and provide further evidence that γ-secretase acts in late endosomes and lysosomes in live/intact cells.

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Kellenberger, Colleen A., Stephen C. Wilson, Jade Sales-Lee i Ming C. Hammond. "RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messengers Cyclic di-GMP and Cyclic AMP-GMP". Journal of the American Chemical Society 135, nr 13 (21.03.2013): 4906–9. http://dx.doi.org/10.1021/ja311960g.

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Yamada, Soichiro, i W. James Nelson. "Localized zones of Rho and Rac activities drive initiation and expansion of epithelial cell–cell adhesion". Journal of Cell Biology 178, nr 3 (23.07.2007): 517–27. http://dx.doi.org/10.1083/jcb.200701058.

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Spatiotemporal coordination of cell–cell adhesion involving lamellipodial interactions, cadherin engagement, and the lateral expansion of the contact is poorly understood. Using high-resolution live-cell imaging, biosensors, and small molecule inhibitors, we investigate how Rac1 and RhoA regulate actin dynamics during de novo contact formation between pairs of epithelial cells. Active Rac1, the Arp2/3 complex, and lamellipodia are initially localized to de novo contacts but rapidly diminish as E-cadherin accumulates; further rounds of activation and down-regulation of Rac1 and Arp2/3 occur at the contacting membrane periphery, and this cycle repeats as a restricted membrane zone that moves outward with the expanding contact. The cortical bundle of actin filaments dissolves beneath the expanding contacts, leaving actin bundles at the contact edges. RhoA and actomyosin contractility are activated at the contact edges and are required to drive expansion and completion of cell–cell adhesion. We show that zones of Rac1 and lamellipodia activity and of RhoA and actomyosin contractility are restricted to the periphery of contacting membranes and together drive initiation, expansion, and completion of cell–cell adhesion.

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Masia, Ricard, William J. McCarty, Carolina Lahmann, Jay Luther, Raymond T. Chung, Martin L. Yarmush i Gary Yellen. "Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices". American Journal of Physiology-Gastrointestinal and Liver Physiology 314, nr 1 (1.01.2018): G97—G108. http://dx.doi.org/10.1152/ajpgi.00093.2017.

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Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD+ ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD+ ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD+ ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD+ ratio, and this increase is mitigated by the presence of NAD+-generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD+ ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD+ ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real-time measurements of the ratio in living, functioning liver cells, overcoming many limitations of previous methods for measuring this important redox parameter. The feasibility of using Peredox in liver slices is particularly attractive because slices allow preservation of hepatic microanatomy and metabolic zonation of hepatocytes.

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Gökerküçük, Elif Begüm, Marc Tramier i Giulia Bertolin. "Imaging Mitochondrial Functions: From Fluorescent Dyes to Genetically-Encoded Sensors". Genes 11, nr 2 (23.01.2020): 125. http://dx.doi.org/10.3390/genes11020125.

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Mitochondria are multifunctional organelles that are crucial to cell homeostasis. They constitute the major site of energy production for the cell, they are key players in signalling pathways using secondary messengers such as calcium, and they are involved in cell death and redox balance paradigms. Mitochondria quickly adapt their dynamics and biogenesis rates to meet the varying energy demands of the cells, both in normal and in pathological conditions. Therefore, understanding simultaneous changes in mitochondrial functions is crucial in developing mitochondria-based therapy options for complex pathological conditions such as cancer, neurological disorders, and metabolic syndromes. To this end, fluorescence microscopy coupled to live imaging represents a promising strategy to track these changes in real time. In this review, we will first describe the commonly available tools to follow three key mitochondrial functions using fluorescence microscopy: Calcium signalling, mitochondrial dynamics, and mitophagy. Then, we will focus on how the development of genetically-encoded fluorescent sensors became a milestone for the understanding of these mitochondrial functions. In particular, we will show how these tools allowed researchers to address several biochemical activities in living cells, and with high spatiotemporal resolution. With the ultimate goal of tracking multiple mitochondrial functions simultaneously, we will conclude by presenting future perspectives for the development of novel genetically-encoded fluorescent biosensors.

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Wang, Weijia, Yang Zhang, Philip Dettinger, Andreas Reimann, Tobias Kull, Dirk Loeffler, Markus G. Manz, Claudia Lengerke i Timm Schroeder. "Cytokine combinations for human blood stem cell expansion induce cell-type– and cytokine-specific signaling dynamics". Blood 138, nr 10 (14.05.2021): 847–57. http://dx.doi.org/10.1182/blood.2020008386.

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Abstract How hematopoietic stem cells (HSCs) integrate signals from their environment to make fate decisions remains incompletely understood. Current knowledge is based on either averages of heterogeneous populations or snapshot analyses, both missing important information about the dynamics of intracellular signaling activity. By combining fluorescent biosensors with time-lapse imaging and microfluidics, we measured the activity of the extracellular-signal–regulated kinase (ERK) pathway over time (ie, dynamics) in live single human umbilical cord blood HSCs and multipotent progenitor cells (MPPs). In single cells, ERK signaling dynamics were highly heterogeneous and depended on the cytokines, their combinations, and cell types. ERK signaling was activated by stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand in HSCs but SCF, interleukin 3, and granulocyte colony-stimulating factor in MPPs. Different cytokines and their combinations led to distinct ERK signaling dynamics frequencies, and ERK dynamics in HSCs were more transient than those in MPPs. A combination of 5 cytokines recently shown to maintain HSCs in long-term culture, had a more-than-additive effect in eliciting sustained ERK dynamics in HSCs. ERK signaling dynamics also predicted future cell fates. For example, CD45RA expression increased more in HSC daughters with intermediate than with transient or sustained ERK signaling. We demonstrate heterogeneous cytokine- and cell-type–specific ERK signaling dynamics, illustrating their relevance in regulating hematopoietic stem and progenitor (HSPC) cell fates.

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Warren, Sean, Anca Margineanu, Matilda Katan, Chris Dunsby i Paul French. "Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells". International Journal of Molecular Sciences 16, nr 12 (30.06.2015): 14695–716. http://dx.doi.org/10.3390/ijms160714695.

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Li, Lei, Changcheng Zhang, Peng Wang, Aoxue Wang, Jiasheng Zhou, Guoqing Chen, Jianhua Xu i in. "Imaging the Redox States of Live Cells with the Time-Resolved Fluorescence of Genetically Encoded Biosensors". Analytical Chemistry 91, nr 6 (19.02.2019): 3869–76. http://dx.doi.org/10.1021/acs.analchem.8b04292.

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Murata, Michael M., Xiangduo Kong, Emmanuel Moncada, Yumay Chen, Hiromi Imamura, Ping Wang, Michael W. Berns, Kyoko Yokomori i Michelle A. Digman. "NAD+ consumption by PARP1 in response to DNA damage triggers metabolic shift critical for damaged cell survival". Molecular Biology of the Cell 30, nr 20 (15.09.2019): 2584–97. http://dx.doi.org/10.1091/mbc.e18-10-0650.

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DNA damage signaling is critical for the maintenance of genome integrity and cell fate decision. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor rapidly activated in a damage dose- and complexity-dependent manner playing a critical role in the initial chromatin organization and DNA repair pathway choice at damage sites. However, our understanding of a cell-wide consequence of its activation in damaged cells is still limited. Using the phasor approach to fluorescence lifetime imaging microscopy and fluorescence-based biosensors in combination with laser microirradiation, we found a rapid cell-wide increase of the bound NADH fraction in response to nuclear DNA damage, which is triggered by PARP-dependent NAD+ depletion. This change is linked to the metabolic balance shift to oxidative phosphorylation (oxphos) over glycolysis. Inhibition of oxphos, but not glycolysis, resulted in parthanatos due to rapid PARP-dependent ATP deprivation, indicating that oxphos becomes critical for damaged cell survival. The results reveal the novel prosurvival response to PARP activation through a change in cellular metabolism and demonstrate how unique applications of advanced fluorescence imaging and laser microirradiation-induced DNA damage can be a powerful tool to interrogate damage-induced metabolic changes at high spatiotemporal resolution in a live cell.

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Vicente, Manuel, Jussep Salgado-Almario, Michelle M. Collins, Antonio Martínez-Sielva, Masafumi Minoshima, Kazuya Kikuchi, Beatriz Domingo i Juan Llopis. "Cardioluminescence in Transgenic Zebrafish Larvae: A Calcium Imaging Tool to Study Drug Effects and Pathological Modeling". Biomedicines 9, nr 10 (22.09.2021): 1294. http://dx.doi.org/10.3390/biomedicines9101294.

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Zebrafish embryos and larvae have emerged as an excellent model in cardiovascular research and are amenable to live imaging with genetically encoded biosensors to study cardiac cell behaviours, including calcium dynamics. To monitor calcium ion levels in three to five days post-fertilization larvae, we have used bioluminescence. We generated a transgenic line expressing GFP-aequorin in the heart, Tg(myl7:GA), and optimized a reconstitution protocol to boost aequorin luminescence. The analogue diacetylh-coelenterazine enhanced light output and signal-to-noise ratio. With this cardioluminescence model, we imaged the time-averaged calcium levels and beat-to-beat calcium oscillations continuously for hours. As a proof-of-concept of the transgenic line, changes in ventricular calcium levels were observed by Bay K8644, an L-type calcium channel activator and with the blocker nifedipine. The β-adrenergic blocker propranolol decreased calcium levels, heart rate, stroke volume, and cardiac output, suggesting that larvae have a basal adrenergic tone. Zebrafish larvae treated with terfenadine for 24 h have been proposed as a model of heart failure. Tg(myl7:GA) larvae treated with terfenadine showed bradycardia, 2:1 atrioventricular block, decreased time-averaged ventricular calcium levels but increased calcium transient amplitude, and reduced cardiac output. As alterations of calcium signalling are involved in the pathogenesis of heart failure and arrhythmia, the GFP-aequorin transgenic line provides a powerful platform for understanding calcium dynamics.

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Wagner, Teresa R., i Ulrich Rothbauer. "Nanobodies Right in the Middle: Intrabodies as Toolbox to Visualize and Modulate Antigens in the Living Cell". Biomolecules 10, nr 12 (21.12.2020): 1701. http://dx.doi.org/10.3390/biom10121701.

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In biomedical research, there is an ongoing demand for new technologies to elucidate disease mechanisms and develop novel therapeutics. This requires comprehensive understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, post-translational modifications and dynamic interactions of cellular components. Traceable intracellular binding molecules provide new opportunities for real-time cellular diagnostics. Most prominently, intrabodies derived from antibody fragments of heavy-chain only antibodies of camelids (nanobodies) have emerged as highly versatile and attractive probes to study and manipulate antigens within the context of living cells. In this review, we provide an overview on the selection, delivery and usage of intrabodies to visualize and monitor cellular antigens in living cells and organisms. Additionally, we summarize recent advances in the development of intrabodies as cellular biosensors and their application to manipulate disease-related cellular processes. Finally, we highlight switchable intrabodies, which open entirely new possibilities for real-time cell-based diagnostics including live-cell imaging, target validation and generation of precisely controllable binding reagents for future therapeutic applications.

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Mensching, Leonore, Sebastian Rading, Viacheslav Nikolaev i Meliha Karsak. "Monitoring Cannabinoid CB2 -Receptor Mediated cAMP Dynamics by FRET-Based Live Cell Imaging". International Journal of Molecular Sciences 21, nr 21 (23.10.2020): 7880. http://dx.doi.org/10.3390/ijms21217880.

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G-protein coupled cannabinoid CB2 receptor signaling and function is primarily mediated by its inhibitory effect on adenylate cyclase. The visualization and monitoring of agonist dependent dynamic 3′,5′-cyclic adenosine monophosphate (cAMP) signaling at the single cell level is still missing for CB2 receptors. This paper presents an application of a live cell imaging while using a Förster resonance energy transfer (FRET)-based biosensor, Epac1-camps, for quantification of cAMP. We established HEK293 cells stably co-expressing human CB2 and Epac1-camps and quantified cAMP responses upon Forskolin pre-stimulation, followed by treatment with the CB2 ligands JWH-133, HU308, β-caryophyllene, or 2-arachidonoylglycerol. We could identify cells showing either an agonist dependent CB2-response as expected, cells displaying no response, and cells with constitutive receptor activity. In Epac1-CB2-HEK293 responder cells, the terpenoid β-caryophyllene significantly modified the cAMP response through CB2. For all of the tested ligands, a relatively high proportion of cells with constitutively active CB2 receptors was identified. Our method enabled the visualization of intracellular dynamic cAMP responses to the stimuli at single cell level, providing insights into the nature of heterologous CB2 expression systems that contributes to the understanding of Gαi-mediated G-Protein coupled receptor (GPCR) signaling in living cells and opens up possibilities for future investigations of endogenous CB2 responses.

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Frei, Michelle S., Miroslaw Tarnawski, M. Julia Roberti, Birgit Koch, Julien Hiblot i Kai Johnsson. "Engineered HaloTag variants for fluorescence lifetime multiplexing". Nature Methods 19, nr 1 (16.12.2021): 65–70. http://dx.doi.org/10.1038/s41592-021-01341-x.

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AbstractSelf-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications.

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Houser, Mei C. Q., Yuliia Turchyna, Florian Perrin, Lori Chibnik, Oksana Berezovska i Masato Maesako. "Limited Substrate Specificity of PS/γ-Secretase Is Supported by Novel Multiplexed FRET Analysis in Live Cells". Biosensors 11, nr 6 (26.05.2021): 169. http://dx.doi.org/10.3390/bios11060169.

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Presenilin (PS)/γ-secretase is an aspartyl protease that processes a wide range of transmembrane proteins such as the amyloid precursor protein (APP) and Notch1, playing essential roles in normal biological events and diseases. However, whether there is a substrate preference for PS/γ-secretase processing in cells is not fully understood. Structural studies of PS/γ-secretase enfolding a fragment of APP or Notch1 showed that the two substrates engage the protease in broadly similar ways, suggesting the limited substrate specificity of PS/γ-secretase. In the present study, we developed a new multiplexed imaging platform that, for the first time, allowed us to quantitatively monitor how PS/γ-secretase processes two different substrates (e.g., APP vs. Notch1) in the same cell. In this assay, we utilized the recently reported, spectrally compatible visible and near-infrared (NIR)-range Förster resonance energy transfer (FRET) biosensors that permit quantitative recording of PS/γ-secretase activity in live cells. Here, we show that, overall, PS/γ-secretase similarly cleaves Notch1 N100, wild-type APP C99, and familial Alzheimer’s disease (FAD)-linked APP C99 mutants in Chinese hamster ovary (CHO) cells, which further supports the limited PS/γ-secretase substrate specificity. On the other hand, a cell-by-cell basis analysis demonstrates a certain degree of variability in substrate recognition and processing by PS/γ-secretase among different cells. Our new multiplexed FRET assay could be a useful tool to better understand how PS/γ-secretase processes its multiple substrates in normal and disease conditions in live, intact cells.

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Konishi, Yoshinobu, Kenta Terai, Takaya Abe, Yoko Hamazaki, Akifumi Takaori-Kondo i Michiyuki Matsuda. "Live-Cell FRET Imaging Reveals a Role of ERK Activity Dynamics in Thymocyte Motility". Blood 132, Supplement 1 (29.11.2018): 861. http://dx.doi.org/10.1182/blood-2018-99-116044.

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Abstract Thymocyte motility is key to orchestrating migration between different thymic microenvironments at the appropriate developmental stage. Several lines of evidence suggest that extracellular signal-regulated kinase (ERK) signaling pathways plays critical roles in T cell development. Nevertheless, the dynamics of ERK activity and its role in regulating cell motility remain largely unknown due to technical difficulties. Therefore, there is an increasing demand for genetic reporter systems that provide the information of thymocyte motility and ERK signaling under physiological condition in real time. To meet the demand, we developed transgenic mice expressing fluorescence resonance energy transfer (FRET) based biosensor for ERK incorporated into the ROSA26 locus conditionally. EKAREV is a genetically encoded intramolecular FRET biosensor for monitoring ERK activity in living cells. We introduced cDNAs of EKAREV into the ROSA26 locus to generate mouse lines named R26-EKAREV-NLSfl/+. In these mouse lines, EKAREV will be expressed in the nucleus after Cre-mediated excision of the loxP-flanked tdKeima-coding sequence. To study ERK activity dynamics in T cells, R26-EKAREV-NLSfl/+ were crossed with Lck-Cre mice to generate R26-EKAREV-NLSlck/+. R26-EKAREV-NLSlck/+ showed uniform and high-level expression of EKAREV in lymphocytes and allowed us to examine the ERK activity of T cells in vivo. After initial characterization of R26-EKAREV-NLSlck/+, we attempted to unravel the potential crosstalk between ERK activity dynamics and cell motility within the thymic microenvironment. Long-term in vivo imaging of thymus was difficult due to the anatomical location abutting the heart. To circumvent this problem, thymocytes obtained from R26-EKAREV-NLSlck/+ were overlaid on C57BL/6 (WT) thymic slices and observed under two-photon excitation microscopy. During the course of experiments, we noticed a negative correlation between ERK activity and migration speed of thymocytes. To confirm this observation, thymocytes were cultured in the presence or absence of an mitogen-activated protein kinase/ERK kinase (MEK) inhibitor prior to transfer onto thymic slices. Treatment of thymocytes with MEK inhibitor increased averaged migration speed in both double-positive (DP) and single-positive (SP) subsets. Moreover, time-lapse imaging of each subsets of thymocytes revealed that the deviation of ERK activity from the average of individual cells regulated cell motility in CD4-SP, while the absolute value of ERK activity regulated cell motility in DP and CD8-SP. Collectively, these results suggest that CD4-SP is unique in that the ERK activity dynamics negatively regulate cell motility. Which signal regulates ERK activity and cell motility of CD4-SP in the medulla? Interaction between T cell receptor (TCR) and major histocompatibility complex (MHC) is known to regulate ERK activity and cell motility of T cells, but their direct relationship remains unknown in tissues. To examine this possibility, CD4-SP thymocytes were overlaid onto WT or MHC class II knockout (KO) thymic slices. When overlaid onto KO thymic slices, the average migration speed was significantly increased, indicating that CD4-SP cells arrest upon TCR-MHC II interaction. Moreover, the variance of ERK activity, but not the average of it, in CD4-SP cells on KO thymic slices was decreased, suggesting that TCR-MHC II interaction contribute the variance of ERK activity in CD4-SP. Our findings unravel that the deviation of ERK activity induced by TCR-MHC interactions negatively regulate cell motility of CD4-SP in the medulla. The live-cell FRET imaging of ERK activity will open a new window to understand the dynamic nature and the diverse functions of ERK signaling in T cell biology. Disclosures Takaori-Kondo: Janssen Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Celgene: Honoraria, Research Funding.

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Potocký, Martin, Roman Pleskot, Přemysl Pejchar, Nicolas Vitale, Benedikt Kost i Viktor Žárský. "Live-cell imaging of phosphatidic acid dynamics in pollen tubes visualized by Spo20p-derived biosensor". New Phytologist 203, nr 2 (22.04.2014): 483–94. http://dx.doi.org/10.1111/nph.12814.

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Limsakul, Praopim, Qin Peng, Yiqian Wu, Molly E. Allen, Jing Liang, Albert G. Remacle, Tyler Lopez i in. "Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface". Cell Chemical Biology 25, nr 4 (kwiecień 2018): 370–79. http://dx.doi.org/10.1016/j.chembiol.2018.01.002.

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Scalisi, Silvia, Francesca Pennacchietti, Sandeep Keshavan, Nathan D. Derr, Alberto Diaspro, Dario Pisignano, Agnieszka Pierzynska-Mach, Silvia Dante i Francesca Cella Zanacchi. "Quantitative Super-Resolution Microscopy to Assess Adhesion of Neuronal Cells on Single-Layer Graphene Substrates". Membranes 11, nr 11 (15.11.2021): 878. http://dx.doi.org/10.3390/membranes11110878.

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Single Layer Graphene (SLG) has emerged as a critically important nanomaterial due to its unique optical and electrical properties and has become a potential candidate for biomedical applications, biosensors, and tissue engineering. Due to its intrinsic 2D nature, SLG is an ideal surface for the development of large-area biosensors and, due to its biocompatibility, can be easily exploited as a substrate for cell growth. The cellular response to SLG has been addressed in different studies with high cellular affinity for graphene often detected. Still, little is known about the molecular mechanism that drives/regulates the cellular adhesion and migration on SLG and SLG-coated interfaces with respect to other substrates. Within this scenario, we used quantitative super-resolution microscopy based on single-molecule localization to study the molecular distribution of adhesion proteins at the nanoscale level in cells growing on SLG and glass. In order to reveal the molecular mechanisms underlying the higher affinity of biological samples on SLG, we exploited stochastic optical reconstruction microscopy (STORM) imaging and cluster analysis, quantifying the super-resolution localization of the adhesion protein vinculin in neurons and clearly highlighting substrate-related correlations. Additionally, a comparison with an epithelial cell line (Chinese Hamster Ovary) revealed a cell dependent mechanism of interaction with SLG.

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Dmitriev, Ruslan I., Xavier Intes i Margarida M. Barroso. "Luminescence lifetime imaging of three-dimensional biological objects". Journal of Cell Science 134, nr 9 (1.05.2021): 1–17. http://dx.doi.org/10.1242/jcs.254763.

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ABSTRACT A major focus of current biological studies is to fill the knowledge gaps between cell, tissue and organism scales. To this end, a wide array of contemporary optical analytical tools enable multiparameter quantitative imaging of live and fixed cells, three-dimensional (3D) systems, tissues, organs and organisms in the context of their complex spatiotemporal biological and molecular features. In particular, the modalities of luminescence lifetime imaging, comprising fluorescence lifetime imaging (FLI) and phosphorescence lifetime imaging microscopy (PLIM), in synergy with Förster resonance energy transfer (FRET) assays, provide a wealth of information. On the application side, the luminescence lifetime of endogenous molecules inside cells and tissues, overexpressed fluorescent protein fusion biosensor constructs or probes delivered externally provide molecular insights at multiple scales into protein–protein interaction networks, cellular metabolism, dynamics of molecular oxygen and hypoxia, physiologically important ions, and other physical and physiological parameters. Luminescence lifetime imaging offers a unique window into the physiological and structural environment of cells and tissues, enabling a new level of functional and molecular analysis in addition to providing 3D spatially resolved and longitudinal measurements that can range from microscopic to macroscopic scale. We provide an overview of luminescence lifetime imaging and summarize key biological applications from cells and tissues to organisms.

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Aoki, Kazuhiro, Yuji Kamioka i Michiyuki Matsuda. "Fluorescence resonance energy transfer imaging of cell signaling fromin vitrotoin vivo: Basis of biosensor construction, live imaging, and image processing". Development, Growth & Differentiation 55, nr 4 (7.02.2013): 515–22. http://dx.doi.org/10.1111/dgd.12039.

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Li, Dandan, Xiaohe Tian, Aidong Wang, Lijuan Guan, Jun Zheng, Fei Li, Shengli Li, Hongping Zhou, Jieying Wu i Yupeng Tian. "Nucleic acid-selective light-up fluorescent biosensors for ratiometric two-photon imaging of the viscosity of live cells and tissues". Chemical Science 7, nr 3 (2016): 2257–63. http://dx.doi.org/10.1039/c5sc03956h.

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Mesnard, Daniel, i Daniel B. Constam. "Imaging proprotein convertase activities and their regulation in the implanting mouse blastocyst". Journal of Cell Biology 191, nr 1 (27.09.2010): 129–39. http://dx.doi.org/10.1083/jcb.201005026.

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Axis formation and allocation of pluripotent progenitor cells to the germ layers are governed by the TGF-β–related Nodal precursor and its secreted proprotein convertases (PCs) Furin and Pace4. However, when and where Furin and Pace4 first become active have not been determined. To study the distribution of PCs, we developed a novel cell surface–targeted fluorescent biosensor (cell surface–linked indicator of proteolysis [CLIP]). Live imaging of CLIP in wild-type and Furin- and Pace4-deficient embryonic stem cells and embryos revealed that Furin and Pace4 are already active at the blastocyst stage in the inner cell mass and can cleave membrane-bound substrate both cell autonomously and nonautonomously. CLIP was also cleaved in the epiblast of implanted embryos, in part by a novel activity in the uterus that is independent of zygotic Furin and Pace4, suggesting a role for maternal PCs during embryonic development. The unprecedented sensitivity and spatial resolution of CLIP opens exciting new possibilities to elucidate PC functions in vivo.

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Grisan, Francesca, Martina Spacci, Carlotta Paoli, Andrea Costamagna, Marco Fantuz, Miriam Martini, Konstantinos Lefkimmiatis i Alessandro Carrer. "Cholesterol Activates Cyclic AMP Signaling in Metaplastic Acinar Cells". Metabolites 11, nr 3 (26.02.2021): 141. http://dx.doi.org/10.3390/metabo11030141.

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Cholesterol is a non-essential metabolite that exerts both structural and signaling functions. However, cholesterol biosynthesis is elevated, and actively supports, pancreatic carcinogenesis. Our previous work showed that statins block the reprogramming of mutant KRAS-expressing acinar cells, that spontaneously undergo a metaplastic event termed acinar-to-ductal metaplasia (ADM) to initiate carcinogenesis. Here we tested the impact of cholesterol supplementation on isolated primary wild-type acinar cells and observed enhanced ductal transdifferentiation, associated with generation of the second messenger cyclic adenosine monophosphate (cAMP) and the induction of downstream protein kinase A (PKA). Inhibition of PKA suppresses cholesterol-induced ADM ex vivo. Live imaging using fluorescent biosensors dissected the temporal and spatial dynamics of PKA activation upon cholesterol addition and showed uneven activation both in the cytosol and on the outer mitochondrial membrane of primary pancreatic acinar cells. The ability of cholesterol to activate cAMP signaling is lost in tumor cells. Qualitative examination of multiple normal and transformed cell lines supports the notion that the cAMP/PKA axis plays different roles during multi-step pancreatic carcinogenesis. Collectively, our findings describe the impact of cholesterol availability on the cyclic AMP/PKA axis and plasticity of pancreatic acinar cells.

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Ma, Hoi Tang, Yiu Huen Tsang, Miriam Marxer i Randy Y. C. Poon. "Cyclin A2-Cyclin-Dependent Kinase 2 Cooperates with the PLK1-SCFβ-TrCP1-EMI1-Anaphase-Promoting Complex/Cyclosome Axis To Promote Genome Reduplication in the Absence of Mitosis". Molecular and Cellular Biology 29, nr 24 (12.10.2009): 6500–6514. http://dx.doi.org/10.1128/mcb.00669-09.

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ABSTRACT Limiting genome replication to once per cell cycle is vital for maintaining genome stability. Inhibition of cyclin-dependent kinase 1 (CDK1) with the specific inhibitor RO3306 is sufficient to trigger multiple rounds of genome reduplication. We demonstrated that although anaphase-promoting complex/cyclosome (APC/C) remained inactive during the initial G2 arrest, it was activated upon prolonged inhibition of CDK1. Using cellular biosensors and live-cell imaging, we provide direct evidence that genome reduplication was associated with oscillation of APC/C activity and nuclear-cytoplasmic shuttling of CDC6 even in the absence of mitosis at the single-cell level. Genome reduplication was abolished by ectopic expression of EMI1 or depletion of CDC20 or CDH1, suggesting the critical role of the EMI1-APC/C axis. In support of this, degradation of EMI1 itself and genome reduplication were delayed after downregulation of PLK1 and β-TrCP1. In the absence of CDK1 activity, activation of APC/C and genome reduplication was dependent on cyclin A2 and CDK2. Genome reduplication was then promoted by a combination of APC/C-dependent destruction of geminin (thus releasing CDT1), accumulation of cyclin E2-CDK2, and CDC6. Collectively, these results underscore the crucial role of cyclin A2-CDK2 in regulating the PLK1-SCFβ-TrCP1-EMI1-APC/C axis and CDC6 to trigger genome reduplication after the activity of CDK1 is suppressed.

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Wall, Adam A., Nicholas D. Condon, Lin Luo i Jennifer L. Stow. "Rab8a localisation and activation by Toll-like receptors on macrophage macropinosomes". Philosophical Transactions of the Royal Society B: Biological Sciences 374, nr 1765 (17.12.2018): 20180151. http://dx.doi.org/10.1098/rstb.2018.0151.

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Macropinocytosis is a prevalent and essential pathway in macrophages where it contributes to anti-microbial responses and innate immune cell functions. Cell surface ruffles give rise to phagosomes and to macropinosomes as multi-functional compartments that contribute to environmental sampling, pathogen entry, plasma membrane turnover and receptor signalling. Rapid, high resolution, lattice light sheet imaging demonstrates the dynamic nature of macrophage ruffling. Pathogen-mediated activation of surface and endosomal Toll-like receptors (TLRs) in macrophages upregulates macropinocytosis. Here, using multiple forms of imaging and microscopy, we track membrane-associated, fluorescently-tagged Rab8a expressed in live macrophages, using a variety of cell markers to demonstrate Rab8a localization and its enrichment on early macropinosomes. Production of a novel biosensor and its use for quantitative FRET analysis in live cells, pinpoints macropinosomes as the site for TLR-induced activation of Rab8a. We have previously shown that TLR signalling, cytokine outputs and macrophage programming are regulated by the GTPase Rab8a with PI3 Kγ as its effector. Finally, we highlight another effector, the phosphatase OCRL, which is located on macropinosomes and interacts with Rab8a, suggesting that Rab8a may operate on multiple levels to modulate phosphoinositides in macropinosomes. These findings extend our understanding of macropinosomes as regulatory compartments for innate immune function in macrophages. This article is part of the Theo Murphy meeting issue ‘Macropinocytosis’.

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Reinhard, Nathalie R., Marieke Mastop, Taofei Yin, Yi Wu, Esmeralda K. Bosma, Theodorus W. J. Gadella, Joachim Goedhart i Peter L. Hordijk. "The balance between Gαi-Cdc42/Rac and Gα12/13-RhoA pathways determines endothelial barrier regulation by sphingosine-1-phosphate". Molecular Biology of the Cell 28, nr 23 (7.11.2017): 3371–82. http://dx.doi.org/10.1091/mbc.e17-03-0136.

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The bioactive sphingosine-1-phosphatephosphate (S1P) is present in plasma, bound to carrier proteins, and involved in many physiological processes, including angiogenesis, inflammatory responses, and vascular stabilization. S1P can bind to several G-protein–coupled receptors (GPCRs) activating a number of different signaling networks. At present, the dynamics and relative importance of signaling events activated immediately downstream of GPCR activation are unclear. To examine these, we used a set of fluorescence resonance energy transfer–based biosensors for different RhoGTPases (Rac1, RhoA/B/C, and Cdc42) as well as for heterotrimeric G-proteins in a series of live-cell imaging experiments in primary human endothelial cells. These experiments were accompanied by biochemical GTPase activity assays and transendothelial resistance measurements. We show that S1P promotes cell spreading and endothelial barrier function through S1PR1-Gαi-Rac1 and S1PR1-Gαi-Cdc42 pathways. In parallel, a S1PR2-Gα12/13-RhoA pathway is activated that can induce cell contraction and loss of barrier function, but only if Gαi-mediated signaling is suppressed. Our results suggest that Gαq activity is not involved in S1P-mediated regulation of barrier integrity. Moreover, we show that early activation of RhoA by S1P inactivates Rac1 but not Cdc42, and vice versa. Together, our data show that the rapid S1P-induced increase in endothelial integrity is mediated by a S1PR1-Gαi-Cdc42 pathway.

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Hanna, Samer, Veronika Miskolci, Dianne Cox i Louis Hodgson. "A New Genetically Encoded Single-Chain Biosensor for Cdc42 Based on FRET, Useful for Live-Cell Imaging". PLoS ONE 9, nr 5 (5.05.2014): e96469. http://dx.doi.org/10.1371/journal.pone.0096469.

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Frey, Wesley D., Ashlyn Anderson, Julie Nguyen, Emma Cowles i James Jackson. "Abstract 1576: Chemotherapy induced cellular cannibalism is mediated by phosphoinositide species and clathrin". Cancer Research 82, nr 12_Supplement (15.06.2022): 1576. http://dx.doi.org/10.1158/1538-7445.am2022-1576.

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Abstract Cancer cells can survive chemotherapy and drive lethal relapse if they avoid cell death in conditions of 1) DNA damage and/or mitotic stress caused by treatment; 2) nutrient depletion. We and others have previously shown that the breast cancers most likely to survive chemotherapy are TP53 wild-type, and these are among the most lethal breast cancers. For instance, chemotherapy treated TNBC patients with TP53 wild-type tumors have a median overall survival of 45 months, contrasting with 263 months for TP53 mutant tumors. TP53 wild type cells survive stress by entering a state of arrest and cellular senescence. But to persist in senescence, cells must survive in limited access to vasculature and nutrient sources while also supporting a high metabolic burden that includes production of cytokines and chemokines that drive pro-tumorigenic phenotypes and relapse. We previously showed a novel cannibalism phenotype of chemotherapy induced senescent cells. Engulfment occurred after exposure to different chemotherapy drugs in vitro, in 9 different cell lines, and in vivo in syngeneic mouse mammary tumor models. Engulfed prey cells were processed to the lysosomes of predator cells and broken down. While we showed the phenotype was unrelated to entosis, and senescent cells expressed many phagocytosis/macrophage related genes, the basic mechanisms of the engulfment are unknown. Here, we use biosensors and live cell imaging to delineate the steps of whole cell engulfment in cells that have entered senescence to survive chemotherapy. We show predator cell filamentous actin was localized to the prey cell throughout the process of engulfment. Biosensors to various phosphoinositide (PI) species showed increased concentration and localization of predator PI(4)P and PI(4,5)P2 at the prey cell during early stages of engulfment, followed by a burst of PI(3)P before internalization. PIK3C2B, an enzyme responsible for generating PI(3)P, was required for complete engulfment. Inhibition or knockdown of Clathrin, known to associate with PIK3C2B and PI(4,5)P2, severely impaired engulfment. In sum, these data demonstrate the mechanism of cellular cannibalism used by breast cancer cells to survive chemotherapy. Citation Format: Wesley D. Frey, Ashlyn Anderson, Julie Nguyen, Emma Cowles, James Jackson. Chemotherapy induced cellular cannibalism is mediated by phosphoinositide species and clathrin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1576.

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Wrobel, Günter, Matthias Höller, Sven Ingebrandt, Sabine Dieluweit, Frank Sommerhage, Hans Peter Bochem i Andreas Offenhäusser. "Transmission electron microscopy study of the cell–sensor interface". Journal of The Royal Society Interface 5, nr 19 (3.07.2007): 213–22. http://dx.doi.org/10.1098/rsif.2007.1094.

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An emerging number of micro- and nanoelectronics-based biosensors have been developed for non-invasive recordings of physiological cellular activity. The interface between the biological system and the electronic devices strongly influences the signal transfer between these systems. Little is known about the nanoscopic structure of the cell–sensor interface that is essential for a detailed interpretation of the recordings. Therefore, we analysed the interface between the sensor surface and attached cells using transmission electron microscopy (TEM). The maximum possible resolution of our TEM study, however, was restricted by the quality of the interface preparation. Therefore, we complemented our studies with imaging ellipsometry. We cultured HEK293 cells on substrates, which had been precoated with different types of proteins. We found that contact geometry between attached cell membrane and substrate was dependent on the type of protein coating used. In the presence of polylysine, the average distance of the membrane–substrate interface was in the range of 35–40 nm. However, the cell membrane was highly protruded in the presence of other proteins like fibronectin, laminin or concanavalin-A. The presented method allows the nanoscopic characterization of the cell–sensor interface.

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