Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However, for scientists wishing to publish obtained images and image-analysis results, there are currently no unified guidelines for best practices. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here, we present community-developed checklists for preparing light microscopy images and describing image analyses for publications. These checklists offer authors, readers and publishers key recommendations for image formatting and annotation, color selection, data availability and reporting image-analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby to heighten the quality and explanatory power of microscopy data.

Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However for scientists wishing to publish the obtained images and image analyses results, there are to date no unified guidelines. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here we present community-developed checklists for preparing light microscopy images and image analysis for publications. These checklists offer authors, readers, and publishers key recommendations for image formatting and annotation, color selection, data availability, and for reporting image analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby heighten the quality and explanatory power of microscopy data is in publications.

Cohesin and CTCF control the dynamics of chromosome folding.

In mammals, interactions between sequences within topologically associating domains enable control of gene expression across large genomic distances. Yet it is unknown how frequently such contacts occur, how long they last and how they depend on the dynamics of chromosome folding and loop extrusion activity of cohesin. By imaging chromosomal locations at high spatial and temporal resolution in living cells, we show that interactions within topologically associating domains are transient and occur frequently during the course of a cell cycle. Interactions become more frequent and longer in the presence of convergent CTCF sites, resulting in suppression of variability in chromosome folding across time. Supported by physical models of chromosome dynamics, our data suggest that CTCF-anchored loops last around 10 min. Our results show that long-range transcriptional regulation might rely on transient physical proximity, and that cohesin and CTCF stabilize highly dynamic chromosome structures, facilitating selected subsets of chromosomal interactions.

QUAREP-LiMi: A community-driven initiative to establish guidelines for quality assessment and reproducibility for instruments and images in light microscopy.

A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist's experiments, while more than half have even failed to reproduce their own experiments. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g. DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility. In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper (1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; (2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers and observers of such; (3) outlines the current actions of the QUAREP-LiMi initiative and (4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.

Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation.

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.

Neutrophil extracellular trap formation requires OPA1-dependent glycolytic ATP production.

Optic atrophy 1 (OPA1) is a mitochondrial inner membrane protein that has an important role in mitochondrial fusion and structural integrity. Dysfunctional OPA1 mutations cause atrophy of the optic nerve leading to blindness. Here, we show that OPA1 has an important role in the innate immune system. Using conditional knockout mice lacking Opa1 in neutrophils (Opa1N∆), we report that lack of OPA1 reduces the activity of mitochondrial electron transport complex I in neutrophils. This then causes a decline in adenosine-triphosphate (ATP) production through glycolysis due to lowered NAD+ availability. Additionally, we show that OPA1-dependent ATP production in these cells is required for microtubule network assembly and for the formation of neutrophil extracellular traps. Finally, we show that Opa1N∆ mice exhibit a reduced antibacterial defense capability against Pseudomonas aeruginosa.

H3K9 methylation extends across natural boundaries of heterochromatin in the absence of an HP1 protein.

Proteins of the conserved HP1 family are elementary components of heterochromatin and are generally assumed to play a central role in the creation of a rigid, densely packed heterochromatic network that is inaccessible to the transcription machinery. Here, we demonstrate that the fission yeast HP1 protein Swi6 exists as a single highly dynamic population that rapidly exchanges in cis and in trans between different heterochromatic regions. Binding to methylated H3K9 or to heterochromatic RNA decelerates Swi6 mobility. We further show that Swi6 is largely dispensable to the maintenance of heterochromatin domains. In the absence of Swi6, H3K9 methylation levels are maintained by a mechanism that depends on polymeric self-association properties of Tas3, a subunit of the RNA-induced transcriptional silencing complex. Our results disclose a surprising role for Swi6 dimerization in demarcating constitutive heterochromatin from neighboring euchromatin. Thus, rather than promoting maintenance and spreading of heterochromatin, Swi6 appears to limit these processes and appropriately confine heterochromatin.

Interfering with the connection between the nucleus and the cytoskeleton affects nuclear rotation, mechanotransduction and myogenesis.

Mechanical stress controls a broad range of cellular functions. The cytoskeleton is physically connected to the extracellular matrix via integrin receptors, and to the nuclear lamina by the LINC complex that spans both nuclear membranes. We asked here how disruption of this direct link from the cytoskeleton to nuclear chromatin affects mechanotransduction. Fibroblasts grown on flexible silicone membranes reacted to cyclic stretch by nuclear rotation. This rotation was abolished by inhibition of actomyosin contraction as well as by overexpression of dominant-negative versions of nesprin or sun proteins that form the LINC complex. In an in vitro model of muscle differentiation, cyclic strain inhibits differentiation and induces proliferation of C2C12 myoblasts. Interference with the LINC complex in these cells abrogated their stretch-induced proliferation, while stretch increased p38 MAPK and NFkappaB phosphorylation and the transcript levels of myogenic transcription factors MyoD and myogenin. We found that the physical link from the cytoskeleton to the nuclear lamina is crucial for correct mechanotransduction, and that disruption of the LINC complex perturbs the mechanical control of cell differentiation.

The pollutant diethylhexyl phthalate regulates hepatic energy metabolism via species-specific PPARalpha-dependent mechanisms.

BACKGROUND: The modulation of energetic homeostasis by pollutants has recently emerged as a potential contributor to the onset of metabolic disorders. Diethylhexyl phthalate (DEHP) is a widely used industrial plasticizer to which humans are widely exposed. Phthalates can activate the three peroxisome proliferator-activated receptor (PPAR) isotypes on cellular models and induce peroxisome proliferation in rodents. OBJECTIVES: In this study, we aimed to evaluate the systemic and metabolic consequences of DEHP exposure that have remained so far unexplored and to characterize the underlying molecular mechanisms of action. METHODS: As a proof of concept and mechanism, genetically engineered mouse models of PPARs were exposed to high doses of DEHP, followed by metabolic and molecular analyses. RESULTS: DEHP-treated mice were protected from diet-induced obesity via PPARalpha-dependent activation of hepatic fatty acid catabolism, whereas the activity of neither PPARbeta nor PPARgamma was affected. However, the lean phenotype observed in response to DEHP in wild-type mice was surprisingly abolished in PPARalpha-humanized mice. These species differences are associated with a different pattern of coregulator recruitment. CONCLUSION: These results demonstrate that DEHP exerts species-specific metabolic actions that rely to a large extent on PPARalpha signaling and highlight the metabolic importance of the species-specific activation of PPARalpha by xenobiotic compounds.

From mechanotransduction to extracellular matrix gene expression in fibroblasts.

Tissue mechanics provide an important context for tissue growth, maintenance and function. On the level of organs, external mechanical forces largely influence the control of tissue homeostasis by endo- and paracrine factors. On the cellular level, it is well known that most normal cell types depend on physical interactions with their extracellular matrix in order to respond efficiently to growth factors. Fibroblasts and other adherent cells sense changes in physical parameters in their extracellular matrix environment, transduce mechanical into chemical information, and integrate these signals with growth factor derived stimuli to achieve specific changes in gene expression. For connective tissue cells, production of the extracellular matrix is a prominent response to changes in mechanical load. We will review the evidence that integrin-containing cell-matrix adhesion contacts are essential for force transmission from the extracellular matrix to the cytoskeleton, and describe novel experiments indicating that mechanotransduction in fibroblasts depends on focal adhesion adaptor proteins that might function as molecular springs. We will stress the importance of the contractile actin cytoskeleton in balancing external with internal forces, and describe new results linking force-controlled actin dynamics directly to the expression of specific genes, among them the extracellular matrix protein tenascin-C. As assembly lines for diverse signaling pathways, matrix adhesion contacts are now recognized as the major sites of crosstalk between mechanical and chemical stimuli, with important consequences for cell growth and differentiation.

Direct protein-protein interaction of 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the endoplasmic reticulum lumen.

Hexose-6-phosphate dehydrogenase (H6PDH) has been shown to stimulate 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1)-dependent local regeneration of active glucocorticoids. Here, we show that coexpression with H6PDH results in a dramatic shift from 11beta-HSD1 oxidase to reductase activity without affecting the activity of the endoplasmic reticular enzyme 17beta-HSD2. Immunoprecipitation experiments revealed coprecipitation of H6PDH with 11beta-HSD1 but not with the related enzymes 11beta-HSD2 and 17beta-HSD2, suggesting a specific interaction between H6PDH and 11beta-HSD1. The use of the 11beta-HSD1/11beta-HSD2 chimera indicates that the N-terminal 39 residues of 11beta-HSD1 are sufficient for interaction with H6PDH. An important role of the N-terminus was indicated further by the significantly stronger interaction of 11beta-HSD1 mutant Y18-21A with H6PDH compared to wild-type 11beta-HSD1. The protein-protein interaction and the involvement of the N-terminus of 11beta-HSD1 were confirmed by Far-Western blotting. Finally, fluorescence resonance energy transfer (FRET) measurements of HEK-293 cells expressing fluorescently labeled proteins provided evidence for an interaction between 11beta-HSD1 and H6PDH in intact cells. Thus, using three different methods, we provide strong evidence that the functional coupling between 11beta-HSD1 and H6PDH involves a direct physical interaction of the two proteins.

Tenascin-C induction by cyclic strain requires integrin-linked kinase.

Induction of tenascin-C mRNA by cyclic strain in fibroblasts depends on RhoA and Rho dependent kinase (ROCK). Here we show that integrin-linked kinase (ILK) is required upstream of this pathway. In ILK-deficient fibroblasts, RhoA was not activated and tenascin-C mRNA remained low after cyclic strain; tenascin-C expression was unaffected by ROCK inhibition. In ILK wild-type but not ILK-/- fibroblasts, cyclic strain-induced reorganization of actin stress fibers and focal adhesions, as well as nuclear translocation of MAL, a transcriptional co-activator that links actin assembly to gene expression. These findings support a role for RhoA in ILK-mediated mechanotransduction. Rescue of ILK -/- fibroblasts by expression of wild-type ILK restored these responses to cyclic strain. Mechanosensation is not entirely abolished in ILK -/- fibroblasts, since cyclic strain activated Erk-1/2 and PKB/Akt, and induced c-fos mRNA in these cells. Conversely, lysophosphatidic acid stimulated RhoA and induced both c-fos and tenascin-C mRNA in ILK -/- cells. Thus, the signaling pathways controlling tenascin-C expression are functional in the absence of ILK, but are not triggered by cyclic strain. Our results indicate that ILK is selectively required for the induction of specific genes by mechanical stimulation via RhoA-mediated pathways.

Ligand-dependent transcriptional activities of four torafugu pufferfish Takifugu rubripes peroxisome proliferator-activated receptors.

Structural and functional properties were investigated for four peroxisome proliferator-activated receptors (PPARs), PPARalpha1, PPARalpha2, PPARbeta, and PPARgamma, from torafugu pufferfish Takifugu rubripes and determined for their transcriptional activity by the reporter assay using reporter plasmids containing three copies of the acyl-CoA oxidase PPAR response element. Although torafugu PPARs showed a high similarity in the primary structure to other vertebrate counterparts, torafugu PPARalpha2 and gamma contained additional sequences of 21 and 28 amino acids, respectively, as in the case of other teleost fish species when compared with African clawed frog counterparts. The transcriptional activity of torafugu PPARalpha1 was enhanced 4.5- and 11.5-fold by Wy-14643 and 5,8,11,14-eicosatetraynoic acid (ETYA) each at 10 microM, respectively, whereas that of PPARalpha2, 4.5- and 7.3-fold at the same concentration of the respective ligands, respectively. The activities of torafugu PPARalpha1 and alpha2 were also enhanced 5.6- and 6.3-fold by ETYA at 1 microM, respectively, but not by Wy-14643 at this concentration. Furthermore, the activities of the two torafugu PPARalphas were enhanced 4.3- and 7.6-fold by arachidonic acid, 4.4- and 5.2-fold by docosahexaenoic acid, and 6.7- and 8.0-fold by eicosapentaenoic acid each at 50 microM, respectively. On the other hand, the activities of torafugu PPARbeta and gamma were not changed by Wy-14643, ETYA, rosiglitazone, nor PUFAs. These results suggest that the activities of torafugu PPARbeta and gamma require undefined ligands. Alternatively, the molecular mechanisms involved in their activation are different from those of other vertebrates.

The endocrine disruptor monoethyl-hexyl-phthalate is a selective peroxisome proliferator-activated receptor gamma modulator that promotes adipogenesis.

The ability of pollutants to affect human health is a major concern, justified by the wide demonstration that reproductive functions are altered by endocrine disrupting chemicals. The definition of endocrine disruption is today extended to broader endocrine regulations, and includes activation of metabolic sensors, such as the peroxisome proliferator-activated receptors (PPARs). Toxicology approaches have demonstrated that phthalate plasticizers can directly influence PPAR activity. What is now missing is a detailed molecular understanding of the fundamental basis of endocrine disrupting chemical interference with PPAR signaling. We thus performed structural and functional analyses that demonstrate how monoethyl-hexyl-phthalate (MEHP) directly activates PPARgamma and promotes adipogenesis, albeit to a lower extent than the full agonist rosiglitazone. Importantly, we demonstrate that MEHP induces a selective activation of different PPARgamma target genes. Chromatin immunoprecipitation and fluorescence microscopy in living cells reveal that this selective activity correlates with the recruitment of a specific subset of PPARgamma coregulators that includes Med1 and PGC-1alpha, but not p300 and SRC-1. These results highlight some key mechanisms in metabolic disruption but are also instrumental in the context of selective PPAR modulation, a promising field for new therapeutic development based on PPAR modulation.

Molecular basis of selective PPARgamma modulation for the treatment of Type 2 diabetes.

Peroxisome proliferator-activated receptors (PPARs) (alpha, beta/delta and gamma) are lipid sensors capable of adapting gene expression to integrate various lipid signals. As such, PPARs are also very important pharmaceutical targets, and specific synthetic ligands exist for the different isotypes and are either currently used or hold promises in the treatment of major metabolic disorders. In particular, compounds of the class of the thiazolinediones (TZDs) are PPARgamma agonists and potent insulin-sensitizers. The specific but still broad expression patterns of PPARgamma, as well as its implication in numerous pathways, constitutes also a disadvantage regarding drug administration, since this potentially increases the chance to generate side-effects through the activation of the receptor in tissues or cells not affected by the disease. Actually, numerous side effects associated with the administration of TZDs have been reported. Today, a new generation of PPARgamma modulators is being actively developed to activate the receptor more specifically, in a cell and time-dependent manner, in order to induce a specific subset of target genes only and modulate a restricted number of metabolic pathways. We will discuss here why and how the development of such selective PPARgamma modulators is possible, and summarize the results obtained with the published molecules.

Combined simulation and mutagenesis analyses reveal the involvement of key residues for peroxisome proliferator-activated receptor alpha helix 12 dynamic behavior.

The dynamic properties of helix 12 in the ligand binding domain of nuclear receptors are a major determinant of AF-2 domain activity. We investigated the molecular and structural basis of helix 12 mobility, as well as the involvement of individual residues with regard to peroxisome proliferator-activated receptor alpha (PPARalpha) constitutive and ligand-dependent transcriptional activity. Functional assays of the activity of PPARalpha helix 12 mutants were combined with free energy molecular dynamics simulations. The agreement between the results from these approaches allows us to make robust claims concerning the mechanisms that govern helix 12 functions. Our data support a model in which PPARalpha helix 12 transiently adopts a relatively stable active conformation even in the absence of a ligand. This conformation provides the interface for the recruitment of a coactivator and results in constitutive activity. The receptor agonists stabilize this conformation and increase PPARalpha transcription activation potential. Finally, we disclose important functions of residues in PPARalpha AF-2, which determine the positioning of helix 12 in the active conformation in the absence of a ligand. Substitution of these residues suppresses PPARalpha constitutive activity, without changing PPARalpha ligand-dependent activation potential.

Association with coregulators is the major determinant governing peroxisome proliferator-activated receptor mobility in living cells.

The nucleus is an extremely dynamic compartment, and protein mobility represents a key factor in transcriptional regulation. We showed in a previous study that the diffusion of peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors regulating major cellular and metabolic functions, is modulated by ligand binding. In this study, we combine fluorescence correlation spectroscopy, dual color fluorescence cross-correlation microscopy, and fluorescence resonance energy transfer to dissect the molecular mechanisms controlling PPAR mobility and transcriptional activity in living cells. First, we bring new evidence that in vivo a high percentage of PPARs and retinoid X receptors is associated even in the absence of ligand. Second, we demonstrate that coregulator recruitment (and not DNA binding) plays a crucial role in receptor mobility, suggesting that transcriptional complexes are formed prior to promoter binding. In addition, association with coactivators in the absence of a ligand in living cells, both through the N-terminal AB domain and the AF-2 function of the ligand binding domain, provides a molecular basis to explain PPAR constitutive activity.

Integrating nuclear receptor mobility in models of gene regulation.

The mode of action of nuclear receptors in living cells is an actively investigated field but much remains hypothetical due to the lack, until recently, of methods allowing the assessment of molecular mechanisms in vivo. However, these last years, the development of fluorescence microscopy methods has allowed initiating the dissection of the molecular mechanisms underlying gene regulation by nuclear receptors directly in living cells or organisms. Following our analyses on peroxisome proliferator activated receptors (PPARs) in living cells, we discuss here the different models arising from the use of these tools, that attempt to link mobility, DNA binding or chromatin interaction, and transcriptional activity.