Search

Glycolipid metabolism occurs in the Golgi apparatus, but the detailed mechanisms have not yet been elucidated. We used fluorescently labeled glycolipids to analyze glycolipid composition and localization changes and shed light on glycolipid metabolism. In a previous study, the fatty chain of lactosyl ceramide was fluorescently labeled with BODIPY (LacCer-BODIPY) before being introduced into cultured cells to analyze the cell membrane glycolipid recycling process. However, imaging analysis of glycolipid recycling is difficult because of limited spatial resolution. Therefore, we examined the microscopic conditions that allow the temporal analysis of LacCer-BODIPY trafficking and localization. We observed that the glycolipid fluorescent probe migrated from the cell membrane to intracellular organelles before returning to the cell membrane. We used confocal microscopy to observe co-localization of the glycolipid probe with endosomes and Golgi markers, demonstrating that it recycles mainly through the trans-Golgi network (TGN). Here, a glycolipid recycling pathway was observed that did not require the lipids to pass through the lysosome. • Visualization of recycling pathways without degradation in lysosomes. • Lysosomal localization of excessive amounts of glycolipid probes. • Time-lapse imaging of glycolipid recycling. • Multicolor imaging of Golgi stacks and trans-Golgi networks. [ABSTRACT FROM AUTHOR]

• Growth factor signaling causes sustained nuclear ERK1/2 activation. • The SCR and BCR/ABL inhibitor dasatinib blocks ERK1/2 and represses cell invasion. • EGF-stimulated cells may escape dasatinib inhibition of invasion through mesenchymal to amoeboid transition. • Combined inhibition of SRC and Rho-kinase signaling is necessary to completely block EGF-induced invasion. Aberrantly activated kinase signaling pathways drive invasion and dissemination in medulloblastoma (MB). A majority of tumor-promoting kinase signaling pathways feed into the mitogen-activated protein kinase (MAPK) extracellular regulated kinase (ERK1/2) pathway. The activation status of ERK1/2 during invasion of MB cells is not known and its implication in invasion control unclear. We established a synthetic kinase activation relocation sensor (SKARS) for the MAPK ERK1/2 pathway in MB cells for real-time measuring of drug response. We used 3D invasion assays and organotypic cerebellum slice culture to test drug effects in a physiologically relevant tissue environment. We found that hepatocyte growth factor (HGF), epidermal growth factor (EGF), or basic fibroblast growth factor (bFGF) caused rapid nuclear ERK1/2 activation in MB cells, which persisted for several hours. Concomitant treatment with the BCR/ABL kinase inhibitor dasatinib completely repressed nuclear ERK1/2 activity induced by HGF and EGF but not by bFGF. Increased nuclear ERK1/2 activity correlated positively with speed of invasion. Dasatinib blocked ERK-associated invasion in the majority of cells, but we also observed fast-invading cells with low ERK1/2 activity. These ERK1/2-low, fast-moving cells displayed a rounded morphology, while ERK-high fast-moving cells displayed a mesenchymal morphology. Dasatinib effectively blocked EGF-induced proliferation while it only moderately repressed tissue invasion, indicating that a subset of cells may evade invasion repression by dasatinib through non-mesenchymal motility. Thus, growth factor-induced nuclear activation of ERK1/2 is associated with mesenchymal motility and proliferation in MB cells and can be blocked with the BCR/ABL kinase inhibitor dasatinib. [ABSTRACT FROM AUTHOR]

Subdiffraction super‐resolution fluorescence microscopy, or nanoscopy, has seen remarkable developments in the last two decades. Yet, for the visualization of plant cells, nanoscopy is still rarely used. In this study, we established RESOLFT nanoscopy on living green plant tissue. Live‐cell RESOLFT nanoscopy requires and utilizes comparatively low light doses and intensities to overcome the diffraction barrier. We generated a transgenic Arabidopsis thaliana plant line expressing the reversibly switchable fluorescent protein rsEGFP2 fused to the mammalian microtubule‐associated protein 4 (MAP4) in order to ubiquitously label the microtubule cytoskeleton. We demonstrate the use of RESOLFT nanoscopy for extended time‐lapse imaging of cortical microtubules in Arabidopsis leaf discs. By combining our approach with fluorescence lifetime gating, we were able to acquire live‐cell RESOLFT images even close to chloroplasts, which exhibit very strong autofluorescence. The data demonstrate the feasibility of subdiffraction resolution imaging in transgenic plant material with minimal requirements for sample preparation. [ABSTRACT FROM AUTHOR]

The mechanical and electrical properties of biomaterials are essential in cell function regulation during cell-biomaterial interaction. However, previous studies focused on probing cell regulation mechanisms under one type of stimulus, and a platform that enables the study of electromechanical coupling effects of a biomaterial on cells is still lacking. Here, we present an in-situ electromechanical testing and loading system to image live cells when co-cultured with electroactive biomaterials. The system can provide accurate and repeatable stretch on biomaterials and cells to mimic in vivo tension microenvironment. Besides, the integrated displacement transducer, force sensor, and electrical signal detector enable the real time detection of electromechanical signals on electroactive biomaterials under various stretch loading. Combined with a microscope, live cell imaging can be realized to probe cell behavior. The feasibility of the system is validated by culturing mesenchymal stem cells on piezoelectric nanofiber and conductive hydrogel. Experiment results show the device as a reliable and accurate tool to investigate electromechanical properties of biomaterials and probe essential features of live cells. Our system provides a way to correlate cell behavior with electromechanical cues directly and is useful for exploration of cell function during cell-biomaterial interaction. [ABSTRACT FROM AUTHOR]

Light sheet fluorescence microscopy (LSFM) allows nondestructive, label‐free and in vivo imaging of large specimen, even at nontransparent surfaces. We show that LSFM can be applied for label‐free analyses of prokaryotes on the example of electroactive biofilms. Biofilm growth is linked to the production of current serving as measure of metabolic activity in vivo by monitoring with high spatial and temporal resolution. After 35 h of exponential growth, a homogeneous biofilm with a thickness of 9 μm was formed. This was followed by a stratification of the biofilm including the formation of 3D structures over the next 100 h. Light reflection was sufficient to visualize the biofilm structure and development over time and the terminal morphology was confirmed using fluorescence staining. This proof of concept on using LSFM for investigation of biofilms opens the door for its application in the entire field of microbial ecology. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry. [ABSTRACT FROM AUTHOR]

Pattern formation, cell proliferation, and directional cell growth, are driving factors of plant organ shape, size, and overall vegetative development. The establishment of vegetative morphogenesis strongly depends on spatiotemporal control and synchronization of formative and proliferative cell division patterns. In this context, the progression of cell division and the regulation of cell division plane orientation are defined by molecular mechanisms converging to the proper positioning and temporal reorganization of microtubule arrays such as the preprophase microtubule band, the mitotic spindle and the cytokinetic phragmoplast. By focusing on the tractable example of primary root development and lateral root emergence in Arabidopsis thaliana , genetic studies have highlighted the importance of mechanisms underlying microtubule reorganization in the establishment of the root system. In this regard, severe alterations of root growth, and development found in extensively studied katanin1 mutants of A. thaliana (fra2, lue1 , and ktn1-2), were previously attributed to defective rearrangements of cortical microtubules and aberrant cell division plane reorientation. How KATANIN1-mediated microtubule severing contributes to tissue patterning and organ morphogenesis, ultimately leading to anisotropy in microtubule organization is a trending topic under vigorous investigation. Here we addressed this issue during root development, using advanced light-sheet fluorescence microscopy (LSFM) and long-term imaging of ktn1-2 mutant expressing the GFP-TUA6 microtubule marker. This method allowed spatial and temporal monitoring of cell division patterns in growing roots. Analysis of acquired multidimensional data sets revealed the occurrence of ectopic cell divisions in various tissues including the calyptrogen and the protoxylem of the main root, as well as in lateral root primordia. Notably the ktn1-2 mutant exhibited excessive longitudinal cell divisions (parallel to the root axis) at ectopic positions. This suggested that changes in the cell division pattern and the occurrence of ectopic cell divisions contributed significantly to pleiotropic root phenotypes of ktn1-2 mutant. LSFM provided evidence that KATANIN1 is required for the spatiotemporal control of cell divisions and establishment of tissue patterns in living A. thaliana roots. [ABSTRACT FROM AUTHOR]

Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations result in a primary immunodeficiency (PID) characterized typically by chronic mucocutaneous candidiasis (CMC), but a wider phenotypic range is reported and remains unexplained from a pathophysiological point-of-view. We hypothesized that different STAT1 GOF mutations may result in distinct molecular mechanisms, possibly explaining the variable phenotypes observed in patients. We selected STAT1 GOF mutants (R274W, R321S, T419R, and N574I) that are spread over the protein and studied their dynamic behavior in vitro in U3A and HeLa cell lines. All GOF mutants showed increased STAT1 phosphorylation compared to STAT1 WT. Real-time imaging demonstrated three underlying mechanisms for STAT1 GOF: (i) R274W showed a faster nuclear accumulation, (ii) both R321S and N574I showed a reduced nuclear mobility and slower dephosphorylation, whereas (iii) T419R was near-immobile in the nucleus, potentially due to enhanced binding to chromatin. [ABSTRACT FROM AUTHOR]

Summary: Programmed cell death (PCD) is the destruction of unwanted cells through an intracellularly mediated process. Perforation formation in the lace plant (Aponogeton madagascariensis) provides an excellent model for studying developmentally regulated PCD. Ca2+ fluxes have previously been identified as important signals for PCD in plants and mammals. The fundamental goal of this project was to determine the influence of Ca2+ on the rate of cell death and perforation formation during leaf development in the lace plant. This was investigated using the application of various known calcium modulators including lanthanum III chloride (LaCl3), ruthenium red and calcium ionophore A23187. Detached lace plant leaves at an early stage of development were treated with these modulators in both short‐ and long‐term exposure assays and analysed using live cell imaging. Results from this study indicate that calcium plays a vital role in developmentally regulated PCD in the lace plant as application of the modulators significantly altered the rate of cell death and perforation formation during leaf development. In conclusion, this study exemplifies the suitability of the lace plant for live cell imaging and detached leaf experiments to study cell death and provides insight into the importance of Ca2+ in developmentally regulated PCD in planta. [ABSTRACT FROM AUTHOR]

Mechanical force regulates a variety of cellular functions through inducing modulations in nuclear chromatin structures and epigenetic landscapes (outside in). The epigenetic modifications, in turn, regulate gene expressions and affect phenotypic outcomes, including cytoskeletal organization and cell–cell/cell–ECM interactions (inside out). While there have been significant advances in the understanding of mechanotransduction in the nucleus, there is still a lack of knowledge on the potential mechanisms through which mechanical cues affect epigenetic and chromatin regulations to determine genetic outcomes. This review firstly focuses on the current understanding of epigenetic regulations and then summarizes how mechanotransduction and epigenetic modification couple together to regulate molecular and cellular functions, eventually causing functional phenotype changes e.g., diseases. Lastly, we introduce related technologies for mechanistic studies, particularly fluorescence resonance energy transfer (FRET) biosensors for the visualization of dynamic epigenetic regulations in single living cells, as well as the applications of FRET biosensors to visualize mechanotransduction events occurring in the nucleus. These studies could provide new insights into epigenetics in regulating the physiological and pathological processes in living cells under different mechanical environments. [ABSTRACT FROM AUTHOR]

Cell culture provides useful model systems used in a wide range of biological applications, but its utility in marine invertebrates is limited due to the lack of immortalised cell lines. Primary cell and tissue cultures are typically used but remain poorly characterised for oysters, which can cause issues with experimental consistency and reproducibility. Improvements to methods of repeatable isolation, culture, and characterisation of oyster cells and tissues are required to help address these issues. In the current study, systematic improvements have been developed to facilitate the culture of primary cells from adult Pacific oyster tissues and identify novel cell morphologies that have not been reported previously. Cultures analysed by light microscopy, qPCR, and live cell imaging demonstrated maintenance of live, metabolically active Pacific oyster cells for several weeks post-explant. Interestingly, whole hearts dissected from adult oysters were found to continue contracting rhythmically up to 8 weeks after being transferred to a tissue culture system. Mantle tissue explants were also actively moving in the culture system. These improvements in primary cell culture of bivalves may be beneficial for research in ecotoxicology, virology, immunology, and genetic resistance to disease. [ABSTRACT FROM AUTHOR]

The structural-functional hallmark of the liver sinusoidal endothelium is the presence of fenestrae grouped in sieve plates. Fenestrae are open membrane bound pores supported by a (sub)membranous cytoskeletal lattice. Changes in number and diameter of fenestrae alter bidirectional transport between the sinusoidal blood and the hepatocytes. Their physiological relevance has been shown in different liver disease models. Although the structural organization of fenestrae has been well documented using different electron microscopy approaches, the dynamic nature of those pores remained an enigma until the recent developments in the research field of four dimensional (4-D) AFM. In this contribution we highlight how AFM as a biophysical nanocharacterization tool enhanced our understanding in the dynamic behaviour of liver sinusoidal endothelial fenestrae. Different AFM probing approaches, including spectroscopy, enabled mapping of topography and nanomechanical properties at unprecedented resolution under live cell imaging conditions. This dynamic biophysical characterization approach provided us with novel information on the 'short' life-span, formation, disappearance and closure of hepatic fenestrae. These observations are briefly reviewed against the existing literature. [ABSTRACT FROM AUTHOR]

Background: Fluorescence microscopy is a powerful tool in cell biology, especially for the study of dynamic processes. Intensive irradiation of bacteria with UV, blue and violet light has been shown to be able to kill cells, but very little information is available on the effect of blue or violet light during live-cell imaging. Results: We show here that in the model bacterium Bacillus subtilis chromosome segregation and cell growth are rapidly halted by standard violet (405 nm) and blue light (CFP) (445–457 nm) excitation, whereas they are largely unaffected by green light (YFP). The stress sigma factor σB and the blue-light receptor YtvA are not involved in growth arrest. Using synchronized B. subtilis cells, we show that the use of blue light for fluorescence microscopy likely induces non-specific toxic effects, rather than a specific cell cycle arrest. Escherichia coli and Caulobacter crescentus cells also stop to grow after 15 one-second exposures to blue light (CFP), but continue growth when imaged under similar conditions in the YFP channel. In the case of E. coli, YFP excitation slows growth relative to white light excitation, whereas CFP excitation leads to cell death in a majority of cells. Thus, even mild violet/blue light excitation interferes with bacterial growth. Analyzing the dose-dependent effects of violet light in B. subtilis, we show that short exposures to low-intensity violet light allow for continued cell growth, while longer exposures do not. Conclusions: Our experiments show that care must be taken in the design of live-cell imaging experiments in that violet or blue excitation effects must be closely controlled during and after imaging. Violet excitation during sptPALM or other imaging studies involving photoactivation has a threshold, below which little effects can be seen, but above which a sharp transition into cell death occurs. YFP imaging proves to be better suited for time-lapse studies, especially when cell cycle or cell growth parameters are to be examined. [ABSTRACT FROM AUTHOR]

Previous studies with various Src family kinase biosensors showed that the nuclear kinase activities are much suppressed compared to those in the cytosol, suggesting that these kinases are regulated differently in the nucleus and in the cytosol. In this study, using Fyn as an example, we first engineered a Fyn biosensor with a light-inducible nuclear localization signal to demonstrate that the Fyn kinase activity is significantly lower in the nucleus than in the cytosol. To understand how different equilibrium states between Fyn and the corresponding phosphatases are maintained in the cytosol and nucleus, we further engineered a Fyn kinase domain with light-inducible nuclear localization signal. The results revealed that the Fyn kinase can be actively transported into the nucleus upon light activation and upregulate the biosensor signals in the nucleus. Our results suggest that there is limited transport or diffusion of Fyn kinase between the cytosol and nucleus in the cells, which is important for the maintenance of different equilibrium states of Fyn in situ. Unlabelled Image • Biosensor studies showed that Src family kinases have suppressed levels of activity in the nucleus. • A Fyn kinase biosensor with light-inducible nuclear localization signal was engineered to provide insights of the dynamic change of Fyn biosensor during nuclear translocation. • A Fyn kinase domain with light-inducible nuclear localization signal was engineered to study the mechanism responsible for maintaining different levels of Fyn activity in cytosol and nucleus. [ABSTRACT FROM AUTHOR]

Plant growth and development relies on the accurate positioning of the cell plate between dividing cells during cytokinesis. The cell plate is synthetized by a specialized structure called the phragmoplast, which contains bipolar microtubules that polymerize to form a framework with the plus ends at or near the division site. This allows the transport of Golgi-derived vesicles toward the plus ends to form and expand the cell plate. Actin filaments play important roles in cell plate expansion and guidance in plant cytokinesis at the late phase, but whether they are involved at the early phase is unknown. To investigate this further, we disrupted the actin filaments in cell cycle-synchronized tobacco BY-2 cells with latrunculin B (LatB), an actin polymerization inhibitor. We observed the cells under a transmission electron microscope or a spinning-disk confocal laser scanning microscope. We found that disruption of actin filaments by LatB caused the membrane vesicles at the equatorial plane of the cell plate to be dispersed rather than form clusters as they did in the untreated cells. The midzone constriction of phragmoplast microtubules also was perturbed in LatB-treated cells. The live cell imaging and kymograph analysis showed that disruption of actin filaments also changed the accumulation timing of NACK1 kinesin, which plays a crucial role in cell plate expansion. This suggests that there are two functionally different types of microtubules in the phragmoplast. Together, our results show that actin filaments regulate phragmoplast microtubules at the initial phase of plant cytokinesis. [ABSTRACT FROM AUTHOR]

Over recent decades, considerable effort has been made to understand how mechanical stress applied to the actin network alters actin assembly and disassembly dynamics. However, there are conflicting reports concerning the issue both in vitro and in cells. In this review, we discuss concerns regarding previous quantitative live-cell experiments that have attempted to evaluate myosin regulation of filamentous actin (F-actin) turnover. In particular, we highlight an error-generating mechanism in quantitative live-cell imaging, namely convection-induced misdistribution of actin-binding probes. Direct observation of actin turnover at the single-molecule level using our improved electroporation-based Single-Molecule Speckle (eSiMS) microscopy technique overcomes these concerns. We introduce our recent single-molecule analysis that unambiguously demonstrates myosin-dependent regulation of F-actin stability in live cells. We also discuss the possible application of eSiMS microscopy in the analysis of actin remodeling in striated muscle cells. [ABSTRACT FROM AUTHOR]

The 3D organization of chromatin plays an important role in genome stability and many other pivotal biological programs. Therefore, the establishment of imaging methods, which enable us to study the dynamics of chromatin in living cells, is necessary. Although primary live cell imaging methods were a breakthrough, there is a need to develop more specific labeling techniques. With the discovery of programmable DNA binding proteins, such zinc finger proteins (ZFP), transcription activator-like effectors (TALE), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a major leap forward was made. Here, we review the applications and potential of fluorescent repressor-operator systems, programmable DNA binding proteins with an emphasis on CRISPR-based chromatin imaging in living and fixed cells, and their potential application in plant science. [ABSTRACT FROM AUTHOR]

Bis-alkylsulfonic acid and polyethylene glycol (PEG)-substituted BF2 azadipyrromethenes have been synthesized by an adaptable and versatile route. Only four synthetic stages were required to produce the penultimate fluorophore compounds, containing either two alcohol or two terminal alkyne substituents. The final synthetic step introduced either sulfonic acid or polyethylene glycol groups to impart aqueous solubility. Sulfonic acid groups were introduced by reaction of the bis-alcohol-substituted fluorophore with sulfur trioxide, and a double Cu(I)-catalyzed cycloaddition reaction between the bis-alkyne fluorophore and methoxypolyethylene glycol azide yielded a neutral bis-pegylated derivative. Both fluorophores exhibited excellent near-infrared (NIR) photophysical properties in methanol and aqueous solutions. Live cell microscopy imaging revealed efficient uptake and intracellular labelling of cells for both fluorophores. Their simple synthesis, with potential for last-step structural modifications, makes the present NIR-active azadipyrromethene derivatives potentially useful as NIR fluorescence imaging probes for live cells. [ABSTRACT FROM AUTHOR]

A furan‐based water soluble fluorescent probe (E)‐N′‐(2,4‐dihydroxybenzylidene)furan‐2‐carbohydrazide (DBF) has been synthesized and characterized by 1H NMR, 13C NMR, ESI‐mass spectroscopy and single crystal X‐ray diffraction techniques. The synthesized probe exhibits a "turn‐on" fluorescence response towards Al3+ in Tris‐HCl buffer (10 mM) with no significant interference from other metal ions. The strong fluorescence in the presence of Al3+ is attributed to the CHEF (chelation enhanced fluorescence), inhibition of PET (photo‐induced electron transfer) and C=N isomerization. 1 : 1 stoichiometric ratio between DBF and Al3+ was rationalized by Job's plot. Binding constant and LOD were calculated to be 1.031×105 M−1 and 6.34×10−8 M, respectively. MTT assay on live A549 cells indicated no serious cytotoxicity in the cells even at higher concentration. Further, DBF was successfully used for the detection of accumulated Al3+ in the cytoplasm of cells. [ABSTRACT FROM AUTHOR]

With this research we presented a ratiometric and mitochondria-target fluorescent probe (Mito-HT) for detection of H 2 O 2 both in vitro and in live cells. Mito-HT was constructed by direct conjugation of aryl boronate to fluorophore with three synthetic steps. The borate group is cleaved from Mito-HT in the presence of H 2 O 2 , resulting in the exposure of the hydroxyl group of the electron donating group. Then the ICT mechanism was turned on, and the fluorescence emission of Mito-HT at 493 nm was red-shifted to 562 nm, thereby achieving radiometric detection of H 2 O 2. Mito-HT exhibited a highly selectivity towards H 2 O 2 , and this interaction can be completed within 40 min. Mito-HT could be used for quantitative detection of H 2 O 2 (0–200 μM) through ratiometric fluorescence signal readout. And limit of detection (LOD) is approximately 0.33 μM. The relatively high stability and medium fluorescence quantum yield of Mito-HT (0.39) and Mito-HT-OH (0.43) enable clear mitochondria localization and dual-channel fluorescence imaging of H 2 O 2 in live cells with confocal microscopy. Image 1 • A new ratiometric and mitochondria-target fluorescent probe (Mito-HT) was constructed. • Mito-HT exhibited a selective response towards H 2 O 2 through ratiometric fluorescence signal readout. • Mito-HT could be used for quantitative detection of H 2 O 2 (0–200 μM). • Mito-HT localized in mitochondria and allows a dual-channel imaging of H 2 O 2 in live cells. [ABSTRACT FROM AUTHOR]

Desmosome-anchored keratin intermediate filaments (KFs) are essential for epithelial coherence. Yet, desmosomal KF attachment and network organization are still unexplored in vivo. We, therefore, monitored KF network morphogenesis in fluorescent keratin 8 knock-in murine embryos revealing keratin enrichment at newly formed desmosomes followed by KF formation, KF elongation and KF fusion. To examine details of this process and its coupling to desmosome formation, we studied fluorescent keratin and desmosomal protein reporter dynamics in the periphery of expanding HaCaT keratinocyte colonies. Less than 3 min after the start of desmosomal proteins clustering non-filamentous keratin enriched at these sites followed by KF formation and elongation. Subsequently, desmosome-anchored KFs merged into stable bundles generating a rim-and-spokes system consisting of subcortical KFs connecting desmosomes to each other and radial KFs connecting desmosomes to the cytoplasmic KF network. We conclude that desmosomes are organizing centers for the KF cytoskeleton with a hitherto unknown nucleation capacity. [ABSTRACT FROM AUTHOR]

Herein, a facile method to synthesize the fluorescence probe 1‐(4‐(1,5‐diphenyl‐4,5‐dihydro‐1H‐pyrazol‐3‐yl)phenyl)‐1H‐pyrrole‐2,5‐dione (probe 10) was reported. The relative quantum yield determined using quinine sulfate for the probe 10 is 42% in 100% acetonitrile (ACN), which was reduced to 6% in 10% ACN‐water system. The turn‐off probe 10 becomes turn‐on selectively in the presence of glutathione (GSH). The Probe 10 is highly sensitive to GSH in the presence of common interfering molecules like cysteine, N‐acetylcysteine, thioglycolic acid, mercaptoethanol, and mercaptopropionoic acid. The limit of detection is 0.06 μM. The practical use of the probe 10 was demonstrated through quantifying GSH (1.68 mM) in the blood serum of healthy person, artificial cerebrospinal fluid, and artificial urine. Probe 10 works on a wide range of pH 2–12. Cells expressing GSH can be imaged using probe 10 and also useful in analysing the percentage of GSH in the cells under oxidative stress. [ABSTRACT FROM AUTHOR]

Purpose: High energetic carbon (C‐) ion beams undergo nuclear interactions with tissue, producing secondary nuclear fragments. Thus, at depth, C‐ion beams are composed of a mixture of different particles with different linear energy transfer (LET) values. We developed a technique to enable isolation of DNA damage response (DDR) in mixed radiation fields using beam line microscopy coupled with fluorescence nuclear track detectors (FNTDs). Methods: We imaged live cells on a coverslip made of FNTDs right after C‐ion, proton or photon irradiation using an in‐house built confocal microscope placed in the beam path. We used the FNTD to link track traversals with DNA damage and separated DNA damage induced by primary particles from fragments. Results: We were able to spatially link physical parameters of radiation tracks to DDR in live cells to investigate spatiotemporal DDR in multi‐ion radiation fields in real time, which was previously not possible. We demonstrated that the response of lesions produced by the high‐LET primary particles associates most strongly with cell death in a multi‐LET radiation field, and that this association is not seen when analyzing radiation induced foci in aggregate without primary/fragment classification. Conclusions: We report a new method that uses confocal microscopy in combination with FNTDs to provide submicrometer spatial‐resolution measurements of radiation tracks in live cells. Our method facilitates expansion of the radiation‐induced DDR research because it can be used in any particle beam line including particle therapy beam lines. Category: Biological Physics and Response Prediction. [ABSTRACT FROM AUTHOR]

The regeneration of lost body parts is a fascinating phenomenon exhibited by some multicellular organisms. In social amoebae, such as Dictyostelium discoideum , the pseudoplasmodium is a temporary migratory multicellular structure with high regeneration ability. It consists of future stalk cells (prestalk cells) at the anterior end and future spore cells (prespore cells) at the posterior end, and if amputated, the remaining cells can rapidly regenerate the lost portion within several hours. Details of this regeneration event have been extensively documented; however, little is known about the behavior of individual cells involved in this process. In this study, we performed live cell imaging of cell behavior during regeneration of the excised anterior prestalk region. We used cells that specifically express GFP in the prestalk cell lineage to examine how the prestalk region is regenerated after this region is excised. The current model of prestalk regeneration suggests that the progenitors of prestalk cells, known as anterior-like cells (ALCs), which are sparsely distributed in the prespore region, are redistributed to form the new prestalk region. However, we found that the regenerated prestalk region was formed mainly by the transdifferentiation of prespore cells surrounding the excised anterior end, with little clustering of pre-existing ALCs. Furthermore, the movement of randomly distributed labeled cells during regeneration revealed that although the posterior end was deformed and rounded in shape, the relative position of cells along the anterior-posterior axis remained largely unchanged. These results suggest that the original anterior-posterior axis is maintained in posterior bodies and that prespore cells at the anterior side transdifferentiate and regenerate the prestalk region. • Social amoebae slug regeneration was analyzed by live cell imaging. • The prestalk region was regenerated mainly by transdifferentiated prespore cells. • Almost no clustering of pre-existing progenitors was observed in the prestalk region. • The relative positions of cells were not much changed during regeneration. [ABSTRACT FROM AUTHOR]

A unique fluorescence chemosensing method has been introduced to explain a new strategy for simultaneous selective determination of cysteine and serine in two different emission channels through an "on‐off‐on" mechanism. Dansyl chloride‐picolylamine conjugate (DNPC) ensembles with Cu2+ (DNPC‐Cu2+) and shows a turn‐on fluorescence while binding with serine or cysteine. Fluorescence, NMR titrations and DFT calculations has been performed in order to understand the sensing mechanism and the electronic properties of the receptor‐donor complex. The chemosensing method has been successfully applied in human cancer cells to detect and quantify cysteine and serine differentially. [ABSTRACT FROM AUTHOR]

High frequency (HF) mechanical vibration has been used in vitro to study the cellular response to mechanical stimulation and induce stem cell differentiation. However, detailed understanding of the effect of the mechanical cues on cellular physiology is lacking. To meet this limitation, we have designed a system, which enables monitoring of living cells by high-resolution light microscopy during mechanical stimulation by HF vibration or mechanical impacts. The system consists of a commercial speaker, and a 3D printed sample vehicle and frame. The speaker moves the sample in the horizontal plane, allowing simultaneous microscopy. The HF vibration (30–200 Hz) performances of two vehicles made of polymer and aluminum were characterized with accelerometer. The mechanical impacts were characterized by measuring the acceleration of the aluminum vehicle and by time lapse imaging. The lighter polymer vehicle produced higher HF vibration magnitudes at 30–50 Hz frequencies than the aluminum vehicle. However, the aluminum vehicle performed better at higher frequencies (60–70 Hz, 90–100 Hz, 150 Hz). Compatibility of the system in live cell experiments was investigated with epithelial cells (MDCKII, expressing Emerald-Occludin) and HF (0.56 Gpeak, 30 Hz and 60 Hz) vibration. Our findings indicated that our system is compatible with high-resolution live cell microscopy. Furthermore, the epithelial cells were remarkable stable under mechanical vibration stimulation. To conclude, we have designed an inexpensive tool for the studies of cellular biophysics, which combines versatile in vivo like mechanical stimuli with live cell imaging, showing a great potential for several cellular applications. [ABSTRACT FROM AUTHOR]

Little is known about the simultaneous uptake of different engineered nanoparticle types, as it can be expected in our daily life. In order to test such co-exposure effects, murine macrophages (J774A.1 cell line) were incubated with gold (AuNPs) and iron oxide nanoparticles (FeOxNPs) either alone or combined. Environmental scanning electron microscopy revealed that single NPs of both types bound within minutes on the cell surface but with a distinctive difference between FeOxNPs and AuNPs. Uptake analysis studies based on laser scanning microscopy, transmission electron microscopy, and inductively coupled plasma optical emission spectrometry revealed intracellular appearance of both NP types in all exposure scenarios and a time-dependent increase. This increase was higher for both AuNPs and FeOxNPs during co-exposure. Cells treated with endocytotic inhibitors recovered after co-exposure, which additionally hinted that two uptake mechanisms are involved. Cross-talk between uptake pathways is relevant for toxicological studies: Co-exposure acts as an uptake accelerant. If the goal is to maximize the cellular uptake, e.g., for the delivery of pharmaceutical agents, this can be beneficial. However, co-exposure should also be taken into account in the case of risk assessment of occupational settings. The demonstration of co-exposure-invoked pathway interactions reveals that synergetic nanoparticle effects, either positive or negative, must be considered for nanotechnology and nanomedicine in particular to develop to its full potential.

The traction forces exerted by an adherent cell on a substrate have been studied only in the two-dimensions (2D) tangential to substrate surface (Txy). We developed a novel technique to measure the three-dimensional (3D) traction forces exerted by live bovine aortic endothelial cells (BAECs) on polyacrylamide deformable substrate. On 3D images acquired by confocal microscopy, displacements were determined with image-processing programs, and traction forces in tangential (XY) and normal (Z) directions were computed by finite element method (FEM). BAECs generated traction force in normal direction (Tz) with an order of magnitude comparable to Txy. Tz is upward at the cell edge and downward under the nucleus, changing continuously with a sign reversal between cell edge and nucleus edge. The method was evaluated regarding accuracy and precision of displacement measurements, effects of FE mesh size, displacement noises, and simple bootstrapping. These results provide new insights into cell-matrix interactions in terms of spatial and temporal variations in traction forces in 3D. This technique can be applied to study live cells to assess their biomechanical dynamics in conjunction with biochemical and functional activities, for investigating cellular functions in health and disease.

Introduction: Arbuscular mycorrhizal (AM) symbiosis between soil fungi and the majority of plants is based on a mutualistic exchange of organic and inorganic nutrients. This takes place inside root cortical cells that harbor an arbuscule: a highly branched intracellular fungal hypha enveloped by an extension of the host cell membrane—the perifungal membrane—which outlines a specialized symbiotic interface compartment. The perifungal membrane develops around each intracellular hypha as the symbiotic fungus proceeds across the root tissues; its biogenesis is the result of an extensive exocytic process and shows a few similarities with cell plate insertion which occurs at the end of somatic cytokinesis. Materials and Methods: We here analyzed the subcellular localization of a GFP fusion with TPLATE, a subunit of the endocytic TPLATE complex (TPC), a central actor in plant clathrin-mediated endocytosis with a role in cell plate anchoring with the parental plasma membrane. Results: Our observations demonstrate that Daucus carota and Medicago truncatula root organ cultures expressing a 35S:: At TPLATE-GFP construct accumulate strong fluorescent green signal at sites of symbiotic interface construction, along recently formed perifungal membranes and at sites of cell-to-cell hyphal passage between adjacent cortical cells, where the perifungal membrane fuses with the plasmalemma. Discussion: Our results strongly suggest that TPC-mediated endocytic processes are active during perifungal membrane interface biogenesis—alongside exocytic transport. This novel conclusion, which might be correlated to the accumulation of late endosomes in the vicinity of the developing interface, hints at the involvement of TPC-dependent membrane remodeling during the intracellular accommodation of AM fungi. [ABSTRACT FROM AUTHOR]

Purpose: BMP signaling has an oncogenic and tumor-suppressing activity in lung cancer that makes the prospective therapeutic utility of BMP signaling in lung cancer treatment complex. A more in-depth analysis of lung cancer subtypes is needed to identify BMP-related therapeutic targets. We sought to examine the influence of BMP signaling on the viability, growth and migration properties of the cell line LCLC-103H, which originates from a large cell lung carcinoma with giant cells and an extended aneuploidy. Methods: We used BMP-4 and LDN-214117 as agonist/antagonist system for the BMP receptor type I signaling. Using flow cytometry, wound healing assay, trans-well assay and spheroid culture, we examined the influence of BMP signaling on cell viability, growth and migration. Molecular mechanisms underlying observed changes in cell migration were investigated via gene expression analysis of epithelial–mesenchymal transition (EMT) markers. Results: BMP signaling inhibition resulted in LCLC-103H cell apoptosis and necrosis 72 h after LDN-214117 treatment. Cell growth and proliferation are markedly affected by BMP signaling inhibition. Chemotactic motility and migratory ability of LCLC-103H cells were clearly hampered by LDN-214117 treatment. Cell migration changes after BMP signaling inhibition were shown to be coupled with considerable down-regulation of transcription factors involved in EMT, especially Snail. Conclusions: BMP signaling inhibition in LCLC-103H cells leads to reduced growth and proliferation, hindered migration and accelerated cell death. The findings contribute to the pool of evidence on BMP signaling in lung cancer with a possibility of introducing BMP signaling inhibition as a novel therapeutic approach for the disease. [ABSTRACT FROM AUTHOR]

We describe a method to obtain the brightness and number of molecules at each pixel of an image stack obtained with a laser scanning microscope. The method is based on intensity fluctuations due to the diffusion of molecules in a pixel. For a detector operating in the analog mode, the variance must be proportional to the intensity. Once this constant has been calibrated, we use the ratio between the variance and the intensity to derive the particle brightness. Then, from the ratio of the intensity to the brightness we obtain the average number of particles in the pixel. We show that the method works with molecules in solution and that the results are comparable to those obtained with fluctuation correlation spectroscopy. We compare the results obtained with the detector operating in the analog and photon counting mode. Although the dynamic range of the detector operating in the photon counting mode is superior, the performance of the analog detector is acceptable under common experimental conditions. Since most commercial laser scanning microscopes operate in the analog mode, the calculation of brightness and number of particles can be applied to data obtained with these instruments, provided that the variance is proportional to the intensity. We demonstrate that the recovered brightness of mEGFP, independent of concentration, is similar whether measured in solution or in two different cell types. Furthermore, we distinguish between mobile and immobile components, and introduce a method to correct for slow variations in intensity.

The process of glycolysis breaks down glycogen stored in muscles, producing lactate through pyruvate to generate energy. Excess lactate is then released into the bloodstream. When lactate reaches the liver, it is converted to glucose, which muscles utilize as a substrate to generate ATP. Although the biochemical study of lactate metabolism in hepatocytes and skeletal muscle cells has been extensive, the spatial and temporal dynamics of this metabolism in live cells are still unknown. We observed the dynamics of metabolism-related molecules in primary cultured hepatocytes and a skeletal muscle cell line upon lactate overload. Our observations revealed an increase in cytoplasmic pyruvate concentration in hepatocytes, which led to glucose release. Skeletal muscle cells exhibited elevated levels of lactate and pyruvate levels in both the cytoplasm and mitochondrial matrix. However, mitochondrial ATP levels remained unaffected, indicating that the increased lactate can be converted to pyruvate but is unlikely to be utilized for ATP production. The findings suggest that excess lactate in skeletal muscle cells is taken up into mitochondria with little contribution to ATP production. Meanwhile, lactate released into the bloodstream can be converted to glucose in hepatocytes for subsequent utilization in skeletal muscle cells. • Glucose is rapidly released into the extracellular space of hepatocytes. • Lactate and pyruvate are elevated in the cytosol of hepatocytes and skeletal muscle. • Increases lactate and pyruvate levels in the skeletal muscle mitochondrial matrix. • Mitochondrial lactate does not contribute to skeletal muscle ATP synthesis. [ABSTRACT FROM AUTHOR]

[Display omitted] • Two novel near-infrared emissive probes were designed and synthesized for mercury ions detection. • CyHg-Cl probe exhibited high photostability, low background fluorescence and biotoxicity. • Mercury ions in living cells and real water samples were successfully monitored. Mercury sensing and imaging in the bio-system is essential for comprehending its toxicity and therapies. Based on the merocyanine scaffold, we designed and synthesized two novel near-infrared (NIR) fluorescent probes for detecting Hg2+. The release of chloro-substituted merocyanine structure on the probe CyHg-Cl enables fluorescence enhancement rapidly by introducing Hg2+. In addition, the probe CyHg-Cl exhibits NIR emission and a low detection limit of 0.59 µM. Finally, the probe CyHg-Cl was used to detect Hg2+ in live cells and real water samples. [ABSTRACT FROM AUTHOR]

Deregulation of the receptor tyrosine kinase MET/hepatocyte growth factor (HGF) pathway results in several pathological processes involved in tumor progression and metastasis. In a different context, MET can serve as an entry point for the bacterium Listeria monocytogenes , when activated by the internalin B (InlB) protein during infection of non-phagocytic cells. We have previously demonstrated that MET requires CD44v6 for its ligand-induced activation. However, the stoichiometry and the steps required for the formation of this complex, are still unknown. In this work, we studied the dynamics of the ligand-induced interaction of CD44v6 with MET at the plasma membrane. Using Förster resonance energy transfer-based fluorescence lifetime imaging microscopy in T-47D cells, we evidenced a direct interaction between MET and CD44v6 promoted by HGF and InlB in live cells. In the absence of MET, fluorescence correlation spectroscopy experiments further showed the dimerization of CD44v6 and the increase of its diffusion induced by HGF and InlB. In the presence of MET, stimulation of the cells by HGF or InlB significantly decreased the diffusion of CD44v6, in line with the formation of a ternary complex of MET with CD44v6 and HGF/InlB. Finally, similarly to HGF/InlB, disruption of liquid-ordered domains (Lo) by methyl-β-cyclodextrin increased CD44v6 mobility suggesting that these factors induce the exit of CD44v6 from the Lo domains. Our data led us to propose a model for MET activation, where CD44v6 dimerizes and diffuses rapidly out of Lo domains to form an oligomeric MET/ligand/CD44v6 complex that is instrumental for MET activation. [Display omitted] • CD44v6 directly interacts with MET upon HGF or InlB induction in live cells. • HGF and InlB promote the dimerization of CD44v6. • MET activation is independent of liquid-ordered domains. [ABSTRACT FROM AUTHOR]

[Display omitted] • Highly fluorescent N, B-doped CNDs discerning interactions with Fe(II) and Fe(III). • The "urn-Off" optical probe relies on a concentration-dependent fluorescence decrease. • The N, B-doped CNDs are selective towards Fe(II) ions in the presence of other metals. • The CNDs can monitor Fe(II) presence in living cells by fluorescent imaging. The interactions between metal ions and fluorescent probes are fundamentals for discerning the metal ions. Co-doped carbon nanodots (CNDs) have demonstrated the capacity to optically detect a wide variety of targets, including metal ions. In this work, the quenching of highly fluorescent, nitrogen (N) and boron (B) co-doped CNDs by Fe(II) ions was discovered and employed to differentiate Fe2+ in water samples and live cells. The fluorescence of the N,B-CNDs was quenched in a concentration-dependent manner with a need of very short reaction time (30 s), and the CNDs show good selectivity towards Fe(II) ions relative to other metal ions including Fe(III). The limit of detection (LOD) was calculated as 1.8 nM and the linear response range was 7 nM to 1 μM. The recovery percentage was determined to be 97%, 101%, and 103% for spiked Fe(II) concentrations of 31, 125, and 1000 nM, respectively, in tap water. The N,B-CNDs were able to monitor the presence of Fe(II) in living cells using the optical imaging of Fe(II) ions, and the quenching in cells was completed within one hour. Regarding the quenching mechanism, the photoinduced electron transfer (PET) procedure is proposed with support of cyclic voltammetry study on the CNDs upon addition of Fe(II). Overall, the N,B-CNDs promise as optical probes for the detection of Fe2+ in water and living cells, and demonstrated a satisfactory level of sensitivity and selectivity. [ABSTRACT FROM AUTHOR]

[Display omitted] • A novel nicotinic hydrazone-based turn-on fluorescence probe 3 has been synthesized for the detection of carbonate ions in an aqueous medium. • The probe showed excellent selectivity, sensitivity, and good reversibility toward CO 3 2– ions. • The deprotonation mechanism of sensing was elucidated by 1H NMR, ESI-MS spectra, and DFT calculations. • An INHIBIT-type molecular logic gate was successfully constructed using probe 3- CO 3 2– and CH 3 COOH. • Probe 3 effectively senses CO 3 2– ions in the real water samples and live cells. A novel turn-on fluorescent probe 3 was synthesized by condensing salicylaldehyde and nicotinic hydrazide for the selective detection of CO 3 2– in aqueous medium. Probe 3 exhibited a turn-on fluorescence response toward CO 3 2– with excellent selectivity, sensitivity (DL = 2.76 μM), and good reversibility. The binding constant (K) of probe 3 with CO 3 2– was calculated to be 5 × 103 M−1 (log K 3.69). The 1:1 stoichiometry of the complex between probe 3 and CO 3 2– ions was confirmed by Job's plot and ESI-MS spectra. Deprotonation and hydrogen-bonding interactions are involved in the recognition of CO 3 2– ion, which was also suggested by 1H NMR, ESI-MS spectra, and Density Functional Theory (DFT) calculations. Moreover, an INHIBIT type molecular logic gate was constructed by using 3:CO 3 2– and CH 3 COOH as inputs and current signal as output. Owing to the practical applications, probe 3 demonstrated its efficiency in quantifying CO 3 2– ion in real water samples through standard addition method, thus showcasing its potential in real environment. Further, the MTT assay indicated very low cytotoxicity (IC 50 = 1 mM) of probe 3 and also the cell imaging experiments demonstrated the effective sensing of CO 3 2– ions with probe 3 in the biological systems. [ABSTRACT FROM AUTHOR]

[Display omitted] Chimeric Antigen Receptor T cell (CAR-T) therapy has emerged as a transformative therapeutic strategy for hematological malignancies. However, its efficacy in treating solid tumors remains limited. An in-depth and comprehensive understanding of CAR-T cell signaling pathways and the ability to track CAR-T cell biodistribution and activation in real-time within the tumor microenvironment will be instrumental in designing the next generation of CAR-T cells for solid tumor therapy. This review summarizes the signaling network and the cellular and molecular imaging tools and platforms that are utilized in CAR-T cell-based immune therapies, covering both in vitro and in vivo studies. Firstly, we provide an overview of the existing understanding of the activation and cytotoxic mechanisms of CAR-T cells, compared to the mechanism of T cell receptor (TCR) signaling pathways. We further describe the commonly employed tools for live cell imaging, coupled with recent research progress, with a focus on genetically encoded fluorescent proteins (FPs) and biosensors. We then discuss the utility of diverse in vivo imaging modalities, including fluorescence and bioluminescence imaging, Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), and photoacoustic (PA) imaging, for noninvasive monitoring of CAR-T cell dynamics within tumor tissues, thereby providing critical insights into therapy's strengths and weaknesses. Lastly, we discuss the current challenges and future directions of CAR-T cell therapy from the imaging perspective. We foresee that a comprehensive and integrative approach to CAR-T cell imaging will enable the development of more effective treatments for solid tumors in the future. [ABSTRACT FROM AUTHOR]

• We review enzyme-free nucleic acid amplification for DNA repair enzyme imaging. • Their sensing mechanisms and practical applications are highlighted. • The performances of different approaches are compared. • We discuss the potential challenges and future perspectives in this field. To maintain the stability and integrity of cellular genomic DNA, DNA repair enzymes catalyze the repair of DNA damage caused by exogenous and endogenous factors through various DNA repair pathways (e.g., base excision repair pathway). Abnormal DNA repair enzyme activity hampers the timely repair of DNA damage, inducing cell growth stagnation, cell death, transformation of normal cells into tumor cells and eventually the occurrence of human diseases including cancer. DNA repair enzymes have become the valuable biomarkers and potential therapeutic targets in biomedical research and clinical diagnosis. To accurately monitor DNA repair enzymes with low expression levels in cells, signal amplification strategies have been introduced to achieve reliable signal sensing and signal gain, facilitating diagnosis and treatment of malignant diseases. Conventional strategies for amplified detection of DNA repair enzymes involve multiple enzymes, washing/separation steps, and thermal cycling, and they are unable to provide spatiotemporal information of intracellular DNA repair enzymes. In recent years, some enzyme-free nucleic acid amplification approaches have been introduced for real-time imaging of intracellular DNA repair enzymes, providing new insights into the dynamic changes of DNA repair enzymes at the single-cell level. Herein, we review the recent advances in enzyme-free nucleic acid amplification-based fluorescent biosensors for the imaging of intracellular DNA repair enzymes. We highlight the mechanisms and applications of these enzyme-free nucleic acid amplification approaches, and further compare the performances of different enzyme-free nucleic acid amplification approaches for DNA repair enzyme imaging. Furthermore, we discuss the potential challenges and future perspectives in this field. [ABSTRACT FROM AUTHOR]

The misfolding and aggregation of specific proteins induced by endogenous oxidation are closely related to some incurable diseases including cancers, diabetes and neurodegenerative diseases. Reactive oxygen species (ROS), as a common oxidizing substance in vivo would induce the oxidation of endogenous biomacromolecules like proteins. However, it remains a challenge to detect protein aggregation induced by specific ROS. Herein, we demonstrate that the developed probe (P2) with a dual function that can simultaneously detect hydrogen peroxide (H 2 O 2) levels and protein aggregates formed by oxidative stress. In this probe, the boronic acid pinacol ester group was conjugated to fluorescent molecular rotor 4-hydroxybenzylidene-imidazolinone (HBI) and served as the H 2 O 2 detection group. Experimental results show that the fluorescence of P2 can be activated by H 2 O 2 and exhibited a positive correlation with the degree of protein aggregation. Moreover, combined with AggTag method P2 was successfully applied to dual-channel imaging of protein aggregation that is stressed by H 2 O 2 in cellular. In general, this work not only provides a potential method to study protein aggregation that associates with oxidation in live cells but also can be generally applied to study other biological processes. • A dual-functional probe activated by hydrogen peroxide (H 2 O 2) and viscosity was designed and synthesized. • The probe responds to H 2 O 2 with high selectivity. • The probe exhibits a cascade response to H 2 O 2 and protein aggregation. • Revealing the process of protein aggregation under H 2 O 2 stress in live cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • A Zn2+ selective turn-on optical sensor (DIH) with ESIPT process was reported. • DIH showed high selectivity and sensitivity Zn2+ over other metal ions tested. • The detection mechanism was elucidated by ESI–MS, NMR and DFT calculations. • The sensor is suitable for imaging Zn2+ in living cells. Zinc ions (Zn2+) play a significant role in our daily lives and production, which is related to human health and environment protection. Therefore, it is crucial in this field to develop sensors with improved analyte selectivity and sensitivity. We prepared a fluorescent sensor, DIH, through the reaction of 5-(tert -butyl)-2-hydroxyisophthalaldehyde with isonicotinohydrazide. DIH, in free form, can undergo excited state intramolecular proton transfer (ESIPT) process upon excitation. Among a wide range of metal ions tested, only Zn2+ inhibited this ESIPT process, resulting in an increase in fluorescence intensity (13.3-fold) with chelation enhancement fluorescence process (CHEF). The complex (DIH/Zn2+) stoichiometry was determined to be 1:1 and the binding constant of the complex was estimated as 3.18 × 107 M−1. DIH could also be applied to detect zinc ions down to a low concentration level of 11.2 nanomolar with fluorescence spectroscopy. Moreover, the cytotoxicity evaluation of DIH against human colon adenocarcinoma cells lines demonstrated that the sensor was nontoxic and could be used successfully in cellular imaging. [ABSTRACT FROM AUTHOR]

Cells are constantly exposed to numerous mutagens that produce diverse types of DNA lesions. Eukaryotic cells have evolved an impressive array of DNA repair mechanisms that are able to detect and repair these lesions, thus preventing genomic instability. The DNA repair process is subjected to precise spatiotemporal coordination, and repair proteins are recruited to lesions in an orderly fashion, depending on their function. Here, we present DNArepairK, a unique open-access database that contains the kinetics of recruitment and removal of 70 fluorescently tagged DNA repair proteins to complex DNA damage sites in living HeLa Kyoto cells. An interactive graphical representation of the data complemented with live cell imaging movies facilitates straightforward comparisons between the dynamics of proteins contributing to different DNA repair pathways. Notably, most of the proteins included in DNArepairK are represented by their kinetics in both nontreated and PARP1/2 inhibitor-treated (talazoparib) cells, thereby providing an unprecedented overview of the effects of anticancer drugs on the regular dynamics of the DNA damage response. We believe that the exclusive dataset available in DNArepairK will be of value to scientists exploring the DNA damage response but, also, to inform and guide the development and evaluation of novel DNA repair-targeting anticancer drugs.

Cell migration is critical for brain development and linked to several neurodevelopmental disorders, including schizophrenia. We have shown previously that cell migration is dysregulated in olfactory neural stem cells from people with schizophrenia. Although they moved faster than control cells on plastic substrates, patient cells were insensitive to regulation by extracellular matrix proteins, which increase the speeds of control cells. As well as speed, cell migration is also described by directional persistence, the straightness of movement. The aim of this study was to determine whether directional persistence is dysregulated in schizophrenia patient cells and whether it is modified on extracellular matrix proteins. Directional persistence in patient-derived and control-derived olfactory cells was quantified from automated live-cell imaging of migrating cells. On plastic substrates, patient cells were more persistent than control cells, with straighter trajectories and smaller turn angles. On most extracellular matrix proteins, persistence increased in patient and control cells in a concentration-dependent manner, but patient cells remained more persistent. Patient cells therefore have a subtle but complex phenotype in migration speed and persistence on most extracellular matrix protein substrates compared to control cells. If present in the developing brain, this could lead to altered brain development in schizophrenia.