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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.

Physical proximity between genomic sequences in mammalian chromosomes controls key biological processes such as transcriptional regulation and DNA repair. Yet it is currently unknown if chromosomal contacts are rare and stable or instead frequent and dynamic, and how they depend on the loop extrusion activity of cohesin or barriers such as CTCF. By imaging chromosomal locations at high spatial and temporal resolution over several hours in living cells, we show that sequences within topological associating domains (TADs) frequently come into physical proximity during the course of a cell cycle and remain close to each other only for a few minutes. Such contacts become nonetheless substantially longer and more frequent in the presence of convergent CTCF sites, resulting in a suppression of variability in chromosome folding in single cells across time. Supported by physical models of chromosome dynamics, our data additionally suggests that individual CTCF-anchored loops last around 10 minutes. The estimates of chromosomal contact dynamics in our study provide a novel quantitative framework to link chromosome structure to function and show that cohesin and CTCF stabilize otherwise highly dynamic chromosome structures to facilitate selected subsets of chromosomal interactions.

RNA degradation ensures appropriate levels of mRNA transcripts within cells and eliminates aberrant RNAs. Detailed studies of RNA degradation dynamics have been heretofore infeasible because of the inherent instability of degradation intermediates due to the high processivity of the enzymes involved. To visualize decay intermediates and to characterize the spatiotemporal dynamics of mRNA decay, we have developed a set of methods that apply XRN1-resistant RNA sequences (xrRNAs) to protect mRNA transcripts from 5'-3' exonucleolytic digestion. To our knowledge, this approach is the only method that can detect the directionality of mRNA degradation and that allows tracking of degradation products in unperturbed cells. Here, we provide detailed procedures for xrRNA reporter design, transfection and cell line generation. We explain how to extract xrRNA reporter mRNAs from mammalian cells, as well as their detection and quantification using northern blotting and quantitative PCR. The procedure further focuses on how to detect and quantify intact reporter mRNAs and XRN1-resistant degradation intermediates using single-molecule fluorescence microscopy. It provides detailed instructions for sample preparation and image acquisition using fixed, as well as living, cells. The procedure puts special emphasis on detailed descriptions of high-throughput image analysis pipelines, which are provided along with the article and were designed to perform spot co-localization, detection efficiency normalization and the quality control steps necessary for interpretation of results. The aim of the analysis software published here is to enable nonexpert readers to detect and quantify RNA decay intermediates within 4-6 d after reporter mRNA expression.

Cellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins. Although the function of SGs remains largely unknown, it is widely assumed they contain exclusively non-translating mRNA. Here, we re-examine this hypothesis using single-molecule imaging of mRNA translation in living cells. Although we observe non-translating mRNAs are preferentially recruited to SGs, we find unequivocal evidence that mRNAs localized to SGs can undergo translation. Our data indicate that SG-associated translation is not rare, and the entire translation cycle (initiation, elongation, and termination) can occur on SG-localized transcripts. Furthermore, translating mRNAs can be observed transitioning between the cytosol and SGs without changing their translational status. Together, these results demonstrate that mRNA localization to SGs is compatible with translation and argue against a direct role for SGs in inhibition of protein synthesis.

It is well established that mRNAs encoding secretory or membrane-bound proteins are translated on the surface of the endoplasmic reticulum (ER). The extent to which mRNAs that encode cytosolic proteins associate with the ER, however, remains controversial. To address this question, we quantified the number of cytosolic protein-encoding mRNAs that co-localize with the ER using single-molecule RNA imaging in fixed and living cells. We found that a small but significant number of mRNAs that encode cytosolic proteins associate with the ER and show that this interaction is translation dependent. Furthermore, we demonstrate that cytosolic protein-encoding transcripts can remain on the ER with dwell times consistent with multiple rounds of translation and have higher ribosome occupancies than transcripts translated in the cytosol. These results advance our understanding of the diversity and dynamics of localized translation on the ER.

Gene expression oscillators can structure biological events temporally and spatially. Different biological functions benefit from distinct oscillator properties. Thus, finite developmental processes rely on oscillators that start and stop at specific times; a poorly understood behavior. Here, we have characterized a massive gene expression oscillator comprising >3,700 genes inC. eleganslarvae. We report that oscillations initiate in embryos, arrest transiently after hatching and in response to perturbation, and cease in adults. Experimental observation of the transitions between oscillatory and non-oscillatory states at a resolution where we can identify bifurcation points reveals an oscillator operating near a Saddle Node on Invariant Cycle (SNIC) bifurcation. These findings constrain the architecture and mathematical models that can represent this oscillator. They also reveal that oscillator arrests occur reproducibly in a specific phase. Since we find oscillations to be coupled to developmental processes, including molting, this characteristic of SNIC bifurcations thus endows the oscillator with the potential to halt larval development at defined intervals, and thereby execute a developmental checkpoint function.

mRNA turnover plays an important role in the regulation of post-transcriptional gene expression. While many protein factors involved in mRNA degradation have been identified, we still lack a basic understanding of the principles that regulate the spatiotemporal dynamics of mRNA turnover within single cells. To overcome this limitation, we have developed the TREAT biosensor, which allows for discrimination of intact reporter transcripts and stabilized decay intermediates using single RNA imaging. Here, we present an image analysis pipeline that performs semiautomated detection and tracking of individual mRNA particles. It colocalizes tracks and applies the colocalization information to quantify the number of intact transcripts and degradation intermediates. Based on the analysis of control data, the workflow further determines detection efficiencies and uses them to correct RNA particle numbers.

mRNA turnover plays an important role in the regulation of post-transcriptional gene expression. While many protein factors involved in mRNA degradation have been identified, we still lack a basic understanding of the principles that regulate the spatiotemporal dynamics of mRNA turnover within single cells. To overcome this limitation, we have developed the TREAT biosensor, which allows for discrimination of intact reporter transcripts and stabilized decay intermediates using single RNA imaging. Here, we present an image analysis pipeline that performs semiautomated detection and tracking of individual mRNA particles. It colocalizes tracks and applies the colocalization information to quantify the number of intact transcripts and degradation intermediates. Based on the analysis of control data, the workflow further determines detection efficiencies and uses them to correct RNA particle numbers.

Quantitative fluorescence microscopy techniques are frequently applied to answer fundamental biological questions. Single-molecule RNA imaging methods have enabled the direct observation of the initial steps of the mRNA life cycle in living cells, however, the dynamic mechanisms that regulate mRNA translation are still poorly understood. We have developed an RNA biosensor that can assess the translational state of individual mRNA transcripts with spatiotemporal resolution in living cells. In this chapter, we describe how to perform a TRICK (translating RNA imaging by coat protein knock-off) experiment and specifically focus on a detailed description of our image processing and data analysis procedure.

Liquid–liquid phase separation (LLPS) of proteins containing intrinsically disordered regions (IDRs) has been proposed as a mechanism underlying the formation of membrane-less organelles. Tight regulation of IDR behavior is essential to ensure that LLPS only takes place when necessary. Here, we report that IDR acetylation/deacetylation regulates LLPS and assembly of stress granules (SGs), membrane-less organelles forming in response to stress. Acetylome analysis revealed that the RNA helicase DDX3X, an important component of SGs, is a novel substrate of the deacetylase HDAC6. The N-terminal IDR of DDX3X (IDR1) can undergo LLPS in vitro, and its acetylation at multiple lysine residues impairs the formation of liquid droplets. We also demonstrated that enhanced LLPS propensity through deacetylation of DDX3X-IDR1 by HDAC6 is necessary for SG maturation, but not initiation. Our analysis provides a mechanistic framework to understand how acetylation and deacetylation of IDRs regulate LLPS spatiotemporally, and impact membrane-less organelle formation in vivo. HDAC6 modulates acetylation at multiple lysine residues in the N-terminal intrinsically disordered region of RNA helicase DDX3X to regulate liquid–liquid phase separation and stress granule maturation.

Quantitative fluorescence microscopy techniques are frequently applied to answer fundamental biological questions. Single-molecule RNA imaging methods have enabled the direct observation of the initial steps of the mRNA life cycle in living cells, however, the dynamic mechanisms that regulate mRNA translation are still poorly understood. We have developed an RNA biosensor that can assess the translational state of individual mRNA transcripts with spatiotemporal resolution in living cells. In this chapter, we describe how to perform a TRICK (translating RNA imaging by coat protein knock-off) experiment and specifically focus on a detailed description of our image processing and data analysis procedure.

Background Pericytes, surrounding the endothelium, fulfill diverse functions that are crucial for vascular homeostasis. The loss of pericytes is associated with pathologies, such as diabetic retinopathy and Alzheimer’s disease. Thus, there exists a need for an experimental system that combines pharmacologic manipulation and quantification of pericyte coverage during sprouting angiogenesis. Here, we describe an in vitro angiogenesis assay that develops lumenized vascular sprouts composed of endothelial cells enveloped by pericytes, with the additional ability to comparatively screen the effect of multiple small molecules simultaneously. For automated analysis, we also present an ImageJ plugin tool we developed to quantify sprout morphology and pericyte coverage. Methods Human umbilical vein endothelial cells and human brain vascular pericytes were coated on microcarrier beads and embedded in fibrin gels in a 96-well plate to form lumenized vascular sprouts. After treatment with pharmacologic compounds, sprouts were fixed, stained, and imaged via optical z-sections over the area of each well. The maximum intensity projections of these images were stitched together to form montages of the wells, and those montages were processed and analyzed. Results Vascular sprouts formed within 4–12 days and contained a patent lumen surrounded by a layer of human endothelial cells and pericytes. Using our workflow and image analysis, pericyte coverage after treatment with various compounds was successfully quantified. Conclusions Here we present a robust in vitro assay using primary human vascular cells that allows researchers to analyze the effects of multiple compounds on sprouting angiogenesis and pericyte coverage. Our ImageJ plugin offers automated evaluation across multiple different vascular parameters, such as sprout length, cell density, branch points, and pericyte coverage. Electronic supplementary material The online version of this article (doi:10.1186/s41232-016-0033-2) contains supplementary material, which is available to authorized users.

Nucleosomes are essential for proper chromatin organization and the maintenance of genome integrity. Histones are post-translationally modified and often evicted at sites of DNA breaks, facilitating the recruitment of repair factors. Whether such chromatin changes are localized or genome-wide is debated. Here we show that cellular levels of histones drop 20-40% in response to DNA damage. This histone loss occurs from chromatin, is proteasome-mediated and requires both the DNA damage checkpoint and the INO80 nucleosome remodeler. We confirmed reductions in histone levels by stable isotope labeling of amino acids in cell culture (SILAC)-based mass spectrometry, genome-wide nucleosome mapping and fluorescence microscopy. Chromatin decompaction and increased fiber flexibility accompanied histone degradation, both in response to DNA damage and after artificial reduction of histone levels. As a result, recombination rates and DNA-repair focus turnover were enhanced. Thus, we propose that a generalized reduction in nucleosome occupancy is an integral part of the DNA damage response in yeast that provides mechanisms for enhanced chromatin mobility and homology search.

SummaryBlood vessels function in the uptake, transport, and delivery of gases and nutrients within the body. A key question is how the central lumen of blood vessels develops within a cord of vascular endothelial cells. Here, we demonstrate that sialic acids of apical glycoproteins localize to apposing endothelial cell surfaces and generate repelling electrostatic fields within an endothelial cell cord. Both in vitro and in vivo experiments show that the negative charge of sialic acids is required for the separation of endothelial cell surfaces and subsequent lumen formation. We also demonstrate that sulfate residues can substitute for sialic acids during lumen initiation. These results therefore reveal a key step in the creation of blood vessels, the most abundant conduits in the vertebrate body. Because negatively charged mucins and proteoglycans are often found on luminal cell surfaces, it is possible that electrostatic repulsion is a general principle also used to initiate lumen formation in other organs.

The Mre11-Rad50-Xrs2 (MRX) complex is related to SMC complexes that form rings capable of holding two distinct DNA strands together. MRX functions at stalled replication forks and double-strand breaks (DSBs). A mutation in the N-terminal OB fold of the 70 kDa subunit of yeast replication protein A, rfa1-t11, abrogates MRX recruitment to both types of DNA damage. The rfa1 mutation is functionally epistatic with loss of any of the MRX subunits for survival of replication fork stress or DSB recovery, although it does not compromise end-resection. High-resolution imaging shows that either the rfa1-t11 or the rad50Δ mutation lets stalled replication forks collapse and allows the separation not only of opposing ends but of sister chromatids at breaks. Given that cohesin loss does not provoke visible sister separation as long as the RPA-MRX contacts are intact, we conclude that MRX also serves as a structural linchpin holding sister chromatids together at breaks.

A hallmark feature of type 1 and type 2 diabetes mellitus is the progressive dysfunction and loss of insulin-producing pancreatic beta cells, and inflammatory cytokines are known to trigger beta cell death. Here we asked whether the anti-oxidant protein DJ-1 encoded by the Parkinson's disease gene PARK7 protects islet cells from cytokine- and streptozotocin-mediated cell death. Wild type and DJ-1 knockout mice (KO) were treated with multiple low doses of streptozotocin (MLDS) to induce inflammatory beta cell stress and cell death. Subsequently, glucose tolerance tests were performed, and plasma insulin as well as fasting and random blood glucose concentrations were monitored. Mitochondrial morphology and number of insulin granules were quantified in beta cells. Moreover, islet cell damage was determined in vitro after streptozotocin and cytokine treatment of isolated wild type and DJ-1 KO islets using calcein AM/ethidium homodimer-1 staining and TUNEL staining. Compared to wild type mice, DJ-1 KO mice became diabetic following MLDS treatment. Insulin concentrations were substantially reduced, and fasting blood glucose concentrations were significantly higher in MLDS-treated DJ-1 KO mice compared to equally treated wild type mice. Rates of beta cell apoptosis upon MLDS treatment were twofold higher in DJ-1 KO mice compared to wild type mice, and in vitro inflammatory cytokines led to twice as much beta cell death in pancreatic islets from DJ-1 KO mice versus those of wild type mice. In conclusion, this study identified the anti-oxidant protein DJ-1 as being capable of protecting pancreatic islet cells from cell death induced by an inflammatory and cytotoxic setting.

In the nervous system, NMDA receptors (NMDARs) participate in neurotransmission and modulate the viability of neurons. In contrast, little is known about the role of NMDARs in pancreatic islets and the insulin-secreting beta cells whose functional impairment contributes to diabetes mellitus. Here we found that inhibition of NMDARs in mouse and human islets enhanced their glucose-stimulated insulin secretion (GSIS) and survival of islet cells. Further, NMDAR inhibition prolonged the amount of time that glucose-stimulated beta cells spent in a depolarized state with high cytosolic Ca(2+) concentrations. We also noticed that, in vivo, the NMDAR antagonist dextromethorphan (DXM) enhanced glucose tolerance in mice, and that in vitro dextrorphan, the main metabolite of DXM, amplified the stimulatory effect of exendin-4 on GSIS. In a mouse model of type 2 diabetes mellitus (T2DM), long-term treatment with DXM improved islet insulin content, islet cell mass and blood glucose control. Further, in a small clinical trial we found that individuals with T2DM treated with DXM showed enhanced serum insulin concentrations and glucose tolerance. Our data highlight the possibility that antagonists of NMDARs may provide a useful adjunct treatment for diabetes.

Elderly patients often suffer from multiple age-related diseases. Here we show that the expression of DJ-1, an antioxidant protein with reduced expression in the central nervous system of patients with Parkinson's disease, is reduced in pancreatic islets of patients with type 2 diabetes mellitus (T2DM). In contrast, under non-diabetic conditions, DJ-1 expression increases in mouse and human islets during aging. In mouse islets, we show that DJ-1 prevents an increase in reactive oxygen species levels as the mice age. This antioxidant function preserves mitochondrial integrity and physiology, prerequisites for glucose-stimulated insulin secretion. Accordingly, DJ-1-deficient mice develop glucose intolerance and reduced β cell area as they age or gain weight. Our data suggest that DJ-1 is more generally involved in age- and lifestyle-related human diseases and show for the first time that DJ-1 plays a key role in glucose homeostasis and might serve as a novel drug target for T2DM.

Endothelial cells are equipped with the intrinsic ability to form tubes and sprouts with a central lumen. However, the mechanisms that allow endothelial cells to form a lumen are largely unknown. We would like to discuss critically current models of vascular lumen formation and point to many unexplored and open questions. We briefly present what vascular researchers can learn from the formation of other cell systems with a lumen, such as cysts formed by Madin-Darby Canine Kidney (MDCK) cells as well as tracheae formed by Drosophila epithelial cells. In addition, we point to a number of questions that need to be addressed to understand better the cell biology that drives the formation of a vascular lumen.

Endothelial cells are equipped with the intrinsic ability to form tubes and sprouts with a central lumen. However, the mechanisms that allow endothelial cells to form a lumen are largely unknown. We would like to discuss critically current models of vascular lumen formation and point to many unexplored and open questions. We briefly present what vascular researchers can learn from the formation of other cell systems with a lumen, such as cysts formed by Madin-Darby Canine Kidney (MDCK) cells as well as tracheae formed by Drosophila epithelial cells. In addition, we point to a number of questions that need to be addressed to understand better the cell biology that drives the formation of a vascular lumen.

SUMMARY In vertebrates, endothelial cells (ECs) form blood vessels in every tissue. Here, we investigated vascular lumen formation in the developing aorta, the first and largest arterial blood vessel in all vertebrates. Comprehensive imaging, pharmacological manipulation, and genetic approaches reveal that, in mouse embryos, the aortic lumen develops extracellularly between adjacent ECs. We show that ECs adhere to each other, and that CD34-sialomucins, Moesin, F-actin, and non-muscle Myosin II localize at the endothelial cell-cell contact to define the luminal cell surface. Resultant changes in EC shape lead to lumen formation. Importantly, VE-Cadherin and VEGF-A act at different steps. VE-Cadherin is required for localizing CD34-sialomucins to the endothelial cell-cell contact, a prerequisite to Moesin and F-actin recruitment. In contrast, VEGF-A is required for F-actin-nm-Myosin II interactions and EC shape change. Based on these data, we propose a molecular mechanism of in vivo vascular lumen formation in developing blood vessels.