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Heterogeneity within specific cell types is common and increasingly apparent with the advent of single cell transcriptomics. Transcriptional and functional cellular specialization has been described for insulin-secreting β cells of the endocrine pancreas, including so-called extreme β cells exhibiting >2-fold higher insulin gene activity. However, it is not yet clear whether β cell heterogeneity is stable or reflects dynamic cellular states. We investigated the temporal kinetics of endogenous insulin gene activity using live cell imaging, with complementary experiments employing FACS and single cell RNA sequencing, in β cells from Ins2GFP knock-in mice. In vivo staining and FACS analysis of islets from Ins2GFP mice confirmed that at a given moment, ~25% of β cells exhibited significantly higher activity at the conserved insulin gene Ins2. Live cell imaging captured Ins2 gene activity dynamics in single β cells over time. Autocorrelation analysis indicated that cells displaying fluctuations in Ins2 gene activity most commonly exhibited a frequency of 17 hours. Increased glucose concentrations stimulated more cells to oscillate and resulted in higher average Ins2 gene activity per cell. Single cell RNA sequencing determined that Ins2(GFP)HIGH β cells were enriched for markers of β cell maturity and had reduced expression of anti-oxidant genes. Ins2(GFP)HIGH β cells were also significantly less viable at all glucose concentrations and in the context of ER stress. Collectively, our results demonstrate that the heterogeneity of insulin production, observed in mouse and human β cells, can be accounted for by dynamic states of insulin gene activity. Our observations define a previously uncharacterized form of β cell plasticity. Understanding the dynamics of insulin production has relevance for understanding the pathobiology of diabetes and for regenerative therapy research.

Evaluating the correlation between hypoxia inducible factor 1 (HIF-1) and nitric oxide (NO) generated under hypoxia is of great significance. In this work, we developed a fluorescent probe for the monitor of HIF-1 activity influenced by NO under hypoxia in hepatoma cells with dual-targeting for hepatocyte and lipid droplet (LD). The probe shows excellent selectivity to NO and high sensitivity with 6000-fold fluorescence enhancement. Live cell imaging experiments revealed the probe's capability of imaging exogenous and endogenous NO with specific in LDs of HepG2 cells. For cells under hypoxia, HIF-1 induced LD level is observed to correlate with NO level. This work provides the in-situ visualization of NO-dependent HIF-1 upregulation through LD accumulation.

ANNEXIN V belongs to a family of phospholipid binding proteins which is able to bind to negatively charged phospholipids such as phosphatidylserine (PS) in the presence of a high affinity Ca2+ ion. When apoptosis occurs, even at early stage, PS will be exposed to the outside of the cell surface from the cytoplasm side of membrane leaflets., Therefore ANNEXIN V has been suggested as a bio-marker for imaging early apoptotic events of various cell death including those in disease conditions. However, most ANNEXIN V-based apoptotic detecting techniques were in vitro approaches. Here, we presented a new BRET (Bioluminescence Resonance Energy Transfer) based genetic coded biosensor by fusing ANNEXIN V and a BRET version of NanoLuc (teLuc) for both in vitro and in vivo apoptosis detection. The BRET feature of this new sensor makes it convenient to be applied to both conventional fluorescent-based in vitro apoptosis detection and bioluminescence-based animal live imaging for in vivo study. Because of its robust bioluminescence signal, it is possible to perform the evaluation of the disease-induced apoptotic damage and recovery process directly at deep tissue level in live animal.

Bone is the major connective tissue maintaining the structural integrity of the human body. However, fracture and many skeletal degenerative diseases can compromise this function. Thus, therapeutics related to bone degeneration are of significant research interest and require good in vitro models for such therapeutic evaluation. Bone is a highly vascularized tissue and incorporation of this feature is significantly important for mimicking the osteogenic microenvironment. In the current study, we developed a vascularized flat bone model via simultaneous mechanical actuation of mechanical strain and fluid shear. The mechanical strain was achieved by static magnetic field actuation of a magnetic nanocomposite scaffold. The fluid shear was generated by developing a micropattern on the magnetic nanocomposite via replica molding and laser-based microfabrication. From the live cell imaging window of the microdevice, both bone and vasculature like cellular morphology was observed. The SEM study showed thick ECM deposition in the dynamic culture. In the PCR study, both osteogenic (Col-1, osteocalcin) and angiogenic phenotypes (PECAM) were observed in the dynamic culture scaffolds while chondrogenic marker (Col-2) was downregulated.

A century ago this year, Pío del Río‐Hortega (1921) coined the term ‘oligodendroglia’ for the ‘interfascicular glia’ with very few processes, launching an extensive discovery effort on his new cell type. One hundred years later, we review his original contributions to our understanding of the system of cytoplasmic channels within myelin in the context of what we observe today using light and electron microscopy of genetically encoded fluorescent reporters and immunostaining. We use the term myelinic channel system to describe the cytoplasm‐delimited spaces associated with myelin; being the paranodal loops, inner and outer tongues, cytoplasm‐filled spaces through compact myelin and further complex motifs associated to the sheath. Using a central nervous system myelinating cell culture model that contains all major neural cell types and produces compact myelin, we find that td‐tomato fluorescent protein delineates the myelinic channel system in a manner reminiscent of the drawings of adult white matter by Río‐Hortega, despite that he questioned whether some cytoplasmic figures he observed represented artefact. Together, these data lead us to propose a slightly revised model of the ‘unrolled’ sheath. Further, we show that the myelinic channel system, while relatively stable, can undergo subtle dynamic shape changes over days. Importantly, we capture an under‐appreciated complexity of the myelinic channel system in mature myelin sheaths., Using fluorescent reporter protein and immunostaining we revisit Pío del Río‐Hortega’s exquisite drawings of the cytoplasm of CNS myelin. Together, these data highlight an under‐appreciated complexity of the myelinic channel system. We employ 3D reconstructions to illustrate some key features.

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.

Spinal cord injury (SCI) is a devastating condition causing the loss of sensory and motor functions. SCI pathology is multifaceted, encompassing inflammation, scarring, neuronal damage, and vascular and tissue remodeling. The dynamics of SCI rapidly transform from acute, sub-acute, and chronic phases. The rapidly changing environment necessitates the real-time monitoring of disease severity. Therefore, in this study, we used the IVIS spectrum, a noninvasive fluorescence imaging modality, to monitor the disease pathology in live animals. We used near-infrared fluorescence imaging agents including Angiosense 750 EX, a probe that detects vascular changes, and Cat B 680 FAST, a probe that detects inflammation at various day points post injury (DPI), that is, DPI-1, DPI-14, and DPI-28. We quantified the pathophysiological changes after SCI using IVIS in live animals. As a result, we observed distinct differences in the disease progression between injured and sham mice. Moreover, live imaging showed a good correlation with behavioral studies, protein expression, and immunohistological analysis. Hence, the goal of this study was to introduce a new optical imaging modality that offers a determination of disease severity and the advantage of accelerated imaging of the correlated biomarkers in a real-time and dynamic manner. This study concluded that Cat B 680 Fast and Angiosense 750 EX could be used to assess the disease severity after SCI. Furthermore, our study suggests that the noninvasive fluorescence optical imaging modality offers a unique approach in monitoring neuroinflammatory diseases in live animals.

Stem cell (SC) exhaustion is a hallmark of aging. However, the process of SC depletion during aging has not been observed in live animals, and the underlying mechanism contributing to tissue deterioration remains obscure. We find that, in aged mice, epithelial cells escape from the hair follicle (HF) SC compartment to the dermis, contributing to HF miniaturization. Single-cell RNA-seq and assay for transposase-accessible chromatin using sequencing (ATAC-seq) reveal reduced expression of cell adhesion and extracellular matrix genes in aged HF-SCs, many of which are regulated by Foxc1 and Nfatc1. Deletion of Foxc1 and Nfatc1 recapitulates HF miniaturization and causes hair loss. Live imaging captures individual epithelial cells migrating away from the SC compartment and HF disintegration. This study illuminates a hitherto unknown activity of epithelial cells escaping from their niche as a mechanism underlying SC reduction and tissue degeneration. Identification of homeless epithelial cells in aged tissues provides a new perspective for understanding aging-associated diseases. Using live imaging, the study shows, in mice, that epithelial cells escape from the hair follicle stem cell compartment during aging. Stem cell escape is associated with reduced cell adhesion and extracellular matrix gene expression and leads to hair follicle miniaturization.

Roles of interstitial tissue in morphogenesis of testicular structures remain less well understood. To analyze the roles of CD34+ cells in the reconstruction of interstitial tissue containing Leydig cells (LCs), and testicular structures, we used 3D-reaggregate culture of dissociated testicular cells from prepubertal mouse. After a week of culture, adult Leydig cells (ALCs) were preferentially incorporated within CD34+ cell-aggregates, but fetal LCs (FLCs) were not. Immunofluorescence studies showed that integrins α4, α9 and β1, and VCAM1, one of the ligands for integrins α4β1 and α9β1, are expressed mainly in CD34+ cells and ALCs, but not in FLCs. Addition of function-blocking antibodies against each integrin and VCAM1 to the culture disturbed the reconstruction of testicular structures. Antibodies against α4 and β1 integrins and VCAM1 robustly inhibited cell-to-cell adhesion between testicular cells and between CD34+ cells. Cell-adhesion assays indicated that CD34+ cells adhere to VCAM1 through the interaction with α4β1 integrin. Live cell imaging showed that CD34+ cells adhered around ALC-aggregates. CD34+ cells on the dish moved toward the aggregates, extending filopodia, and entered into them, which was disturbed by VCAM1 antibody. These results indicate that VCAM1-α4β1 integrin interaction plays pivotal roles in formation of testicular interstitial tissues in vitro and also in vivo.

Since microRNA-205 (miRNA-205) is a predictive biomarker for antiradiation of nasopharyngeal carcinoma (NPC), quantitative detection of miRNA-205 is important for developing personalized strategies for the treatment of NPC. In this investigation, based on the graphene oxide (GO) sensor and hybridization chain reaction (HCR) for fluorescence signal amplification, a highly sensitive and selective detection method for miRNA-205 was designed. A target-recycling mechanism is employed, where a single miRNA-205 target triggers the signal amplification of many DNA signal probes. The biosensor shows the ability to analyze miRNA-205 in solution, and it can detect miRNA-205 at concentrations as low as 311.96 pM. Furthermore, the method is specific in that it distinguishes between a target miRNA and a sequence with single-, double-, and three-base mismatches, as well as other miRNAs. Considering its simplicity and superior sensitivity, it was also verified in 1‰ serum with a detection limit of 111.65 pM. Importantly, the method successfully demonstrated that miRNA-205 could be imaged in living cells, which provided the possibility of localizing target molecules in live cell imaging applications. This method has great clinical application potential in the determination of miRNA-205, a biomarker for radiation-resistant NPC.

Numerous imaging modules are utilized to study changes that occur during cellular processes. Besides qualitative (immunohistochemical) or semiquantitative (Western blot) approaches, direct quantitation method(s) for detecting and analyzing signal intensities for disease(s) biomarkers are lacking. Thus, there is a need to develop method(s) to quantitate specific signals and eliminate noise during live tissue imaging. An increase in reactive oxygen species (ROS) such as superoxide (O2•-) radicals results in oxidative damage of biomolecules, which leads to oxidative stress. This can be detected by dihydroethidium staining in live tissue(s), which does not rely on fixation and helps prevent stress on tissues. However, the signal-to-noise ratio is reduced in live tissue staining. We employ the Drosophila eye model of Alzheimer's disease as a proof of concept to quantitate ROS in live tissue by adapting an unbiased method. The method presented here has a potential application for other live tissue fluorescent images.

Objective: 3D optical coherence tomography (OCT) is used for analyses of human placenta organoids in situ without sample preparation. Methods: The trophoblast organoids analyzed were derived from primary human trophoblast. In this study a custom made ultra-high-resolution spectral domain OCT system with uniform spatial and axial resolution of 2.48 μm in organoid tissue was used. The obtained OCT results align to differentiation status tested via quantitative polymerase chain reaction, Western blot analyses, immunohistochemistry, and immunofluorescence of histological sections. Results: 3D OCT enables a more detailed placenta organoid monitoring compared to brightfield microscopy. Inner architecture with light scattering “bridges” surrounding cavities were visualized and quantified in situ for the first time. The formation of these bridges and cavities is congruent to differentiated trophoblast organoids having developed syncytiotrophoblasts. Conclusion: Using 3D OCT in living placenta organoids is a fast tool to assess the differentiation status and resolve internal structures in situ , which is not possible with standard live cell imaging modality. Significance: Only recently human placenta-derived organoids were established, allowing to have a highly reproducible and stable in vitro model to investigate not only developmental but also physiological and pathophysiological processes during early pregnancy. To our knowledge, this work is the first to analyze living human placenta organoids using 3D OCT. Thereby, the rapid and especially non-endpoint OCT qualitative analyses align to the differentiation stage of organoids, which will aid future advancement in this field.

The present study reports the feasibility and successful production of rabbit cG-CAOMECS, designed to reconstruct corneal epithelium of patients with bilateral limbal stem cell deficiency. To produce a safe, chemically defined and FDA compliant cG-CAOMECS, oral mucosal epithelial cells were isolated from a biopsy of rabbit buccal tissue and seeded on a cGMP-certified cell culture surface coated with GMP-grade extracellular matrix. A newly designed clinical-grade medium (KaFa™ medium) was utilized to carry out cell expansion. Detachment and harvesting of the produced cell sheet was accomplished using collagenase treatment. Live cell imaging and morphological analysis techniques were used to examine cell growth. Cells attached onto the surface and self-assembled into colony-forming units (CFUs). Microscopic examination showed that CFUs formed during the first 5 days, and basal monolayer cell sheet formed in less than 10 days. Cells expanded to form a multilayered epithelial cell sheet that was harvested after 17–19 days in culture. Immunostaining and Western blot analyses showed that deltaNp63 was expressed in the basal cells and K3/K12 was expressed in the apical cells, indicating the presence of corneal epithelial-like cells in the produced cell sheet. Adhesion molecules, E-cadherin, beta-catenin, and Cnx43 were also expressed and exhibited the epithelial integrity of the cell sheet. The expression of integrin-beta1 and beta4 confirmed that the collagenase treatment used for detaching and harvesting the cell sheet did not have adverse effects. Our results showed that the utilization of clinical-grade and FDA-approved reagents successfully supported the production of cG-CAMECS.

Poupartia borbonica is an endemic tree from the Mascarene Islands that belongs to the Anacardiaceae family. The leaves of this plant were phytochemically studied previously, and isolated alkyl cyclohexenone derivatives, poupartones A – C, demonstrated antiplasmodial and antimalarial activities. In addition to their high potency against the Plasmodium sp., high toxicity on human cells was also displayed. The present study aims to investigate in more detail the cytotoxicity and pharmacological interest of poupartone B, one of the most abundant derivatives in the leaves of P. borbonica. For that purpose, real-time live-cell imaging of different human cancer cell lines and normal fibroblasts, treated or not treated with poupartone B, was performed. A potent inhibition of cell proliferation associated with the induction of cell death was observed. A detailed morphological analysis of different adherent cell lines exposed to high concentrations of poupartone B (1 – 2 µg/mL) demonstrated that this compound induced an array of cellular alterations, including a rapid retraction of cellular protrusions associated with cell rounding, massive cytoplasmic vacuolization, loss of plasma membrane integrity, and plasma membrane bubbling, ultimately leading to paraptosis-like cell death. The structure-activity relation of this class of compounds, their selective toxicity, and pharmacological potential are discussed.

RNA-based therapeutics are highly promising for the treatment of numerous diseases, by their ability to tackle the genetic origin in multiple possible ways. RNA molecules are, however, incapable of crossing cell membranes, hence a safe and efficient delivery vehicle is pivotal. Extracellular vesicles (EVs) are endogenously derived nano-sized particles and possess several characteristics which make them excellent candidates as therapeutic RNA delivery agent. This includes the inherent capability to functionally transfer RNAs in a selective manner and an enhanced safety profile compared to synthetic particles. Nonetheless, the fundamental mechanisms underlying this selective inter- and intracellular trafficking and functional transfer of RNAs by EVs are poorly understood. Improving our understanding of these systems is a key element of working towards an EV-based or EV-mimicking system for the functional delivery of therapeutic RNA. In this review, state-of-the-art approaches to detect and visualize RNA in situ and in live cells are discussed, as well as strategies to assess functional RNA transfer, highlighting their potential in studying EV-RNA trafficking mechanisms.

Supplemental Digital Content is available in the text., Objective: Extracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signaling during homeostasis and in disease. Extensive studies have described functional roles for EV populations, including during cardiovascular disease, but the in vivo characterization of endogenously produced EVs is still in its infancy. Because of their genetic tractability and live imaging amenability, zebrafish represent an ideal but under-used model to investigate endogenous EVs. We aimed to establish a transgenic zebrafish model to allow the in vivo identification, tracking, and extraction of endogenous EVs produced by different cell types. Approach and Results: Using a membrane-tethered fluorophore reporter system, we show that EVs can be fluorescently labeled in larval and adult zebrafish and demonstrate that multiple cell types including endothelial cells and cardiomyocytes actively produce EVs in vivo. Cell-type specific EVs can be tracked by high spatiotemporal resolution light-sheet live imaging and modified flow cytometry methods allow these EVs to be further evaluated. Additionally, cryo electron microscopy reveals the full morphological diversity of larval and adult EVs. Importantly, we demonstrate the utility of this model by showing that different cell types exchange EVs in the adult heart and that ischemic injury models dynamically alter EV production. Conclusions: We describe a powerful in vivo zebrafish model for the investigation of endogenous EVs in all aspects of cardiovascular biology and pathology. A cell membrane fluorophore labeling approach allows cell-type specific tracing of EV origin without bias toward the expression of individual protein markers and will allow detailed future examination of their function.

Spatiotemporal changes in general transcription levels play a vital role in the dynamic regulation of various critical activities. Phosphorylation levels at Ser2 in heptad repeats within the C-terminal domain of RNA polymerase II, representing the elongation form, is an indicator of transcription. However, rapid transcriptional changes during tissue development and cellular phenomena are difficult to capture in living organisms. We introduced a genetically encoded system termed modification-specific intracellular antibody (mintbody) into Arabidopsis thaliana. We developed a protein processing- and 2A peptide-mediated two-component system for real-time quantitative measurement of endogenous modification level. This system enables quantitative tracking of the spatiotemporal dynamics of transcription. Using this method, we observed that the transcription level varies among tissues in the root and changes dynamically during the mitotic phase. The approach is effective for achieving live visualization of the transcription level in a single cell and facilitates an improved understanding of spatiotemporal transcription dynamics., Shibuta et al develop a live imaging system that allows them to track the spatiotemporal dynamics of RNA polymerase II modification in single cells in Arabidopsis thaliana. This approach potentially enables a more detailed understanding of transcriptional dynamics in plants.

Accurate recognition of antigens by specific T cells is crucial for adaptive immunity to work properly. The activation of a T-cell antigen-specific response by an antigen-presenting cell (APC) has not been clearly measured at a single T-cell level. It is also unknown whether the cell-extrinsic environment alters antigen recognition by a T cell. To measure the activation probability of a single T cell by an APC, we performed a single-cell live imaging assay and found that the activation probability changes depending not only on the antigens but also on the interactions of other T cells with the APC. We found that the specific reactivity of single naïve T cells was poor. However, their antigen-specific reactivity increased drastically when attached to an APC interacting with activated T cells. Activation of T cells was suppressed when regulatory T cells interacted with the APC. These findings suggest that although the ability of APCs to activate an antigen-specific naïve T cell is low at a single-cell level, the surrounding environment of APCs improves the specificity of the bulk response.

Glioblastoma (GBM) is the most common and lethal brain tumor. GBM has a remarkable degree of motility and is able to infiltrate the healthy brain. In order to perform a rationale-based drug-repositioning study, we have used known inhibitors of two small Rho GTPases, Rac1 and Cdc42, which are upregulated in GBM and are involved in the signaling processes underlying the orchestration of the cytoskeleton and cellular motility. The selected inhibitors (R-ketorolac and ML141 for Cdc42 and R-ketorolac and EHT 1864 for Rac1) have been successfully employed to reduce the infiltration propensity of GBM in live cell imaging studies. Complementarily, all-atom simulations have elucidated the molecular basis of their inhibition mechanism, identifying the binding sites targeted by the inhibitors and dissecting their impact on the small Rho GTPases' function. Our results demonstrate the potential of targeting the Rac1 and Cdc42 proteins with small molecules to contrast GBM infiltration growth and supply precious information for future drug discovery studies aiming to fight GBM and other infiltrative cancer types.

Ruthenium is popular as a metal core for chemotherapeutics, due to versatile molecular coordination. Because new metallodrugs are synthesized at high rates, our studies included assays in zebrafish to expedite the initial evaluation as anticancer agents. Here we evaluated novel metallodrugs (PMC79 and LCR134), and cisplatin, a widely used platinum-based chemotherapeutic. We hypothesized that this model could characterize anticancer properties and recapitulate previous in vitro results in vivo. Our findings suggest anticancer properties of PMC79 and LCR134 were similar with less toxicity than cisplatin. Exposures from 24 to 72 h at or below the LOAELs of PMC79 and LCR134 (3.9 µM and 13.5 µm, respectively), impaired blood vessel development and tailfin regeneration. Blood vessel examination through live imaging of larvae revealed distinct regional antiangiogenic impacts. The significant decrease in gene expression of the VEGF-HIF pathway and beta-actin could explain the morphological effects observed in the whole organism following exposure. Tailfin amputation in larvae exposed to PMC79 or LCR134 inhibited tissue regrowth and cell division, but did not impact normal cell proliferation unlike cisplatin. This suggests Ru drugs may be more selective in targeting cancerous cells than cisplatin. Additionally, in vitro mechanisms were confirmed. PMC79 disrupted cytoskeleton formation in larvae and P-glycoprotein transporters in vivo was inhibited at low doses which could limit off-target effects of chemotherapeutics. Our results demonstrate the value for using the zebrafish in metallodrug research to evaluate mechanisms and off-target effects. In light of the findings reported in this article, future investigation of PMC79 and LCR134 are warranted in higher vertebrate models.

All blood cells originate from hematopoietic stem/progenitor cells (HSPCs). HSPCs are formed from endothelial cells (ECs) of the dorsal aorta (DA), via endothelial-to-hematopoietic transition (EHT). The zebrafish is a primary model organism to study the process in vivo. While the role of mechanical stress in controlling gene expression promoting cell differentiation is actively investigated, mechanisms driving shape changes of the DA and individual ECs remain poorly understood. We address this problem by developing a new DA micromechanical model and applying it to experimental data on zebrafish morphogenesis. The model considers the DA as an isotropic tubular membrane subjected to hydrostatic blood pressure and axial stress. The DA evolution is described as a movement in the dimensionless controlling parameters space: normalized hydrostatic pressure and axial stress. We argue that HSPC production is accompanied by two mechanical instabilities arising in the system due to the plane stress in the DA walls and show how a complex interplay between mechanical forces in the system drives the emerging morphological changes.

Hepatocytes are essential for maintaining the homeostasis of iron and lipid metabolism in mammals. Dysregulation of either iron or lipids has been linked with serious health consequences, including non-alcoholic fatty liver disease (NAFLD). Considered the hepatic manifestation of metabolic syndrome, NAFLD is characterised by dysregulated lipid metabolism leading to a lipid storage phenotype. Mild to moderate increases in hepatic iron have been observed in ∼30% of individuals with NAFLD; however, direct observation of the mechanism behind this increase has remained elusive. To address this issue, we sought to determine the metabolic consequences of iron loading on cellular metabolism using live cell, time-lapse Fourier transform infrared (FTIR) microscopy utilising a synchrotron radiation source to track biochemical changes. The use of synchrotron FTIR is non-destructive and label-free, and allowed observation of spatially resolved, sub-cellular biochemical changes over a period of 8 h. Using this approach, we have demonstrated that iron loading in AML12 cells induced perturbation of lipid metabolism congruent with steatosis development. Iron-loaded cells had approximately three times higher relative ester carbonyl concentration compared with controls, indicating an accumulation of triglycerides. The methylene/methyl ratio qualitatively suggests the acyl chain length of fatty acids in iron-loaded cells increased over the 8 h period of monitoring compared with a reduction observed in the control cells. Our findings provide direct evidence that mild to moderate iron loading in hepatocytes drives de novo lipid synthesis, consistent with a role for iron in the initial hepatic lipid accumulation that leads to the development of hepatic steatosis.

See Silagi and Segal (doi:10.1093/brain/awab196) for a scientific commentary on this article. The chemotherapy drug paclitaxel induces an increase in Nav1.7 channel expression in sensory neurons, enhancing their excitability. Akin et al. use live imaging to visualize trafficking and surface localization of Nav1.7 channels in sensory axons after exposure to paclitaxel, and reveal a role for inflammation in this process., The microtubule-stabilizing chemotherapy drug paclitaxel (PTX) causes dose-limiting chemotherapy-induced peripheral neuropathy (CIPN), which is often accompanied by pain. Among the multifaceted effects of PTX is an increased expression of sodium channel Nav1.7 in rat and human sensory neurons, enhancing their excitability. However, the mechanisms underlying this increased Nav1.7 expression have not been explored, and the effects of PTX treatment on the dynamics of trafficking and localization of Nav1.7 channels in sensory axons have not been possible to investigate to date. In this study we used a recently developed live imaging approach that allows visualization of Nav1.7 surface channels and long-distance axonal vesicular transport in sensory neurons to fill this basic knowledge gap. We demonstrate concentration and time-dependent effects of PTX on vesicular trafficking and membrane localization of Nav1.7 in real-time in sensory axons. Low concentrations of PTX increase surface channel expression and vesicular flux (number of vesicles per axon). By contrast, treatment with a higher concentration of PTX decreases vesicular flux. Interestingly, vesicular velocity is increased for both concentrations of PTX. Treatment with PTX increased levels of endogenous Nav1.7 mRNA and current density in dorsal root ganglion neurons. However, the current produced by transfection of dorsal root ganglion neurons with Halo-tag Nav1.7 was not increased after exposure to PTX. Taken together, this suggests that the increased trafficking and surface localization of Halo-Nav1.7 that we observed by live imaging in transfected dorsal root ganglion neurons after treatment with PTX might be independent of an increased pool of Nav1.7 channels. After exposure to inflammatory mediators to mimic the inflammatory condition seen during chemotherapy, both Nav1.7 surface levels and vesicular transport are increased for both low and high concentrations of PTX. Overall, our results show that PTX treatment increases levels of functional endogenous Nav1.7 channels in dorsal root ganglion neurons and enhances trafficking and surface distribution of Nav1.7 in sensory axons, with outcomes that depend on the presence of an inflammatory milieu, providing a mechanistic explanation for increased excitability of primary afferents and pain in CIPN.

Purpose The aim of this study was to develop transferrin-conjugated nanoemulsions utilizing hydrophobic ion pairing for a targeted cellular uptake. Methods In the lipophilic phase of nanoemulsion composed of 60% oleic acid, 30% Capmul MCM EP and 10% Span 85, 1% cetyltrimethylammonium bromide (CTAB) and 3% phosphatidic acid (PA) were incorporated. After emulsification, the resulting droplets were decorated with human protein transferrin via hydrophobic ion pairing with PA and characterized regarding droplet size and zeta potential. Subsequently, cellular uptake of transferrin-conjugated nanoemulsion was investigated on Caco-2 and HeLa cell lines and determined by flow cytometry, cell lysis method and live cell imaging using confocal laser scanning microscopy. Results The nanoemulsion showed a droplet size of 123.03 ± 2.1 nm and zeta potential of − 54.5 mV that changed because of the surface decoration with transferrin to 182.7 ± 0.2 and + 30.2 mV, respectively. Within the uptake studies utilizing flow cytometry, transferrin-conjugated nanoemulsion showed a 5.2-fold higher uptake in Caco-2 cells and twofold improvement in case of HeLa cells compared with unmodified formulation. The outcome was confirmed visually via live cell imaging. Conclusion According to the results, transferrin-conjugated nanoemulsion might be considered as a promising drug delivery system for a selective receptor-mediated drug delivery.

Paneth cells and Lgr5+ intestinal stem cells (Lgr5+ ISCs) constitute the stem cell niche and maintain small intestinal epithelial integrity by recognizing various niche factors derived from subepithelial cells and external antigens. Although it has been known that interferon-gamma (IFN-gamma), a Th1 cytokine, is associated with intestinal epithelial disruption during inflammation as a niche factor, dynamics of Paneth cells and Lgr5+ ISCs in response to IFN-gamma remain to be understood. Here we show that CAG-tdTomato;Lgr5-EGFP (CT-LE) mice generated in this study enable to identify Paneth cells and Lgr5+ ISCs separately by fluorescence signals. Lgr5+ ISCs underwent cell death a little earlier than Paneth cells in response to IFN-gamma by simultaneous tracking using CT-LE mice. In addition, the timing of cell death in most Paneth cells overlapped with Lgr5+ ISCs, suggesting that Paneth cell depletion is induced directly by IFN-gamma. Taken together, we established a novel simultaneous stem cell niche tracking method and clarified the involvement of both Paneth cells and Lgr5+ ISCs in stem cell niche damage induced by IFN-gamma, further contribute to understanding the mechanism for maintaining intestinal homeostasis by stem cell niche. (C) 2021 The Authors. Published by Elsevier Inc.

The molecules of the cell are compartmentalized into membrane- and non-membrane-bound organelles. Many non-membrane-bound organelles are phase-separated biomolecular condensates with distinct physicochemical properties that can absorb and concentrate specific proteins and nucleic acids involved in discrete biochemical processes. We reasoned that selective condensate partitioning might also occur with small molecule drugs whose targets occur within condensates, and that the therapeutic index and efficacy of such compounds might therefore relate to their ability to partition into condensates. To test this idea, we focused our study on nuclear condensates reported in cell lines, demonstrated they occur in normal human and malignant breast cancer, and developed assays to test clinically active antineoplastic small molecule drugs relative to these condensates.To study the behavior of drugs within condensates, these were modeled in vitro with purified proteins and visualized by fluorescent confocal microscopy. We found that cisplatin, tamoxifen, JQ1, THZ1, and mitoxantrone are concentrated in specific protein condensates in vitro, and that this occurs through physicochemical properties independent of the drug target. For each drug, the small molecule partitioned into the same condensate in vitro in which its established target resides in vivo. A screen of a chemically diverse fluorescent probes and mutant-protein condensates demonstrated that pi-system interactions between aromatic moieties in the protein and small molecule govern concentration in condensates. These results show that clinically important drugs partition into specific protein condensates in vitro by virtue of defined chemical properties, thereby altering their local concentration.Alkylating agents are a class of commonly used antineoplastic compounds, of which cisplatin is a prominent example. In vitro droplet assays revealed that cisplatin is selectively concentrated in transcriptional condensates, and that this ability is required for efficient platination of target DNA. In cell studies revealed that cisplatin preferentially targets DNA contained within MED1 condensates, and disrupts the genetic regulatory elements that compose phase-separated transcriptional condensates. Live cell imaging demonstrated that transcriptional condensates are dissolved by cisplatin, whereas other condensates remain intact. Thus, we conclude that cisplatin preferentially modifies transcriptional condensate-associated DNA in cells, and that this causes selective condensate disruption. The mechanisms that produce drug resistance can provide clues to drug activity in the clinical setting. Investigating the behavior of tamoxifen within ER transcriptional condensates demonstrated that it disrupts these condensates in vitro and on oncogenes in cells; hormonal therapy resistant ESR1 mutations render these condensates resistant. MED1 overexpression, a poorly understood mechanism of tamoxifen resistance, increased the size of ER-MED1 condensates, thereby rending tamoxifen more dilute and ineffective when concentrated therein. This suggest that altering the size and nature of transcription condensates in breast cancer can mediate drug resistance in the clinical setting.Our results show that antineoplastic drugs partition selectively into condensates, that this can occur through physicochemical properties independent of their molecular targets, and that resistance to drugs may occur through condensate altering mechanisms. These results have implications for development of efficacious cancer therapeutics; effective target engagement will depend on factors such as drug partitioning in condensates. Assays of the type described here may thus help optimize condensate partitioning, target engagement, and the therapeutic index of drugs for cancer treatment. Citation Format: Isaac Klein, Ann Boija, Lena Afeyan, Susana Wilson Hawken, Mengyang Fan, Alessandra Dall'Agnese, Ozgur Oksuz, Jonathan Henninger, Krishna Shrinivas, Benjamin Sabari, Ido Sagi, Victoria Clark, Jesse Platt, Mrityunjoy Kar, Patrick McCall, Alicia Zamudio, John Mantiega, Eliot Coffey, Charles Li, Nancy Hannett, Yang Guo, Tim-Michael Decker, Tong Lee, Tinghu Zhang, Jing-Ke Weng, Dylan Taatjes, Arup Chakraborty, Phillip Sharp, Young Tae Chang, Anthony Hyman, Nathanael Gray, Richard Young. Partitioning of cancer therapeutics in nuclear condensates [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS3-10.

During neural circuit formation, axons navigate several choice points to reach their final target. At each one of these intermediate targets, growth cones need to switch responsiveness from attraction to repulsion in order to move on. Molecular mechanisms that allow for the precise timing of surface expression of a new set of receptors that support the switch in responsiveness are difficult to study in vivo. Mostly, mechanisms are inferred from the observation of snapshots of many different growth cones analyzed in different preparations of tissue harvested at distinct time points. However, to really understand the behavior of growth cones at choice points, a single growth cone should be followed arriving at and leaving the intermediate target. Existing ex vivo preparations, like cultures of an “open‐book” preparation of the spinal cord have been successfully used to study floor plate entry and exit, but artifacts prevent the analysis of growth cone behavior at the floor plate exit site. Here, we describe a novel spinal cord preparation that allows for live imaging of individual axons during navigation in their intact environment. When comparing growth cone behavior in our ex vivo system with snapshots from in vivo navigation, we do not see any differences. The possibility to observe the dynamics of single growth cones navigating their intermediate target allows for measuring growth speed, changes in morphology, or aberrant behavior, like stalling and wrong turning. Moreover, observation of the intermediate target—the floor plate—revealed its active participation and interaction with commissural axons during midline crossing., We describe a novel method to culture the intact spinal cord of the chicken embryo for live imaging of axon guidance at the midline. The stability of this ex vivo method allowed us to characterize key aspects of midline crossing, such as axonal growth, timing of crossing, dynamic morphological changes of the growth cones, and growth cone‐floor plate cell interactions.

Quantitative methods for assessing differentiative potency of adipose-derived stem/stromal cells may lead to improved clinical application of this multipotent stem cell, by advancing our understanding of specific processes such as adipogenic differentiation. Conventional cell staining methods are used to determine the formation of adipose areas during adipogenesis as a qualitative representation of adipogenic potency. Staining methods such as oil-red-O are quantifiable using absorbance measurements, but these assays are time and material consuming. Detection methods for cell characteristics using advanced image analysis by machine learning are emerging. Here, live-cell imaging was combined with a deep learning-based detection tool to quantify the presence of adipose areas and lipid droplet formation during adipogenic differentiation of adipose-derived stem/stromal cells. Different detection masks quantified adipose area and lipid droplet formation at different time points indicating kinetics of adipogenesis and showed differences between individual donors. Whereas CEBPA and PPARG expression seems to precede the increase in adipose area and lipid droplets, it might be able to predict expression of ADIPOQ. The applied method is a proof of concept, demonstrating that deep learning methods can be used to investigate adipogenic differentiation and kinetics in vitro using specific detection masks based on algorithm produced from annotation of image data.

Fluorescent labels and probes constitute an important class of organic materials used in the development of sensor systems and imaging platforms for various chemical and molecular biology applications. In the development of fluorescent labels and probes, multichromic environment-sensitive fluorophores are very important. In this work, novel triad systems, including subphthalocyanine, BODIPY and thiophene/thianaphthene units, were prepared and their molecular structures were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 1H and 13C NMR spectroscopy. The optical properties of the subphthalocyanine–BODIPY–thiophene/thianaphthene triads were determined using UV-vis absorption and fluorescence (2D and 3D) spectroscopic methods and quantum chemical calculations. The data were analyzed by comparing the results of the final properties to those of the BODIPY- and subphthalocyanine-alone units. The compounds were also evaluated in live cell imaging using a breast cancer cell line. The novel subphthalocyanine–BODIPY–thiophene/thianaphthene triads displayed dual color emission, which is critically important for the development of novel systems particularly in biomedical applications such as bioimaging and cellular sensor systems.

Background Cancer cell aggregation is a key process involved in the formation of tumor cell clusters. It has recently been shown that clusters of circulating tumor cells (CTCs) have an increased metastatic potential compared to isolated circulating tumor cells. Several widely used chemotherapeutic agents that target the cytoskeleton microtubules and cause cell cycle arrest at mitosis have been reported to modulate CTC number or the size of CTC clusters. Results In this study, we investigated in vitro the impact of mitotic arrest on the ability of breast tumor cells to form clusters. By using live imaging and quantitative image analysis, we found that MCF-7 cancer cell aggregation is compromised upon incubation with paclitaxel or vinorelbine, two chemotherapeutic drugs that target microtubules. In line with these results, we observed that MCF-7 breast cancer cells experimentally synchronized and blocked in metaphase aggregated poorly and formed loose clusters. To monitor clustering at the single-cell scale, we next developed and validated an in vitro assay based on live video-microscopy and custom-designed micro-devices. The study of cluster formation from MCF-7 cells that express the fluorescent marker LifeAct-mCherry using this new assay allowed showing that substrate anchorage-independent clustering of MCF-7 cells was associated with the formation of actin-dependent highly dynamic cell protrusions. Metaphase-synchronized and blocked cells did not display such protrusions, and formed very loose clusters that failed to compact. Conclusions Altogether, our results suggest that mitotic arrest induced by microtubule-targeting anticancer drugs prevents cancer cell clustering and therefore, could reduce the metastatic potential of circulating tumor cells.

Rationale: Invariant natural killer T (iNKT) cells and Kupffer cells represent major hepatic populations of innate immune cells. However, their roles in steatohepatitis remain poorly understood. To elucidate their functions in steatohepatitis development, real-time, in vivo analysis is necessary to understand the pathophysiological events in the dynamic interactions between them during diet-induced steatohepatitis. Methods: We used a steatohepatitis animal model induced by a methionine-choline-deficient (MCD) diet. Multi-photon confocal live imaging and conventional experimental techniques were employed to investigate the hepatic pathological microenvironment of iNKT and Kupffer cells, interactions between them, and the biological effects of these interactions in steatohepatitis. Results: We found that iNKT cells were recruited and aggregated into small clusters and interacted dynamically with Kupffer cells in the early stage of steatohepatitis. Most significantly, the iNKT cells in the cluster cleared free lipids released by necrotic hepatocytes and presented a non-classical activation state with high IFN-γ expression. Furthermore, the Kupffer cells in the cell cluster were polarized to type M1. The transcriptome sequencing of iNKT cells showed upregulation of genes related to phagocytosis and lipid processing. Adoptive transfer of iNKT cells to Jα18-/- mice showed that iNKT and Kupffer cell clusters were essential for balancing the liver and peripheral lipid levels and inhibiting liver fibrosis development. Conclusions: Our study identified an essential role for dynamic interactions between iNKT cells and Kupffer cells in promoting lipid phagocytosis and clearance by iNKT cells during early liver steatohepatitis. Therefore, modulating iNKT cells is a potential therapeutic strategy for early steatohepatitis.

Lysosomes, the acidic degradation compartments of eukaryotic cells, play an essential role in many physiological processes. Their dysfunction is associated with a number of diseases, which are often related to an altered localization or luminal pH. Thus, the in-depth characterization of lysosomes within the intact eukaryotic cell is of utmost interest. For microscopic evaluation of lysosomal distribution and acidity, a number of labels have been developed, but many showed poor organelle specificity or rapid clearing from lysosomes, rendering them unsuitable for long-term observations. Here, we describe the synthesis and spectroscopic properties of a novel small molecule marker for lysosomes based on naphthalene monoimide with reversible, pH-dependent spectral shifts in both the absorption and the emission spectrum and acidity-associated changes in fluorescence lifetime. The dye can be excited either with single- or two-photon excitation and appears to be very stably associated with lysosomes for several days. We used this chromophore to detect chemically-induced changes of lysosomal pH in HeLa cells by ratiometric and FLIM imaging.

Live cell imaging is used to track the dynamic adaptation of cell size and motility to various external factors. Bright-field configuration can be used for these experiments; however, the analysis can be challenging and difficult to automate. In this direction, a superior alternative is represented by the use of live cell dyes, which provide intense fluorescence from subcellular structures of living cells. Yet, the potential chemo- and photo-toxicity of the fluorophores poses the necessity of an accurate protocol optimization to avoid artefacts. Toxicity studies generally focus on cell proliferation and apoptosis, neglecting the cellular activities under investigation. Here, we present the case of SYTO 13 in combination with primary endothelial cells. The optimization of the staining procedure is tested comparing cell proliferation and motility rate. In addition, the combined effect of staining and fluorescent illumination, reporting for photochemical toxicity, is evaluated. We demonstrate that while cell viability and proliferation are mainly unaffected by the staining and imagining protocols, a significant reduction of the motility rate is induced both by the chemical dye alone and in combination with fluorescent illumination. The general implications for this procedure are discussed.

Cell migration is the main driver of the evolutionarily conserved process of gastrulation, which shapes metazoan embryo morphology. The molecular and cellular mechanisms of cell migration during gastrulation though well researched lacks an understanding of the contribution of cell sizes to collective cell migration. This is especially important during the early phase of metazoan development, which is dominated by constantly changing cell sizes in the background of which cells migrate en mass to shape the embryo. Here we investigate this phenomenon in zebrafish embryos, a model system in which early cell divisions causes cell sizes to decrease naturally over time as cells migrate collectively to sculpt the embryonic body plan. Because mutations that can perturb cell sizes so early in development do not exist, we generate haploid and tetraploid zebrafish embryos and show that cell sizes in such embryos are smaller and larger than the diploid norm, respectively. Cells in embryos made of smaller or larger than normal cells migrate sub-optimally, leading to gastrulation defects. Gene expression analysis suggests that the observed defects originate from altered cell size, and not from pleiotropic effects of altered ploidy. This interpretation is strengthened when gastrulation defects are rescued by increasing cell sizes in embryos wherein cell sizes are smaller than normal. We show that the migration defects are cell-autonomous by live imaging migrating haploid and tetraploid cells during gastrulation in chimeric diploid embryos. Analysis of membrane protrusion dynamics in single cells shows that cells normally extend protrusions non-uniformly during migration, a phenomenon which is perturbed when cell sizes deviate from the norm. Thus, an optimal range of developmental stage-specific cell sizes appears necessary for collective cell migration to correctly position cells in space and time to shape an amorphous ball of blastoderm into an embryo.

Pyroptotic cell death is a phenomenon that runs through all life activities and plays an important role in physiological and pathological processes of the body's metabolism. It is of big biological significance to understand the phenomenon and nature of cell pyroptosis. In the process of cell pyroptosis, the pore-forming effector gasdermin D (GSDMD) is cleaved to form oligomers, which are inserted into the cell membrane, causing rapid cell death. However, the effective cell death induced by GSDMD complicates our ability to understand the behavior of pyroptosis. In this work, we performed molecular mutagenesis to develop a genetically encoded pyroptotic reporter, where a secreted Gaussia luciferase (Gluc) was strategically placed in the p30-p20 tolerated junction of GSDMD to support natural pyrophosphorylation and promote live imaging of cell pyroptosis. In addition, we demonstrated that this fused Gluc-GSDMD reporter executed inflammatory body-dependent pyroptosis in response to extracellular stimuli, and that the lysed p30-GSDMD can be secreted out of the cell and can be detected in the culture medium and animal blood. Therefore, our study provides a valuable tool that not only noninvasive and real-time monitoring of cell pyroptosis, but also affords a high-throughput functional screening of pyroptosis-targeted compounds in cultured cells and animal models.