Single Plane Illumination Microscopy (SPIM) revolutionized time lapse imaging of live cells and organisms due to its high speed and reduced photodamage. Quantitative mapping of molecular (co-)mobility by fluorescence (cross-)correlation spectroscopy (F(C )CS) in a Single Plane Illumination Microscope (SPIM) has been introduced to reveal molecular diffusion and binding. A complementary aspect of interactions is proximity, which can be studied by Förster resonance energy transfer (FRET). Here, we extend SPIM-FCCS by alternating laser excitation, which reduces false positive cross-correlation, and facilitates co-mapping of FRET. Thus, different aspects of interacting systems can be studied simultaneously, and molecular subpopulations can be discriminated by multiparameter analysis. After demonstrating the benefits of the method on the AP-1 transcription factor, the dimerization and DNA binding behavior of retinoic acid receptor (RAR) and retinoid X receptor (RXR) is revealed and an extension of the molecular switch model of nuclear receptor action is proposed. Our data imply that RAR agonist enhances RAR-RXR heterodimerization, and chromatin binding/dimerization are positively correlated. We also propose a ligand induced conformational change bringing the N-termini of RAR and RXR closer together. RXR agonist increased homodimerization of RXR suggesting that RXR may act as an autonomous transcription factor.
Celiac disease (CeD) is an autoimmune disorder in which ingestion of dietary gluten triggers an immune reaction in the small intestine. The CeD lesion is characterized by crypt hyperplasia, villous atrophy and chronic inflammation with accumulation of leukocytes both in the lamina propria (LP) and in the epithelium, which eventually leads to destruction of the intestinal epithelium and subsequent digestive complications and higher risk of non-hodgkin lymphoma. A lifetime gluten-free diet is currently the only available treatment. Gluten-specific LP CD4 T cells and cytotoxic intraepithelial CD8+ T cells are thought to be central in disease pathology, however, CeD is a complex immune-mediated disorder and to date the findings are mostly based on analysis of heterogeneous cell populations and on animal models. Here, we comprehensively explore the cellular heterogeneity of CD45+ immune cells in human small intestine using index-sorting single-cell RNA-sequencing. We find that myeloid and mast cell transcriptomes are reshaped in CeD. We observe extensive changes in the proportion and transcriptomes of CD4+ and CD8+ T cells and define a CD3zeta expressing NK-T-like cell population present in the control LP and epithelial layers that is absent and replaced in CeD. Our findings show that the immune landscape is dramatically changed in active CeD which provide new insights and considerably extend the current knowledge of CeD immunopathology.
RATIONALE: Mycobacterium vaccae (NCTC 11659) is an environmental saprophytic bacterium with anti-inflammatory, immunoregulatory, and stress resilience properties. Previous studies have shown that whole, heat-killed preparations of M. vaccae prevent allergic airway inflammation in a murine model of allergic asthma. Recent studies also demonstrate that immunization with M. vaccae prevents stress-induced exaggeration of proinflammatory cytokine secretion from mesenteric lymph node cells stimulated ex vivo, prevents stress-induced exaggeration of chemically induced colitis in a model of inflammatory bowel disease, and prevents stress-induced anxiety-like defensive behavioral responses. Furthermore, immunization with M. vaccae induces anti-inflammatory responses in the brain and prevents stress-induced exaggeration of microglial priming. However, the molecular mechanisms underlying anti-inflammatory effects of M. vaccae are not known.
OBJECTIVES: Our objective was to identify and characterize novel anti-inflammatory molecules from M. vaccae NCTC 11659.
METHODS: We have purified and identified a unique anti-inflammatory triglyceride, 1,2,3-tri [Z-10-hexadecenoyl] glycerol, from M. vaccae and evaluated its effects in freshly isolated murine peritoneal macrophages.
RESULTS: The free fatty acid form of 1,2,3-tri [Z-10-hexadecenoyl] glycerol, 10(Z)-hexadecenoic acid, decreased lipopolysaccharide-stimulated secretion of the proinflammatory cytokine IL-6 ex vivo. Meanwhile, next-generation RNA sequencing revealed that pretreatment with 10(Z)-hexadecenoic acid upregulated genes associated with peroxisome proliferator-activated receptor alpha (PPARα) signaling in lipopolysaccharide-stimulated macrophages, in association with a broad transcriptional repression of inflammatory markers. We confirmed using luciferase-based transfection assays that 10(Z)-hexadecenoic acid activated PPARα signaling, but not PPARγ, PPARδ, or retinoic acid receptor (RAR) α signaling. The effects of 10(Z)-hexadecenoic acid on lipopolysaccharide-stimulated secretion of IL-6 were prevented by PPARα antagonists and absent in PPARα-deficient mice.
CONCLUSION: Future studies should evaluate the effects of 10(Z)-hexadecenoic acid on stress-induced exaggeration of peripheral inflammatory signaling, central neuroinflammatory signaling, and anxiety- and fear-related defensive behavioral responses.
Studying the dynamics of intracellular processes and investigating the interaction of individual macromolecules in live cells is one of the main objectives of cell biology. These macromolecules move, assemble, disassemble, and reorganize themselves in distinct manners under specific physiological conditions throughout the cell cycle. Therefore, in vivo experimental methods that enable the study of individual molecules inside cells at controlled culturing conditions have proved to be powerful tools to obtain insights into the molecular roles of these macromolecules and how their individual behavior influence cell physiology. The importance of controlled experimental conditions is enhanced when the investigated phenomenon covers long time periods, or perhaps multiple cell cycles. An example is the detection and quantification of proteins during bacterial DNA replication. Wide-field microscopy combined with microfluidics is a suitable technique for this. During fluorescence experiments, microfluidics offer well-defined cellular orientation and immobilization, flow and medium interchangeability, and high-throughput long-term experimentation of cells. Here we present a protocol for the combined use of wide-field microscopy and microfluidics for the study of proteins of the Escherichia coli DNA replication process. We discuss the preparation and application of a microfluidic device, data acquisition steps, and image analysis procedures to determine the stoichiometry and dynamics of a replisome component throughout the cell cycle of live bacterial cells.
Proteins acting on DNA need to penetrate a dense network of chromatin and associated macromolecules in the cell nucleus to access their target sites. Intracellular mobility of proteins is characterized by diffusion coefficients of the order of 1-100 μm2/s, leading to millisecond time scales for movement on the submicrometer scale. Typical microscopic methods used for characterizing intracellular protein mobility are, e.g., fluorescence photobleaching recovery (FRAP) and fluorescence correlation spectroscopy (FCS). Of these, FRAP can image protein mobility in entire two-dimensional sections of live cells, but is typically limited to the time resolution of confocal image series, some frames per second. FCS, on the other hand, has fast time resolution but so far has been limited to single-point measurements in the focus of a laser beam. Although we have demonstrated first protein mobility maps by point-to-point FCS, this method is extremely time-consuming and not very feasible for live cell measurements. Here we show results from single plane illumination microscopy based fluorescence correlation spectroscopy (SPIM-FCS), a new method for imaging FCS in 3D samples that combines the fast time resolution of FCS with the possibility of acquiring the mobility data in parallel on an entire twodimensional cross-section. This will then provide diffusion coefficients, flow velocities and concentrations in an imaging mode. Extending this technique to two-color fluorescence cross-correlation spectroscopy (SPIM-FCCS) also allows one to measure molecular interactions in an imaging mode
Retinoid X receptor (RXR) is a promiscuous nuclear receptor forming heterodimers with several other receptors, which activate different sets of genes. Upon agonist treatment, the occupancy of its genomic binding regions increased, but only a modest change in the number of sites was revealed by chromatin immunoprecipitation followed by sequencing, suggesting a rather static behavior. However, such genome-wide and biochemical approaches do not take into account the dynamic behavior of a transcription factor. Therefore, we characterized the nuclear dynamics of RXR during activation in single cells on the subsecond scale using live-cell imaging. By applying fluorescence recovery after photobleaching and fluorescence correlation spectroscopy (FCS), techniques with different temporal and spatial resolutions, a highly dynamic behavior could be uncovered which is best described by a two-state model (slow and fast) of receptor mobility. In the unliganded state, most RXRs belonged to the fast population, leaving ∼ 15% for the slow, chromatin-bound fraction. Upon agonist treatment, this ratio increased to ∼ 43% as a result of an immediate and reversible redistribution. Coactivator binding appears to be indispensable for redistribution and has a major contribution to chromatin association. A nuclear mobility map recorded by light sheet microscopy-FCS shows that the ligand-induced transition from the fast to the slow population occurs throughout the nucleus. Our results support a model in which RXR has a distinct, highly dynamic nuclear behavior and follows hit-and-run kinetics upon activation.
Glucocorticoid-induced apoptosis of thymocytes is one of the first recognized forms of programmed cell death. It was shown to require gene activation induced by the glucocorticoid receptor (GR) translocated into the nucleus following ligand binding. In addition, the necessity of the glucocorticoid-induced, but transcription-independent phosphorylation of phosphatidylinositol-specific phospholipase C (PI-PLC) has also been shown. Here we report that retinoic acids, physiological ligands for the nuclear retinoid receptors, enhance glucocorticoid-induced death of mouse thymocytes both in vitro and in vivo. The effect is mediated by retinoic acid receptor (RAR) alpha/retinoid X receptor (RXR) heterodimers, and occurs when both RARα and RXR are ligated by retinoic acids. We show that the ligated RARα/RXR interacts with the ligated GR, resulting in an enhanced transcriptional activity of the GR. The mechanism through which this interaction promotes GR-mediated transcription does not require DNA binding of the retinoid receptors and does not alter the phosphorylation status of Ser232, known to regulate the transcriptional activity of GR. Phosphorylation of PI-PLC was not affected. Besides thymocytes, retinoids also promoted glucocorticoid-induced apoptosis of various T-cell lines, suggesting that they could be used in the therapy of glucocorticoid-sensitive T-cell malignancies.
The retinoic acid receptor (RAR) is a member of the nuclear receptor superfamily. This ligand-inducible transcription factor binds to DNA as a heterodimer with the retinoid X receptor (RXR) in the nucleus. The nucleus is a dynamic compartment and live-cell imaging techniques make it possible to investigate transcription factor action in real-time. We studied the diffusion of EGFP–RAR by fluorescence correlation spectroscopy (FCS) to uncover the molecular interactions determining receptor mobility. In the absence of ligand, we identified two distinct species with different mobilities. The fast component has a diffusion coefficient of D1=1.8–6.0 μm2/second corresponding to small oligomeric forms, whereas the slow component with D2=0.05–0.10 μm2/second corresponds to interactions of RAR with the chromatin or other large structures. The RAR ligand-binding-domain fragment also has a slow component, probably as a result of indirect DNA-binding through RXR, with lower affinity than the intact RAR–RXR complex. Importantly, RAR-agonist treatment shifts the equilibrium towards the slow population of the wild-type receptor, but without significantly changing the mobility of either the fast or the slow population. By using a series of mutant forms of the receptor with altered DNA- or coregulator-binding capacity we found that the slow component is probably related to chromatin binding, and that coregulator exchange, specifically the binding of the coactivator complex, is the main determinant contributing to the redistribution of RAR during ligand activation.
The purpose of this RFC is to describe a method that allows the design of protein domain based parts, starting with gene centered information and translate these informations into BBF RFC 25 compatible part. The method is designed to be used in mammalian expression systems.
The activator protein-1 transcription factor is a heterodimer containing one of each of the Fos and Jun subfamilies of basic-region leucine-zipper proteins. We have previously shown by fluorescence cross-correlation spectroscopy (FCCS) that the fluorescent fusion proteins Fos-EGFP and Jun-mRFP1, cotransfected in HeLa cells, formed stable complexes in situ. Here we studied the relative position of the C-terminal domains via fluorescence resonance energy transfer (FRET) measured by flow cytometry and confocal microscopy. To get a more detailed insight into the conformation of the C-terminal domains of the complex we constructed C-terminal labeled full-length and truncated forms of Fos. We developed a novel iterative evaluation method to determine accurate FRET efficiencies regardless of relative protein expression levels, using a spectral- or intensity-based approach. The full-length C-terminal-labeled Jun and Fos proteins displayed a FRET-measured average distance of 8 +/- 1 nm. Deletion of the last 164 amino acids at the C-terminus of Fos resulted in a distance of 6.1 +/- 1 nm between the labels. FCCS shows that Jun-mRFP1 and the truncated Fos-EGFP also interact stably in the nucleus, although they bind to nuclear components with lower affinity. Thus, the C-terminal end of Fos may play a role in the stabilization of the interaction between activator protein-1 and DNA. Molecular dynamics simulations predict a dye-to-dye distance of 6.7 +/- 0.1 nm for the dimer between Jun-mRFP1 and the truncated Fos-EGFP, in good agreement with our FRET data. A wide variety of models could be developed for the full-length dimer, with possible dye-to-dye distances varying largely between 6 and 20 nm. However, from our FRET results we can conclude that more than half of the occurring dye-to-dye distances are between 6 and 10 nm.
Nuclear receptors are transcription factors that regulate gene expression in a ligand-dependent manner. They play key roles in several basic biological processes such as growth, differentiation and maintenance of homeostasis. According to the current general model of nuclear receptor action, in the absence of ligand, co-repressor and other members of the repressor complex are bound to the receptor, keeping the target gene silent. However, in the presence of an agonist ligand, co-regulator exchange takes place, which means that the repressor complex is released and co-activator with members of the activator complex takes its place. This regulator exchange results in activation of target gene expression. This process is viewed as the molecular switch model, which represents two distinct states of a rather static system. Due to modern biophysics and the evolution of microscope technology, new applications became available in the field of molecular biology, which made it possible to investigate transcriptional regulation at increasingly higher time resolution. As the result of these experiments the earlier static model is being replaced by a rather dynamic one.
ABCG2, a member of the ATP-binding cassette transporters has been identified as a protective pump against endogenous and exogenous toxic agents. ABCG2 was shown to be expressed at high levels in stem cells and variably regulated during cell differentiation. Here we demonstrate that functional ABCG2 is expressed in human monocyte-derived dendritic cells by the activation of a nuclear hormone receptor, PPARgamma. We identified and characterized a 150-base pair long conserved enhancer region, containing three functional PPAR response elements (PPARE), upstream of the human ABCG2 gene. We confirmed the binding of the PPARgamma x RXR heterodimer to this enhancer region, suggesting that PPARgamma directly regulates the transcription of ABCG2. Consistent with these results, elevated expression of ABCG2 mRNA was coupled to enhanced protein production, resulting in increased xenobiotic extrusion capacity via ABCG2 in PPARgamma-activated cells. Furthermore PPARgamma instructed dendritic cells showed increased Hoechst dye extrusion and resistance to mitoxantrone. Collectively, these results uncovered a mechanism by which up-regulation of functional ABCG2 expression can be achieved via exogenous or endogenous activation of the lipid-activated transcription factor, PPARgamma. The increased expression of the promiscuous ABCG2 transporter can significantly modify the xenobiotic and drug resistance of human myeloid dendritic cells.