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In vitro wound healing assays are a suitable application to verify the efficiency of pharmaceuticals or growth factors that will be incorporated in or immobilized to e.g. electrospun biomaterials for wound dressings or other biological devices in advance. Thereby, various factors like culture conditions or cell density influence the specific cell proliferation. Hence, to establish a wound healing assay for various cell types, a stepwise adaptation of cell numbers was done for better estimation and comparison of cell density for the validation of the influence of drugs on the wound healing process. Cell proliferation of different tissue relevant cell types was evaluated by impedance measurements and live cell imaging. Cell numbers could be successfully adapted for assay specific cell densities. In general, a universal comparison of biological or chemical materials and agents in vitro may require the creation of appropriate ISO or OECD standards for a consistent and cell specific adaptation or demand of initial cell density.

Adoptive cell therapy (ACT) using ex vivo engineered/expanded immune cells demonstrated poor efficacy against solid tumors, despite its great success in treating various hematopoietic malignancies. To improve ACT for solid tumors, it is crucial to comprehend how the numerous components of the tumor microenvironment (TME) surrounding solid tumor cells influence killing ability of immune cells. In this study, we sought to determine the effects of extracellular adhesion provided by extracellular matrix (ECM) of TME on immune cell cytotoxicity by devising microwell arrays coated with proteins either preventing or promoting cell adhesion. Solid tumor cells in bovine serum albumin (BSA)-coated microwells did not attach to the surfaces and exhibited a round morphology, but solid tumor cells in fibronectin (FN)-coated microwells adhered firmed to the substrates with a flat shape. The seeding densities of solid tumor cells and immune cells were tuned to maximize one-to-one pairing within a single microwell, and live cell imaging was performed to examine dynamic cell-cell interactions and immune cell cytotoxicity at a single cell level. Both natural killer (NK) cells and T cells showed higher cytotoxicity against round tumor cells in BSA-coated microwells compared to flat tumor cells in FN-coated microwells, suggesting that extracellular adhesion-mediated firm adhesion of tumor cells made them more resistant to immune cell-mediated killing. Additionally, NK cells and T cells in FN-coated microwells exhibited divergent dynamic behaviors, indicating that two distinct subsets of cytotoxic lymphocytes respond differentially to extracellular adhesion cues during target cell recognition.

Microtubule-targeting (MT) drugs taxanes and vinca alkaloids are widely used as chemotherapeutic agents against different tumors for more than 30 years because of their ability to block mitotic progression by disrupting the mitotic spindle and activating the spindle assembly checkpoint (SAC) for a prolonged period of time. However, responses to mitotic arrest are different—some cells die during mitotic arrest, whereas others undergo mitotic slippage and survive becoming able for proliferation. Using normal fibroblasts and several cancer cell types we determined two critical doses, T1 and T2, of mitotic inhibitors (nocodazole, Taxol, and vinorelbine). T1 is the maximal dose cells can tolerate undergoing normal division, and T2 is the minimal mitostatic dose, wherein > 90% of mitotic cells are arrested in mitosis. In all studied cell lines after treatment with mitotic inhibitors in a dose above T2 cells had entered mitosis either die or undergo mitotic slippage. We show that for all three drugs used cell death during mitotic arrest and after slippage proceeded via mitochondria-dependent apoptosis. We determined two types of cancer cells: sensitive to mitotic arrest, that is, undergoing death in mitosis (DiM) frequently, and resistant to mitotic arrest, that is, undergoing mitotic slippage followed by prolonged survival. We then determined that inhibition of Bcl-xL, but not other anti-apoptotic proteins of the Bcl-2 group that regulate MOMP, make resistant cells susceptible to DiM induced by mitotic inhibitors. Combined treatment with MT drugs and highly specific Bcl-xL inhibitors A-1155643 or A-1331852 allows achieving 100% DiM in a time significantly shorter than maximal duration of mitotic arrest in all types of cultured cells tested. We further examined efficacy of sequential treatment of cultured cells using mitotic inhibitors followed by inhibitors of Bcl-xL anti-apoptotic protein and for the first time show that sensitivity to Bcl-xL inhibitors rapidly declines after mitotic slippage. Thus sequential use of mitotic inhibitors and inhibitors of Bcl-xL anti-apoptotic protein will be efficient only if the Bcl-xL inhibitor will be added before mitotic slippage occurs or soon afterward. The combined treatment proposed might be an efficient approach to anti-cancer therapy.

In the SARS-CoV-2 lifecycle, papain-like protease PLpro cuts off the non-structural proteins nsp1, nsp2, and nsp3 from a large polyprotein. This is the earliest viral enzymatic activity, which is crucial for all downstream steps. Here, we designed two genetically encoded fluorescent sensors for the real-time detection of PLpro activity in live cells. The first sensor was based on the Förster resonance energy transfer (FRET) between the red fluorescent protein mScarlet as a donor and the biliverdin-binding near-infrared fluorescent protein miRFP670 as an acceptor. A linker with the PLpro recognition site LKGG in between made this FRET pair sensitive to PLpro cleavage. Upon the co-expression of mScarlet-LKGG-miRFP670 and PLpro in HeLa cells, we observed a gradual increase in the donor fluorescence intensity of about 1.5-fold. In the second sensor, both PLpro and its target—green mNeonGreen and red mScarletI fluorescent proteins separated by an LKGG-containing linker—were attached to the endoplasmic reticulum (ER) membrane. Upon cleavage by PLpro, mScarletI diffused from the ER throughout the cell. About a two-fold increase in the nucleus/cytoplasm ratio was observed as a result of the PLpro action. We believe that the new PLpro sensors can potentially be used to detect the earliest stages of SARS-CoV-2 propagation in live cells as well as for the screening of PLpro inhibitors. [ABSTRACT FROM AUTHOR]

Cyclic adenosine monophosphate (cAMP) is a canonical intracellular messenger playing diverse roles in cell functions. In neurons, cAMP promotes axonal growth during early development, and mediates sensory transduction and synaptic plasticity after maturation. The molecular cascades of cAMP are well documented, but its spatiotemporal profiles associated with neuronal functions remain hidden. Hence, we developed a genetically encoded cAMP indicator based on a bacterial cAMP-binding protein. This indicator “gCarvi” monitors [cAMP]i at 0.2 to 20 µM with a subsecond time resolution and a high specificity over cyclic guanosine monophosphate (cGMP). gCarvi can be converted to a ratiometric probe for [cAMP]i quantification and its expression can be specifically targeted to various subcellular compartments. Monomeric gCarvi also enables simultaneous multisignal monitoring in combination with other indicators. As a proof of concept, simultaneous cAMP/Ca2+ imaging in hippocampal neurons revealed a tight linkage of cAMP to Ca2+ signals. In cerebellar presynaptic boutons, forskolin induced nonuniform cAMP elevations among boutons, which positively correlated with subsequent increases in the size of the recycling pool of synaptic vesicles assayed using FM dye. Thus, the cAMP domain in presynaptic boutons is an important determinant of the synaptic strength. [ABSTRACT FROM AUTHOR]

Huntingtin is a 3144 amino acid protein defined as a scaffold protein with many intracellular locations that suggest functions in these compartments. Expansion of the CAG DNA tract in the huntingtin first exon is the cause of Huntington's disease. An important tool in understanding the biological functions of huntingtin is molecular imaging at the single-cell level by microscopy and nanoscopy. The evolution of these technologies has accelerated since the Nobel Prize in Chemistry was awarded in 2014 for super-resolution nanoscopy. We are in a new era of light imaging at the single-cell level, not just for protein location, but also for protein conformation and biochemical function. Large-scale microscopy-based screening is also being accelerated by a coincident development of machine-based learning that offers a framework for truly unbiased data acquisition and analysis at very large scales. This review will summarize the newest technologies in light, electron, and atomic force microscopy in the context of unique challenges with huntingtin cell biology and biochemistry. [ABSTRACT FROM AUTHOR]

The transcription factor family of nuclear factor kappa B (NF-κB) proteins is widely recognized as a key player in inflammation and the immune responses, where it plays a fundamental role in translating external inflammatory cues into precise transcriptional programs, including the timely expression of a wide variety of cytokines/chemokines. Live cell imaging in single cells showed approximately 15 years ago that the canonical activation of NF-κB upon stimulus is very dynamic, including oscillations of its nuclear localization with a period close to 1.5 hours. This observation has triggered a fruitful interdisciplinary research line that has provided novel insights on the NF-κB system: how its heterogeneous response differs between cell types but also within homogeneous populations; how NF-κB dynamics translate external cues into intracellular signals and how NF-κB dynamics affects gene expression. Here we review the main features of this live cell imaging approach to the study of NF-κB, highlighting the key findings, the existing gaps of knowledge and hinting towards some of the potential future steps of this thriving research field. [ABSTRACT FROM AUTHOR]

Basic neuroscience research employs numerous forms of antibodies as key reagents in diverse applications. While the predominant use of antibodies is as immunolabeling reagents, neuroscientists are making increased use of intracellular antibodies or intrabodies. Intrabodies are recombinant antibodies genetically encoded for expression within neurons. These can be used to target various cargo (fluorescent proteins, reporters, enzymes, etc.) to specific molecules and subcellular domains to report on and manipulate neuronal function with high precision. Intrabodies have the advantages inherent in all genetically encoded recombinant antibodies but represent a distinct subclass in that their structure allows for their expression and function within cells. The high precision afforded by the ability to direct their expression to specific cell types, and the selective binding of intrabodies to targets within these allows intrabodies to offer unique advantages for neuroscience research, given the tremendous molecular, cellular and morphological complexity of brain neurons. Intrabodies expressed within neurons have been used for a variety of purposes in basic neuroscience research. Here I provide a general background to intrabodies and their development, and examples of their emerging utility as valuable basic neuroscience research tools. [ABSTRACT FROM AUTHOR]

Muscle regeneration is essential for proper muscle homeostasis and relies primarily on muscle stem cells (MuSC). MuSC are maintained quiescent in their niche and can be activated following muscle injury. Using an in vitro model of primary human quiescent MuSC (called reserve cells, RC), we analyzed their Ca2+ response following their activation by fetal calf serum and assessed the role of Ca2+ in the processes of RC activation and migration. The results showed that RC displayed a high response heterogeneity in a cell-dependent manner following serum stimulation. Most of these responses relied on inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release associated with Ca2+ influx, partly due to store-operated calcium entry. Our study further found that blocking the IP3 production, Ca2+ influx, or both did not prevent the activation of RC. Intra- or extracellular Ca2+ chelation did not impede RC activation. However, their migration potential depended on Ca2+ responses displayed upon stimulation, and Ca2+ blockers inhibited their movement. We conclude that the two major steps of muscle regeneration, namely the activation and migration of MuSC, differently rely on Ca2+ signals. [ABSTRACT FROM AUTHOR]

Single-cell imaging has emerged as a powerful means to study viral replication dynamics and identify sites of virus–host interactions. Multivariate aspects of viral replication cycles yield challenges inherent to handling large, complex imaging datasets. Herein, we describe the design and implementation of an automated, imaging-based strategy, "Human Immunodeficiency Virus Red-Green-Blue" (HIV RGB), for deriving comprehensive single-cell measurements of HIV-1 unspliced (US) RNA nuclear export, translation, and bulk changes to viral RNA and protein (HIV-1 Rev and Gag) subcellular distribution over time. Differentially tagged fluorescent viral RNA and protein species are recorded using multicolor long-term (>24 h) time-lapse video microscopy, followed by image processing using a new open-source computational imaging workflow dubbed "Nuclear Ring Segmentation Analysis and Tracking" (NR-SAT) based on ImageJ plugins that have been integrated into the Konstanz Information Miner (KNIME) analytics platform. We describe a typical HIV RGB experimental setup, detail the image acquisition and NR-SAT workflow accompanied by a step-by-step tutorial, and demonstrate a use case wherein we test the effects of perturbing subcellular localization of the Rev protein, which is essential for viral US RNA nuclear export, on the kinetics of HIV-1 late-stage gene regulation. Collectively, HIV RGB represents a powerful platform for single-cell studies of HIV-1 post-transcriptional RNA regulation. Moreover, we discuss how similar NR-SAT-based design principles and open-source tools might be readily adapted to study a broad range of dynamic viral or cellular processes. [ABSTRACT FROM AUTHOR]

Advances in fluorescence microscopy can greatly facilitate research in regenerative health. Specifically, live cell imaging is a powerful tool to understand the underlying mechanisms of tissue regeneration, which is characterised by a dynamic interplay at cellular and molecular level. Recent advances in microscopy have aimed to overcome some of the most challenging limitations, such as slow acquisition speed, the resolution limit of light, low signal to noise ratio in thick samples, as well as photobleaching and phototoxicity. In applications such as lightsheet fluorescence microscopy and intra-vital multi-photon microscopy, improved deep tissue imaging have been achieved, and super-resolution technologies have shown to improve optical resolution far beyond the diffraction limit of light for better visualisation at the cellular and molecular level. By combining certain techniques, researchers can now image live samples at much higher resolution for a prolonged time. Advances in analytical technologies will enable researchers to gain an even better understanding of the processes involved to ultimately translate stem cell research into therapeutic interventions. • Advances in fluorescence microscopy allows for in vivo imaging of live animals. • 3D deep tissue imaging is a leap from the conventional 2D applications. • Super–resolution microscopy continues to improve. • Combining microscopy technologies overcome limitations of live cell imaging. • Increased imaging speed of thick samples at sub-resolution level is now possible. [ABSTRACT FROM AUTHOR]

Background: Accurately detecting and quantifying tumor-related microRNAs (miRNAs) in living cells is of great value for early cancer diagnosis. Herein, we present poly-adenine (polyA)-mediated spherical nucleic acid (SNA) nanoprobes for intracellular miRNA imaging in living cells. Methods and results: polyA-mediated spherical nucleic acid (pASNA) nanoprobes consist of gold nanoparticles (AuNPs) anchored with fluorophore-labeled DNA molecules pre-hybridized with recognition sequences and polyA tails. The detection performance for miRNAs in vitro was studied to confirm the feasibility of pASNA nanoprobes for imaging live cell miRNAs. Before the pASNA nanoprobes were used for imaging intracellular miRNAs in MCF-7, HeLa, and LO2 cells, the stability and non-cytotoxicity were investigated using Dnase I and a standard colorimetric CCK8 assay. Flow cytometry, qRT-PCR analyses were conducted to confirm the different expression levels of miR-155 in live cells. Results showed that the pASNA nanoprobes had good detection sensitivity and specificity, excellent stability, and low toxicity. After incubating with pASNA nanoprobes, noticeable fluorescence signal enhancement could be clearly observed in MCF-7 and HeLa cells but not LO2 cells by confocal microscopy. Flow cytometry analysis and qRT-PCR indicated that MCF-7 and HeLa cells had higher miR-155 expression levels compared to LO2 cells. Conclusions: The pASNA nanoprobes we developed had good sensitivity and specificity, excellent nuclease stability and low toxicity, thus representing a new approach to exquisitely reveal the distribution of endogenous miRNAs in live cells. [ABSTRACT FROM AUTHOR]

Co-culture spheroids mimic tumor architecture more accurately than traditional 2D cell cultures, but non-invasive, long-term tracking of live cells within these 3D models remains a challenge. This study addresses this critical need by developing a novel approach for live cell imaging in U-87/HUF co-culture spheroids. We introduce water-soluble, biocompatible red carbon dots (R-CDs) exhibiting exceptional stability and brightness (21% quantum yield) specifically designed for imaging within these 3D models. Furthermore, we designed a microfluidic chip with ellipsoid-shaped microwells to efficiently generate two distinct co-culture spheroid types: direct mixing and core-shell. R-CDs enabled non-invasive tracking of U-87 cancer cell location within these 3D models demonstrating their efficacy for long-term monitoring of live cells in cancer research. This R-CD and microfluidic technology has the potential to accelerate cancer drug discovery by enabling live cell studies in 3D tumor models. [Display omitted] [ABSTRACT FROM AUTHOR]

Human serum albumin (HSA) levels in the body are crucial for the accurate diagnosis of various health issues, necessitating continuous monitoring and novel detection methods. This study focused on the development and synthesis of two novel D-π-A small molecule near-infrared fluorescent probes, PTPA-B and CPTPA-B. The probes demonstrated selective detection of HSA with a rapid response time of 1 min and high sensitivity, featuring limits of detection (LOD) of 0.102 μM and 1.702 μM. Under physiological conditions, the intrinsic fluorescence of these probes is minimal due to intramolecular charge transfer (TICT). Upon entering the hydrophobic cavity of HSA, the probes exhibit red fluorescence by inhibiting the TICT process. Additionally, the probes exhibited enhanced biocompatibility in living cells and enabled fluorescence imaging of L-02 live cells. Therefore, the PTPA-B fluorescent probe serves as an effective tool for serum detection, revealing its potential applications in live cell imaging and precise clinical diagnosis. • Designed and synthesized two novel D-π-A near-infrared fluorescent probes with high selectivity and sensitivity for HSA detection. • The probes provide rapid HSA detection with a 1-min response time and low detection limits (0.102 μM for PTPA-B and 1.702 μM for CPTPA-B). • The probes are effective in live cell imaging, offering enhanced biocompatibility and potential for clinical diagnostics. [ABSTRACT FROM AUTHOR]

[Display omitted] • A new fluorescent probe for ratiometric detection of Zn2+ ion at physiological pH. • The probe displays red emission, large stokes shift and fast response time. • Vivid color change for naked-eye recognition. • Capable of imaging exogenous/endogenous Zn2+ levels in live cells. A novel ratiometric fluorescent probe, namely 5-[(3-dicyanoylidene −5.5-dimethyl) cyclohexenyl-1-ethenyl] salicylaldehyde-3′-hydroxybenzohydrazone (DCSH) is presented for the selective sensing of Zn2+ ion in acetonitrile/water (2/3, pH 7.4) solution. Introducing Zn2+ ions notably caused the peak emission of DCSH to shift from 560 nm to 646 nm, accompanied with a significant enhancement of its intensity. A vivid change in fluorescence color from yellow to red facilitated the immediate identification of Zn2+ ions by visual observation. DCSH exhibits substantial Stokes shifts (110 and 196 nm), rapid detection capability (within 10 s) and high sensitivity to Zn2+ ions, achieving a limit of detection of 31.2 nM. The response mechanism is supposed to involve the block of C = N bond isomerization and excited state intramolecular proton transfer (ESIPT) along with the enhancement of fluorescence through chelation (CHEF) effect. DCSH was effectively utilized for ratiometric fluorescence imaging to monitor exogenous Zn2+ concentrations in HeLa cells. Significantly, DCSH is capable of monitoring elevated levels of Zn2+ ion during apoptosis induced by L-Buthionine sulfoximine (BSO). [ABSTRACT FROM AUTHOR]

For proper mammalian brain development and functioning, the translation of many neuronal mRNAs needs to be repressed without neuronal activity stimulations. We have discovered that the expression of a subclass of neuronal proteins essential for neurodevelopment and neuron plasticity is co-regulated at the translational level by TDP-43 and the Fragile X Syndrome protein FMRP. Using molecular, cellular and imaging approaches, we show that these two RNA-binding proteins (RBP) co-repress the translation initiation of Rac1, Map1b and GluR1 mRNAs, and consequently the hippocampal spinogenesis. The co-repression occurs through binding of TDP-43 to mRNA(s) at specific UG/GU sequences and recruitment of the inhibitory CYFIP1-FMRP complex by its glycine-rich domain. This novel regulatory scenario could be utilized to silence a significant portion of around 160 common target mRNAs of the two RBPs. The study establishes a functional/physical partnership between FMRP and TDP-43 that mechanistically links several neurodevelopmental disorders and neurodegenerative diseases.

The transcription factor family of nuclear factor kappa B (NF-κB) proteins is widely recognized as a key player in inflammation and the immune responses, where it plays a fundamental role in translating external inflammatory cues into precise transcriptional programs, including the timely expression of a wide variety of cytokines/chemokines. Live cell imaging in single cells showed approximately 15 years ago that the canonical activation of NF-κB upon stimulus is very dynamic, including oscillations of its nuclear localization with a period close to 1.5 hours. This observation has triggered a fruitful interdisciplinary research line that has provided novel insights on the NF-κB system: how its heterogeneous response differs between cell types but also within homogeneous populations; how NF-κB dynamics translate external cues into intracellular signals and how NF-κB dynamics affects gene expression. Here we review the main features of this live cell imaging approach to the study of NF-κB, highlighting the key findings, the existing gaps of knowledge and hinting towards some of the potential future steps of this thriving research field.

The cornea is exposed daily to a number of mechanical stresses including shear stress from tear film and blinking. Over time, these stressors can lead to changes in the extracellular matrix that alter corneal stiffness, cell-substrate structures, and the integrity of cell-cell junctions. We hypothesized that changes in tissue stiffness of the cornea with age may alter calcium signaling between cells after injury, and the downstream effects of this signaling on cellular motility and wound healing. Nanoindentation studies revealed that there were significant differences in the stiffness of the corneal epithelium and stroma between corneas of 9- and 27-week mice. These changes corresponded to differences in the timeline of wound healing and in cell signaling. Corneas from 9-week mice were fully healed within 24 h. However, the wounds on corneas from 27-week mice remained incompletely healed. Furthermore, in the 27-week cohort there was no detectable calcium signaling at the wound in either apical or basal corneal epithelial cells. This is in contrast to the young cohort, where there was elevated basal cell activity relative to background levels. Cell culture experiments were performed to assess the roles of P2Y2, P2X7, and pannexin-1 in cellular motility during wound healing. Inhibition of P2Y2, P2X7, or pannexin-1 all significantly reduce wound closure. However, the inhibitors all have different effects on the trajectories of individual migrating cells. Together, these findings suggest that there are several significant differences in the stiffness and signaling that underlie the decreased wound healing efficacy of the cornea in older mice.

Abstract Plant cell wall-derived biomass serves as a renewable source of energy and materials with increasing importance. The cell walls are biomacromolecular assemblies defined by a fine arrangement of different classes of polysaccharides, proteoglycans, and aromatic polymers and are one of the most complex structures in Nature. One of the most challenging tasks of cell biology and biomass biotechnology research is to image the structure and organization of this complex matrix, as well as to visualize the compartmentalized, multiplayer biosynthetic machineries that build the elaborate cell wall architecture. Better knowledge of the plant cells, cell walls, and whole tissue is essential for bioengineering efforts and for designing efficient strategies of industrial deconstruction of the cell wall-derived biomass and its saccharification. Cell wall-directed molecular probes and analysis by light microscopy, which is capable of imaging with a high level of specificity, little sample processing, and often in real time, are important tools to understand cell wall assemblies. This review provides a comprehensive overview about the possibilities for fluorescence label-based imaging techniques and a variety of probing methods, discussing both well-established and emerging tools. Examples of applications of these tools are provided. We also list and discuss the advantages and limitations of the methods. Specifically, we elaborate on what are the most important considerations when applying a particular technique for plants, the potential for future development, and how the plant cell wall field might be inspired by advances in the biomedical and general cell biology fields.

[Display omitted] • The review explores the versatile capabilities of fluorocolorimetric probes developed in Dr S Velmathi's lab at NIT Trichy for analyte detection, imaging, and real-time applications. • They exhibit distinctive visual signals when interacting with specific targets, marking a significant advancement in molecular interaction analysis. • The probes cover a broad spectrum of analytes, including metal ions, anions, biomolecules, ROS, RSS, toxic agents, and nanoparticles. • The review emphasizes the profound impact of fluorocolorimetric probes on developing analytical potential. • These probes contribute to innovative imaging techniques and expand the possibilities of real-time applications. Fluorescent and colorimetric probes are gaining interest in analyte detection, imaging, and real-time applications. However, it faces several challenges in achieving high sensitivity and selectivity, maintaining a strong signal-to-noise ratio, detecting analytes at extremely low concentrations, ensuring stability under different conditions and addressing issues related to cost, accessibility and instrumentation. Additionally, ensuring biological compatibility and enabling real-time monitoring are also challenges that need to overcome. We have investigated the sensing application of probe molecules towards various analytes like metal ions, anions, biomolecules, reactive oxygen species (ROS), reactive sulphur species (RSS), toxic agents and nanoparticles performed by our group together with critically comparing with the existing reports. This featured article provides the impact of fluorescent and colorimetric probes in advancing analytical techniques, imaging and real-time applications that transcend traditional boundaries. [ABSTRACT FROM AUTHOR]

Micro- and nanoplastics (MNPs) are ubiquitous in the environment, resulting in the uptake of MNPs by a variety of organisms, including humans, leading to particle-cell interaction. Human macrophages derived from THP-1 cell lines take up Polystyrene (PS), a widespread plastic. The question therefore arises whether primary human macrophages also take up PS micro- and nanobeads (MNBs) and how they react to this stimulation. Major aim of this study is to visualize this uptake and to validate the isolation of macrophages from peripheral blood mononuclear cells (PBMCs) to assess the impact of MNPs on human macrophages. Uptake of macrophages from THP-1 cell lines and PBMCs was examined by transmission electron microscopy (TEM), scanning electron microscopy and live cell imaging. In addition, the reaction of the macrophages was analyzed in terms of metabolic activity, cytotoxicity, production of reactive oxygen species (ROS) and macrophage polarization. This study is the first to visualize PS MNBs in primary human cells using TEM and live cell imaging. Metabolic activity was size- and concentration-dependent, necrosis and ROS were increased. The methods demonstrated in this study outline an approach to assess the influence of MNP exposure on human macrophages and help investigating the consequences of worldwide plastic pollution. [Display omitted] • Human monocyte-derived macrophages as novel in vitro model to study effects of MNP. • Concentration-dependent cytoplasmic uptake of PS MNBs visualized by TEM and SEM. • Uptake of PS MNBs by primary human macrophages is visible in live cell imaging. • Effects of different sized, non-fluorescent PS-MNBs in physiological concentration. • PS MNBs induce nitric oxide formation and cytotoxic effects in human macrophages. [ABSTRACT FROM AUTHOR]

DNA-functionalized nanomaterials represent the cutting edge of the convergence between nanoscience and life sciences. The gold nanoparticles (AuNPs), with their versatile attributes, serve as multifunctional carriers in complex environments, supporting diverse physiological activities of DNA. Concurrently, DNA enhances the target specificity and stability of nanomaterials, significantly improving or fine-tuning their performance. This review explores key aspects of DNA-functionalized AuNPs, including their synthesis methods, properties, and biosensing applications. It starts by delving deeply into the interactions between DNA and AuNPs, emphasizing their role in material customization and highlighting the advantages and applicability of each technique. Furthermore, the review thoroughly evaluates the latest synthesis methods developed from shielding the charger repulsion and extremely compressing the physical space. It showcases the potential of DNA-functionalized AuNPs in addressing complex challenges for biosensing applications. This synergistic relationship has propelled advancements in nanotechnology, facilitating the integration of life sciences, medicine, and nanotechnology, and expanding the application domains of these novel biomimetic nanocomposite materials. Therefore, there is an urgent need for in-depth research into the synthesis, structure and function of DNA-functionalized nanomaterials to unravel their pivotal mechanisms. Illustration of the application of DNA-functionalized gold nanomaterials in the field of biosensing. [Display omitted] • Mechanisms behind those milestones during the evolution of synthesizing DNA-functionalized AuNPs have been extensively discussed. • Engineering DNA-AuNPs enables biosensing of biomolecules for more intuitive and accurate access to spatiotemporal information. • Continuous innovation of synthesis enhances the efficiency in designing more functionalized nanomaterials across diverse fields. [ABSTRACT FROM AUTHOR]

Emerging studies demonstrates that mitochondrial G-quadruplex (G4) may be a prospective diagnostic and therapeutic target for cancer. Fluorescent probes targeting mitochondrial G4 are crucial for in-depth research on G4 function and mechanism and developing new diagnostic techniques. Here, we report a benzothiazole derivative named ThT-E, which can be prepared with high yield through only one step of reaction. ThT-E exhibits superior detection sensitivity compared to existing probes, and also demonstrates reliable performance for in situ characterization of mitochondrial G4 in living cells. Interestingly, ThT-E has been used to differentiate tumor cells from normal cells by quantifying the formation of mitochondrial G4s, implicating that ThT-E may have wide applications in tumor diagnosis. • A benzothiazole derivative for monitoring mitochondrial G-quadruplexes. • More pronounced fluorescence enhancement in tumor cells than normal cells. • More G-quadruplex formation in tumor mitochondria. • Mitochondrial G-quadruplex as a potential cancer biomarker. • New probe tool for tumor diagnosis. [ABSTRACT FROM AUTHOR]

Determining the activity of lysosomal β-hexosaminidase in cells is of great importance for understanding the roles that these enzymes play in pathophysiological events. Herein, we designed the new fluorescent probe, βGalNAc-Rhod-CM(NEt 2), which consisted of a βGalNAc-linked rhodol unit serving as a β-hexosaminidase reactive fluorogenic moiety and a N,N′ -diethylaminocoumarin (CM(NEt 2)) group acting as a fluorescence marker for determining the degree of cell permeabilization. Treatment of βGalNAc-Rhod-CM(NEt 2) with β-hexosaminidase promoted generation of Rhod-CM(NEt 2), thereby leading to an increase in the intensity of fluorescence of Rhod. However, this probe did not respond to the functionally related glycosidase, O-GlcNAcase. The detection limit of βGalNAc-Rhod-CM(NEt 2) for β-hexosaminidase was determined to be 0.52 nM, indicating that it has high sensitivity for this enzyme. Furthermore, the probe functioned as an excellent fluorogenic substrate for β-hexosaminidase with k cat and K m values of 17 sec−1 and 22 μM, respectively. The results of cell studies using βGalNAc-Rhod-CM(NEt 2) showed that levels of β-hexosaminidase activity in cells can be determined by measuring the intensity of fluorescence arising from Rhod and that the intensity of fluorescence of CM(NEt 2) can be employed to determine the degree of cell permeabilization of the probe. Utilizing the new probe, we assessed β-hexosaminidase activities in several types of cells and evaluated the effect of glucose concentrations in culture media on the activity of this enzyme. [Display omitted] • Development of the sensitive and selective fluorescent probe for β-hexosaminidase. • Determination of levels of β-hexosaminidase activities in several types of cells. • Evaluation of effect of glucose concentrations in media on β-hexosaminidase activity. [ABSTRACT FROM AUTHOR]

Enterovirus consists of a variety of viruses that could cause a wide range of illness in human. The pathogenesis of these viruses remains incompletely understood and no specific treatment is available. Better methods to study enterovirus infection in live cells will help us better understand the pathogenesis of these viruses and might contribute to antiviral development. Here in this study, we developed fluorescent cell-based reporter systems that allow sensitive distinction of individual cells infected with enterovirus 71 (EV71). More importantly, these systems could be easily used for live-cell imaging by monitoring viral-induced fluorescence translocation after EV71 infection. We further demonstrated that these reporter systems could be used to study other enterovirus-mediated MAVS cleavage and they are sensitive for antiviral activity testing. Therefore, integration of these reporters with modern image-based analysis has the potential to generate new insights into enterovirus infection and facilitate antiviral development.

Plastic in oceans degrades to microplastics and nanoplastics, causing various problems for marine fauna and flora. Recently, microplastic has been detected in blood, breast milk and placenta, underlining their ability to enter the human body with still unknown effects. In addition, plastic contains other compounds such as plasticizers, antioxidants or lubricants, whose impact on human health is also elusive. On the cellular level, two transporters involved in cell protection and detoxification of xenobiotic compounds are the ABC-transporters P-glycoprotein (P-gp, MDR1, ABCB1) and breast cancer resistance protein (BCRP, ABCG2). Despite the great importance of these proteins to maintain the correct cellular balance, their interaction with plastic and related products is evasive. In this study, the possible interaction between different plastic-related compounds and these two transporters was investigated. Applying virtual compound screening and molecular docking of more than 1000 commercially available plastic compounds, we identified candidates most probably interacting with these two transporters. Cytotoxicity and uptake assays confirmed their toxic interaction on P-glycoprotein-overexpressing CEM/ADR5000 and BCRP-overexpressing MDA-MD-231-BCRP cell lines. To specifically visualize the results obtained on the P-glycoprotein inhibitor 2,2’-methylenebis(6-tert-butyl-4-methylphenol), we performed live cell time-lapse microscopy. Confocal fluorescence microscopy was used to understand the behavior of the molecule and the consequences that it has on the uptake of the well-known substrate doxorubicin and, in comparison, with the known inhibitor verapamil. Based on the results, we provide evidence that the compound in question is an inhibitor of the P-glycoprotein. Moreover, it is also possible that 2,2’-methylenebis(6-tert-butyl-4-methylphenol), together with three other compounds, may also inhibit the breast cancer resistance protein. This discovery implies that plastic-related compounds can not only harm the human body but can also inhibit detoxifying efflux pumps, which increases their toxic potential as these transporters lose their physiological functions.

The free calcium (Ca2+) levels in pancreatic beta cell organelles have been the subject of many recent investigations. Under pathophysiological conditions, disturbances in these pools have been linked to altered intracellular communication and cellular dysfunction. To facilitate studies of subcellular Ca2+ signaling in beta cells and, particularly, signaling between the endoplasmic reticulum (ER) and mitochondria, we designed a novel dual Ca2+ sensor which we termed DS-1. DS-1 encodes two stoichiometrically fluorescent proteins within a single plasmid, G-CEPIA-er, targeted to the ER and R-CEPIA3-mt, targeted to mitochondria. Our goal was to simultaneously measure the ER and mitochondrial Ca2+ in cells in real time. The Kds of G-CEPIA-er and R-CEPIA3-mt for Ca2+ are 672 and 3.7 μM, respectively. Confocal imaging of insulin-secreting INS-1 832/13 expressing DS-1 confirmed that the green and red fluorophores correctly colocalized with organelle-specific fluorescent markers as predicted. Further, we tested whether DS-1 exhibited the functional properties expected by challenging an INS-1 cell to glucose concentrations or drugs having well-documented effects on the ER and mitochondrial Ca2+ handling. The data obtained were consistent with those seen using other single organelle targeted probes. These results taken together suggest that DS-1 is a promising new approach for investigating Ca2+ signaling within multiple organelles of the cell.

Time-lapse light microscopy combined with in vitro neuronal cultures has provided a significant contribution to the field of Developmental Neuroscience. The establishment of the neuronal polarity, i.e., formation of axons and dendrites, key structures responsible for inter-neuronal signaling, was described in 1988 by Dotti, Sullivan and Banker in a milestone paper that continues to be cited 30 years later. In the following decades, numerous fluorescently labeled tags and dyes were developed for live cell imaging, providing tremendous advancements in terms of resolution, acquisition speed and the ability to track specific cell structures. However, long-term recordings with fluorescence-based approaches remain challenging because of light-induced phototoxicity and/or interference of tags with cell physiology (e.g., perturbed cytoskeletal dynamics) resulting in compromised cell viability leading to cell death. Therefore, a label-free approach remains the most desirable method in long-term imaging of living neurons. In this paper we will focus on label-free high-resolution methods that can be successfully used over a prolonged period. We propose novel tools such as scanning ion conductance microscopy (SICM) or digital holography microscopy (DHM) that could provide new insights into live cell dynamics during neuronal development and regeneration after injury.

M onoxenous Eimeria species are widespread enteropathogenic apicomplexan protozoa with a high economic impact on livestock. In cattle, tenacious oocysts shed by E. bovis-infected animals are ubiquitously found and making infection of calves almost inevitable. To become infectious oocysts, exogenous oxygen-dependent E. bovis sporogony must occur leading to the formation of sporulated oocysts containing four sporocysts each harboring two sporozoites. Investigations on sporogony by live cell imaging techniques of ruminant Eimeria species are still absent in literature as commonly used fluorescent dyes do not penetrate resistant oocyst bi-layered wall. Sporogonial oocysts were daily analyzed by a 3D Cell Explorer Nanolive microscope to explore ongoing aerobic-dependent sporogony as close as possible to an in vivo situation. Subsequently, 3D holotomographic images of sporulating E. bovis oocysts were digitally stained based on refractive indices (RI) of oocyst bi-layered wall and sub-compartments of circumplasm using STEVE software (Nanolive), and the cellular morphometric parameters were obtained. Overall, three different E. bovis sporogony phases, each of them divided into two sub-phases, were documented: (i) sporoblast/sporont transformation into sporogonial stages, (ii) cytokinesis followed by nuclear division, and finally (iii) formation of four sporocysts with two fully developed sporozoites. Approximately 60% of sporulating E. bovis oocysts accomplished aerobic sporogony in a synchronized manner. E. bovis sporogony was delayed (i.e., 6 days) when compared to an in vivo situation where 2–3 days are required but under optimal environmental conditions. Live cell 3D holotomography analysis might facilitate the evaluation of either novel disinfectants- or anti-coccidial drug-derived effects on ruminant/avian Eimeria sporogony in vitro as discrimination of sporogony degrees based on compactness, and dry mass was here successfully achieved. Main changes were observed in the oocyst area, perimeter, compactness, extent, and granularity suggesting those parameters as an efficient tool for a fast evaluation of the sporulation degree. [ABSTRACT FROM AUTHOR]

Bacteria are unicellular organisms whose length is usually around a few micrometers. Advances in microfabrication techniques have enabled the design and implementation of microdevices to confine and observe bacterial colony growth. Microstructures hosting the bacteria and microchannels for nutrient perfusion usually require separate microfabrication procedures due to different feature size requirements. This fact increases the complexity of device integration and assembly process. Furthermore, long-term imaging of bacterial dynamics over tens of hours requires stability in the microscope focusing mechanism to ensure less than one-micron drift in the focal axis. In this work, we design and fabricate an integrated multi-level, hydrodynamically-optimized microfluidic chip to study long-term Escherichia coli population dynamics in confined microchannels. Reliable long-term microscopy imaging and analysis has been limited by focus drifting and ghost effect, probably caused by the shear viscosity changes of aging microscopy immersion oil. By selecting a microscopy immersion oil with the most stable viscosity, we demonstrate successful captures of focally stable time-lapse bacterial images for ≥72 h. Our fabrication and imaging methodology should be applicable to other single-cell studies requiring long-term imaging. [ABSTRACT FROM AUTHOR]

Determining the position of cells in a microscope image is essential in investigating cell behaviors such as migration and differentiation. When observing cells over a time period over many ROIs (region of interests), it is difficult to reposition the sample and find the same cell once the FOV (field of view) is changed. This study describes a novel approach to obtain accurate position data in live-cell time-lapse imaging and large-area stitched images using a Petri-dish with embedded microstructures and a simple microscope. We integrated a braille-like pattern underneath the Petri-dish, a coordinate indicator for microscope image registration to simplify imaging of multiple ROIs at different time points without expensive equipment such as motorized stages or enclosed cell culture incubators. In this study, we micromolded microscopic structures with sizes between 5–10 μm spaced closely (~ 40 μm) in non-repeating pattern underneath the petri-dish. Image processing technique was used to determine the position and rotation of the petri-dish with respect to the microscope FOV. Using this methods, we demonstrated successful imaging of time-lapse images for cell differentiation and migration. Large area patterned cell cultures were imaged over several days and the images were stitched without a motorized microscope. [ABSTRACT FROM AUTHOR]

ESIPT active PBI-keto/enol assemblies have been developed which show 'on-on' optical response towards 2,6- dichloro-4-nitroaniline (DCN) due to a combined ESIPT-AIEE phenomenon with a detection limit of 1.65 nM. The potential of PBI-keto/enol assemblies to detect DCN has also been explored in grape juice/grape residue and soil for six consecutive days. Further, the biological applications of PBIketo/ enol assemblies to detect DCN in blood serum, in MG- 63 cell lines and their ability to restrict the DCN-induced cell death have been demonstrated. [ABSTRACT FROM AUTHOR]

Single molecule localization (SML) and tracking (SPT) techniques, such as (spt)PALM, (u/DNA)PAINT and quantum dot tracking, have given unprecedented insight into the nanoscale molecular organization and dynamics in living cells. They allow monitoring individual proteins with millisecond temporal resolution and high spatial resolution (

Hepatosteatosis is a common metabolic disorder of dairy cows, especially during early lactation. Currently, there are a few models of bovine hepatic steatosis available, including primary hepatocytes, liver slices, and animal models. Studies that elucidate the influence of single fatty acids on lipid classes, fatty acid pattern, gene expression, and phenotypic changes are still limited. Hence, we investigated the suitability of the fetal bovine hepatocyte-derived cell line BFH12 as a model for hepatosteatosis. To create a steatotic environment, we treated BFH12 with stearic acid, palmitic acid, or oleic acid in non-toxic doses. Thin-layer chromatography and gas chromatography were used to analyze lipid classes and fatty acid pattern, and qPCR was used to quantify gene expression of relevant target genes. Lipid droplets were visualized with confocal laser scanning microscopy and evaluated for number and size. Treatment with oleic acid increased triglycerides, as well as lipid droplet count per cell and upregulated carnitine palmitoyl transferase 1, which correlates with findings of in vivo models. Oleic acid was largely incorporated into triglycerides, phospholipids, and non-esterified fatty acids. Stearic acid was found mainly in non-esterified fatty acids and triglycerides, whereas palmitic acid was mainly desaturated to palmitoleic acid. All three fatty acids downregulated stearyl-CoA-desaturase 1. In conclusion, BFH12 can acquire a steatotic phenotype by incorporating and accumulating fatty acids. Oleic acid is particularly suitable to produce hepatosteatosis. Therefore, BFH12 may be a useful in vitro model to study bovine hepatosteatosis and its underlying molecular mechanisms.

During anaphase B, molecular motors slide interpolar microtubules to elongate the mitotic spindle, contributing to the separation of chromosomes. However, sliding of antiparallel microtubules reduces their overlap, which may lead to spindle breakage, unless microtubules grow to compensate sliding. How sliding and growth are coordinated is still poorly understood. In this study, we have used the fission yeast S. pombe to measure microtubule dynamics during anaphase B. We report that the coordination of microtubule growth and sliding relies on promoting rescues at the midzone edges. This makes microtubules stable from pole to midzone, while their distal parts including the plus ends alternate between assembly and disassembly. Consequently, the midzone keeps a constant length throughout anaphase, enabling sustained sliding without the need for a precise regulation of microtubule growth speed. Additionally, we found that in S. pombe, which undergoes closed mitosis, microtubule growth speed decreases when the nuclear membrane wraps around the spindle midzone.

An extensive arsenal of biosensing tools has been developed based on the clustered regularly interspaced short palindromic repeat (CRISPR) platform, including those that detect specific DNA sequences both in vitro and in live cells. To date, DNA imaging approaches have traditionally used full fluorescent reporter-based fusion probes. Such “always-on” probes differentiate poorly between bound and unbound probe and are unable to sensitively detect unique copies of a target sequence in individual cells. Herein we describe a DNA biosensor that provides a sensitive readout for such low-copy DNA sequences through proximity-mediated reassembly of two independently optimized fragments of NanoLuc luciferase (NLuc), a small, bright luminescent reporter. Applying this “turn-on” probe in live cells, we demonstrate an application not easily achieved by fluorescent reporter-based probes, detection of individual endogenous genomic loci using standard epifluorescence microscopy. This approach could enable detection of gene edits during ex vivo editing procedures and should be a useful platform for many other live cell DNA biosensing applications.

The immune system is a complex network of various cellular components that must differentiate between pathogenic bacteria and the commensal bacteria of the human microbiome, where misrecognition is linked to inflammatory disorders. Fragments of bacterial cell wall peptidoglycan bind to pattern recognition receptors within macrophages, leading to immune activation. To study this complex process, a methodology to remodel and label the bacterial cell wall of two different species of bacteria was established using copper (I) catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC). Additionally, an approach for three-dimensional (3D) culture of human macrophages and their invasion with relevant bacteria in a well-defined hydrogel-based synthetic matrix inspired by the microenvironment of the gut was established. Workflows were developed for human monocyte encapsulation and differentiation into macrophages in 3D culture with high viability. Bacteria invaded into macrophages permitted in situ peptidoglycan labeling. Macrophages exhibited biologically-relevant cytokine release in response to bacteria. This molecularly engineered, multi-dimensional bacteria-macrophage co-culture system will prove useful in future studies to observe immunostimulatory, bacterial fragment production and localization in the cell at the carbohydrate level for insights into how the immune system properly senses bacteria.

Kinetoplastid protozoa possess properties that are highly divergent from the mammalian, yeast and bacterial cells more commonly used in synthetic biology and represent a tantalisingly untapped source of bioengineering potential. Trypanosoma brucei brucei (T. b. brucei), an established model organism for studying the Kinetoplastida, is non-pathogenic to humans and provides an interesting test case for establishing synthetic biology in this phylogenetic class. To demonstrate further the tractability of Kinetoplastida to synthetic biology, we sought to construct and demonstrate a Goodwin oscillator, the simplest oscillatory gene network, in T. b. brucei for the first time. We report one completed iteration of the archetypal synthetic biology Design–Build–Test–Learn (DBTL) cycle; firstly, using Ab initio mathematical modelling of the behaviour a theoretical, oscillatory, trypanosomal synthetic gene network (SGN) to inform the design of a plasmid encoding that network. Once assembled, the plasmid was then used to generate a stable transfectant T. b. brucei cell line. To test the performance of the oscillatory SGN, a novel experimental setup was established to capture images of the fluorescent signal from motion-restricted live cells. Data captured were consistent with oscillatory behaviour of the SGN, with cellular fluorescence observed to oscillate with a period of 50 min, with varying amplitude and linear growth trend. This first DBTL cycle establishes a foundation for future cycles in which the SGN design and experimental monitoring setup can be further refined.

How a cell changes from one stable phenotype to another one is a fundamental problem in developmental and cell biology. Mathematically, a stable phenotype corresponds to a stable attractor in a generally multi-dimensional state space, which needs to be destabilized so the cell relaxes to a new attractor. Two basic mechanisms for destabilizing a stable fixed point, pitchfork and saddle-node bifurcations, have been extensively studied theoretically; however, direct experimental investigation at the single-cell level remains scarce. Here, we performed live cell imaging studies and analyses in the framework of dynamical systems theories on epithelial-to-mesenchymal transition (EMT). While some mechanistic details remain controversial, EMT is a cell phenotypic transition (CPT) process central to development and pathology. Through time-lapse imaging we recorded single cell trajectories of human A549/Vim-RFP cells undergoing EMT induced by different concentrations of exogenous TGF-β in a multi-dimensional cell feature space. The trajectories clustered into two distinct groups, indicating that the transition dynamics proceeds through parallel paths. We then reconstructed the reaction coordinates and the corresponding quasi-potentials from the trajectories. The potentials revealed a plausible mechanism for the emergence of the two paths where the original stable epithelial attractor collides with two saddle points sequentially with increased TGF-β concentration, and relaxes to a new one. Functionally, the directional saddle-node bifurcation ensures a CPT proceeds towards a specific cell type, as a mechanistic realization of the canalization idea proposed by Waddington.

Cell division is a tightly regulatedmechanism, notably in tissueswheremalfunctions can lead to tumour formation or developmental defects. This is particularly true in land plants, where cells cannot relocate and therefore cytokinesis determines tissue topology. In plants, cell division is executed in radically different manners than in animals, with the appearance of new structures and the disappearance of ancestral mechanisms. Whilst F-actin and microtubules closely coexist, recent studies mainly focused on the involvement of microtubules in this key process. Here, we used a root tracking system to image the spatio-temporal dynamics of both F-actin reporters and cell division markers in dividing cells embedded in their tissues. In addition to the F-actin accumulation at the phragmoplast, we observed and quantified a dynamic apico-basal enrichment of F-actin fromthe prophase/metaphase transition until the end of the cytokinesis. [ABSTRACT FROM AUTHOR]

The laser scanning confocal microscope (LSCM) has been widely used in the imaging of fixed and live cell samples. We introduced the basic principle of LSCM and its application in live cell imaging. Take FV10-ASW Viewer4.2 software as an example, we summarized the optimization and recommended parameter settings of live cell imaging system from the aspect of scanning speed, resolution, noise reduction, photomultiplier, multi-parameter collaborative optimization, imaging quality evaluation and post-processing. This work may provide some reference for live cell experiment. [ABSTRACT FROM AUTHOR]

Temperature is a key parameter in most biological and physical systems. It is, however, an inherent technological challenge to precisely track and control the temperature of small sample volumes when using high- and super-resolution microscopes. Various effects can cause a deviation of the sample temperature from its expected value, specifically in the observed volume or field-of-view. These effects include direct thermal contact of the objective lens and the coverslip via immersion medium, illumination-induced heating, evaporative cooling, or triggered phase transitions. In this paper, we shed light on these phenomena and introduce our newly developed device VAHEAT, which follows a microscopic approach for controlling and measuring the sample temperature. Our system overcomes traditional limitations of temperature sensing and controlling in high-sensitivity microscopy applications, leading to more reproducible experimental results and conquering new experimental parameter ranges. [ABSTRACT FROM AUTHOR]

Background: The integrity of microtubule filament networks is essential for the roles in diverse cellular functions, and disruption of its structure or dynamics has been explored as a therapeutic approach to tackle diseases such as cancer. Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt microtubules. However, due to associated side effects on healthy cells, there is a need to develop safer drug regimens that still retain clinical efficacy. Currently, many questions remain open regarding the extent of effects on cellular physiology of microtubule-interacting drugs at clinically relevant and low doses. Here, we use super-resolution microscopies (single-molecule localization and optical fluctuation based) to reveal the initial microtubule dysfunctions caused by nanomolar concentrations of colcemid. Results: We identify previously undetected microtubule (MT) damage caused by clinically relevant doses of colcemid. Short exposure to 30–80 nM colcemid results in aberrant microtubule curvature, with a trend of increased curvature associated to increased doses, and curvatures greater than 2 rad/μm, a value associated with MT breakage. Microtubule fragmentation was detected upon treatment with ≥ 100 nM colcemid. Remarkably, lower doses (< 20 nM after 5 h) led to subtle but significant microtubule architecture remodelling characterized by increased curvature and suppression of microtubule dynamics. Conclusions: Our results support the emerging hypothesis that microtubule-interacting drugs induce non-mitotic effects in cells, and establish a multi-modal imaging assay for detecting and measuring nanoscale microtubule dysfunction. The sub-diffraction visualization of these less severe precursor perturbations compared to the established antimitotic effects of microtubule-interacting drugs offers potential for improved understanding and design of anticancer agents. [ABSTRACT FROM AUTHOR]

Inhibitory GABAergic interneurons migrate over long distances from their extracortical origin into the developing cortex. In humans, this process is uniquely slow and prolonged, and it is unclear whether guidance cues unique to humans govern the various phases of this complex developmental process. Here, we use fused cerebral organoids to identify key roles of neurotransmitter signaling pathways in guiding the migratory behavior of human cortical interneurons. We use scRNAseq to reveal expression of GABA, glutamate, glycine, and serotonin receptors along distinct maturation trajectories across interneuron migration. We develop an image analysis software package, TrackPal, to simultaneously assess 48 parameters for entire migration tracks of individual cells. By chemical screening, we show that different modes of interneuron migration depend on distinct neurotransmitter signaling pathways, linking transcriptional maturation of interneurons with their migratory behavior. Altogether, our study provides a comprehensive quantitative analysis of human interneuron migration and its functional modulation by neurotransmitter signaling. SYNOPSIS: Using an interneuron‐specific reporter to track migrating cortical interneurons in human cerebral organoid fusions, this study provides a comprehensive quantitative analysis of human interneuron migration and its functional modulation by diverse neurotransmitter signaling pathways. Single‐cell human GABAergic lineage reconstruction identifies neurotransmitter receptor expression in migrating human cortical interneurons.Newly developed software package, TrackPal, quantifies large‐scale, live‐imaging based tracking of migrating human cortical interneurons.Small‐molecule screening identifies divergent roles of neurotransmitter receptors in regulating intrinsic interneuron migration behavior.Neurotransmitter signaling modulates distinct modes of interneuron migration. [ABSTRACT FROM AUTHOR]

The interrogation of metabolic parameters like pH in live‐cell experiments using optical super‐resolution microscopy (SRM) remains challenging. This is due to a paucity of appropriate metabolic probes enabling live‐cell SRM‐based sensing. Here, ultrasmall fluorescent core–shell aluminosilicate nanoparticle sensors (FAM–ATTO647N aC′ dots) that covalently encapsulate a reference dye (ATTO647N) in the core and a pH‐sensing moiety (FAM) in the shell are introduced. Only the reference dye exhibits optical blinking enabling live‐cell stochastic optical reconstruction microscopy (STORM). Using data from cells incubated for 60 min with FAM–ATTO647N aC′ dots, pixelated information from total internal reflection fluorescence (TIRF) microscopy‐based ratiometric sensing can be combined with that from STORM‐based localizations via the blinking reference dye in order to enhance the resolution of ratiometric pH sensor maps beyond the optical diffraction limit. A nearest‐neighbor interpolation methodology is developed to quantitatively address particle compositional heterogeneity as determined by separate single‐particle fluorescence imaging methods. When combined with STORM‐based estimates of the number of particles per vesicle, vesicle size, and vesicular motion as a whole, this analysis provides detailed live‐cell spatial and functional information, paving the way to a comprehensive mapping and understanding of the spatiotemporal evolution of nanoparticle processing by cells important, e.g., for applications in nanomedicine. [ABSTRACT FROM AUTHOR]

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.

In this timely review, we discuss the important attributes of various chalcogen-containing small-molecule probes that have been synthesized in the Molecular Logic Gate Laboratory at the Korea Advanced Institute of Science and Technology. Specifically, we discuss some of the important chemical and photophysical properties of these probes, including reversibility, responsiveness (response time), cellular localization, sensitivity to analytes, selectivity (toward a specific analyte in question), and some bioavailability criteria. Chalcogenides undergo reversible redox-type reactions with reactive oxygen species (ROS). These controlled solution reactions enable a sensible and clear response as they act to immediately affect the chemical and electronic properties of the chalcogen moiety. Often, the lone pair belongs to chalcogens, which communicate electronically with the rest of the probes. Importantly, chemically oxidized chalcogenides can revert to their original reduced (divalent) form through the addition of natural or unnatural biothiols (or other reductants). This phenomenon is considered reversibility from the standpoint of probes. It can also be called “resetability.” In this manner, a variety of fluorophore frameworks can be used to detect ROS and thiols. Further studies can help experimentally determine the lipophilicity and even the cellular localization of probes, which are important in assessing their value as diagnostic agents in biological sciences and their possible therapeutic potential.

Abstract 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.

Live cell imaging uniquely enables the measurement of dynamic events in single cells, but it has not been used often in the study of gene regulatory networks. Network components can be examined in relation to one another by quantitative live cell imaging of fluorescent protein reporter cell lines that simultaneously report on more than one network component. A series of dual-reporter cell lines would allow different combinations of network components to be examined in individual cells. Dynamical information about interacting network components in individual cells is critical to predictive modeling of gene regulatory networks, and such information is not accessible through omics and other end point techniques. Achieving this requires that gene-edited cell lines are appropriately designed and adequately characterized to assure the validity of the biological conclusions derived from the expression of the reporters. In this brief review we discuss what is known about the importance of dynamics to network modeling and review some recent advances in optical microscopy methods and image analysis approaches that are making the use of quantitative live cell imaging for network analysis possible. We also discuss how strategies for genetic engineering of reporter cell lines can influence the biological relevance of the data.