Transcriptomic analysis of intestine following administration of a transglutaminase 2 inhibitor to prevent gluten-induced intestinal damage in celiac disease.

Transglutaminase 2 (TG2) plays a pivotal role in the pathogenesis of celiac disease (CeD) by deamidating dietary gluten peptides, which facilitates antigenic presentation and a strong anti-gluten T cell response. Here, we elucidate the molecular mechanisms underlying the efficacy of the TG2 inhibitor ZED1227 by performing transcriptional analysis of duodenal biopsies from individuals with CeD on a long-term gluten-free diet before and after a 6-week gluten challenge combined with 100 mg per day ZED1227 or placebo. At the transcriptome level, orally administered ZED1227 effectively prevented gluten-induced intestinal damage and inflammation, providing molecular-level evidence that TG2 inhibition is an effective strategy for treating CeD. ZED1227 treatment preserved transcriptome signatures associated with mucosal morphology, inflammation, cell differentiation and nutrient absorption to the level of the gluten-free diet group. Nearly half of the gluten-induced gene expression changes in CeD were associated with the epithelial interferon-γ response. Moreover, data suggest that deamidated gluten-induced adaptive immunity is a sufficient step to set the stage for CeD pathogenesis. Our results, with the limited sample size, also suggest that individuals with CeD might benefit from an HLA-DQ2/HLA-DQ8 stratification based on gene doses to maximally eliminate the interferon-γ-induced mucosal damage triggered by gluten.

Build and operation of a custom 3D, multicolor, single-molecule localization microscope.

Single-molecule localization microscopy (SMLM) enables imaging scientists to visualize biological structures with unprecedented resolution. Particularly powerful implementations of SMLM are capable of three-dimensional, multicolor and high-throughput imaging and can yield key biological insights. However, widespread access to these technologies is limited, primarily by the cost of commercial options and complexity of de novo development of custom systems. Here we provide a comprehensive guide for interested researchers who wish to establish a high-end, custom-built SMLM setup in their laboratories. We detail the initial configuration and subsequent assembly of the SMLM, including the instructions for the alignment of all the optical pathways, the software and hardware integration, and the operation of the instrument. We describe the validation steps, including the preparation and imaging of test and biological samples with structures of well-defined geometries, and assist the user in troubleshooting and benchmarking the system's performance. Additionally, we provide a walkthrough of the reconstruction of a super-resolved dataset from acquired raw images using the Super-resolution Microscopy Analysis Platform. Depending on the instrument configuration, the cost of the components is in the range US$95,000-180,000, similar to other open-source advanced SMLMs, and substantially lower than the cost of a commercial instrument. A builder with some experience of optical systems is expected to require 4-8 months from the start of the system construction to attain high-quality three-dimensional and multicolor biological images.

Setting up a light microscopy core facility: Facility design.

The successful operation of a light microscopy core facility depends also on the initial setup of its infrastructure. This article covers the aspects of location selection and room planning and what environmental factors need to be considered. These include light, temperature, vibrations as well as the basic installations needed for microscope operation.

The Oral Transglutaminase 2 Inhibitor ZED1227 Accumulates in the Villous Enterocytes in Celiac Disease Patients during Gluten Challenge and Drug Treatment.

The enzyme transglutaminase 2 (TG2) plays a key role in celiac disease (CeD) pathogenesis. Active TG2 is located mainly extracellularly in the lamina propria but also in the villous enterocytes of the duodenum. The TG2 inhibitor ZED1227 is a promising drug candidate for treating CeD and is designed to block the TG2-catalyzed deamidation and crosslinking of gliadin peptides. Our aim was to study the accumulation of ZED1227 after oral administration of the drug. We studied duodenal biopsies derived from a phase 2a clinical drug trial using an antibody that detects ZED1227 when bound to the catalytic center of TG2. Human epithelial organoids were studied in vitro for the effect of ZED1227 on the activity of TG2 using the 5-biotin-pentylamine assay. The ZED1227-TG2 complex was found mainly in the villous enterocytes in post-treatment biopsies. The signal of ZED1227-TG2 was strongest in the luminal epithelial brush border, while the intensity of the signal in the lamina propria was only ~20% of that in the villous enterocytes. No signal specific to ZED1227 could be detected in pretreatment biopsies or in biopsies from patients randomized to the placebo treatment arm. ZED1227-TG2 staining co-localized with total TG2 and native and deamidated gliadin peptides on the enterocyte luminal surface. Inhibition of TG2 activity by ZED1227 was demonstrated in epithelial organoids. Our findings suggest that active TG2 is present at the luminal side of the villous epithelium and that inhibition of TG2 activity by ZED1227 occurs already there before gliadin peptides enter the lamina propria.

CSPP1 stabilizes growing microtubule ends and damaged lattices from the luminal side.

Microtubules are dynamic cytoskeletal polymers, and their organization and stability are tightly regulated by numerous cellular factors. While regulatory proteins controlling the formation of interphase microtubule arrays and mitotic spindles have been extensively studied, the biochemical mechanisms responsible for generating stable microtubule cores of centrioles and cilia are poorly understood. Here, we used in vitro reconstitution assays to investigate microtubule-stabilizing properties of CSPP1, a centrosome and cilia-associated protein mutated in the neurodevelopmental ciliopathy Joubert syndrome. We found that CSPP1 preferentially binds to polymerizing microtubule ends that grow slowly or undergo growth perturbations and, in this way, resembles microtubule-stabilizing compounds such as taxanes. Fluorescence microscopy and cryo-electron tomography showed that CSPP1 is deposited in the microtubule lumen and inhibits microtubule growth and shortening through two separate domains. CSPP1 also specifically recognizes and stabilizes damaged microtubule lattices. These data help to explain how CSPP1 regulates the elongation and stability of ciliary axonemes and other microtubule-based structures.

Super-Resolution Microscopy of the Synaptonemal Complex within the Caenorhabditis elegans Germline.

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures the interaction of homologous chromosomes is the assembly of the synaptonemal complex (SC) in meiotic prophase I. Even though there is little sequence homology between protein components within the SC among different species, the general structure of the SC has been highly conserved during evolution. In electron micrographs, the SC appears as a tripartite, ladder-like structure composed of lateral elements or axes, transverse filaments, and a central element. However, precisely identifying the localization of individual components within the complex by electron microscopy to determine the molecular structure of the SC remains challenging. By contrast, fluorescence microscopy allows for the identification of individual protein components within the complex. However, since the SC is only ~100 nm wide, its substructure cannot be resolved by diffraction-limited conventional fluorescence microscopy. Thus, determining the molecular architecture of the SC requires super-resolution light microscopy techniques such as structured illumination microscopy (SIM), stimulated-emission depletion (STED) microscopy, or single-molecule localization microscopy (SMLM). To maintain the structure and interactions of individual components within the SC, it is important to observe the complex in an environment that is close to its native environment in the germ cells. Therefore, we demonstrate an immunohistochemistry and imaging protocol that enables the study of the substructure of the SC in intact, extruded Caenorhabditis elegans germline tissue with SMLM and STED microscopy. Directly fixing the tissue to the coverslip reduces the movement of the samples during imaging and minimizes aberrations in the sample to achieve the high resolution necessary to visualize the substructure of the SC in its biological context.

Photobleaching and Sensitized Emission-Based Methods for the Detection of Förster Resonance Energy Transfer.

Förster resonance energy transfer (FRET) is a non-radiative interaction between two molecules that happens at distances in the range of a few nanometers. Using FRET interactions between suitably selected fluorophores allows to study molecular interactions or conformational changes of single molecules on fluorescence microscopes even though the optical resolution of the microscope is limited to distances that are almost two orders of magnitude higher.In this chapter several variants of FRET detection methods are described that are based either on the targeted photobleaching of one of the participating molecule species or on the direct detection of the fluorescence signal that is created as a result of the FRET interactions.

Mammalian HP1 Isoforms Have Specific Roles in Heterochromatin Structure and Organization.

HP1 is a structural component of heterochromatin. Mammalian HP1 isoforms HP1α, HP1ß, and HP1γ play different roles in genome stability, but their precise role in heterochromatin structure is unclear. Analysis of Hp1α-/-, Hp1ß-/-, and Hp1γ-/- MEFs show that HP1 proteins have both redundant and unique functions within pericentric heterochromatin (PCH) and also act globally throughout the genome. HP1α confines H4K20me3 and H3K27me3 to regions within PCH, while its absence results in a global hyper-compaction of chromatin associated with a specific pattern of mitotic defects. In contrast, HP1ß is functionally associated with Suv4-20h2 and H4K20me3, and its loss induces global chromatin decompaction and an abnormal enrichment of CTCF in PCH and other genomic regions. Our work provides insight into the roles of HP1 proteins in heterochromatin structure and genome stability.

STED imaging performance estimation by means of Fourier transform analysis.

Due to relatively high powers used in STED, biological samples may be affected by the illumination in the process of image acquisition. Similarly, the performance of the system may be limited by the sample itself. Optimization of the STED parameters taking into account the sample itself is therefore a complex task as there is no clear methodology that can determine the image improvement in an objective and quantitative manner. In this work, a method based on Fourier transform formalism is presented to analyze the performance of a STED system. The spatial frequency distribution of pairs of confocal and STED images are compared to obtain an objective parameter, the Azimuth Averaged Spectral Content Spread (AASCS), that is related to the performance of the system in which the sample is also considered. The method has been first tested on samples of beads, and then applied to cell samples labeled with multiple fluorescent dyes. The results show that a single parameter, the AASCS, can be used to determine the optimal settings for STED image acquisition in an objective way, only by using the information provided by the images from the sample themselves. The AASCS also helps minimize the depletion power, for better preservation of the samples.

TANGO1 assembles into rings around COPII coats at ER exit sites.

TANGO1 (transport and Golgi organization 1) interacts with CTAGE5 and COPII components Sec23/Sec24 and recruits ERGIC-53 (endoplasmic reticulum [ER]-Golgi intermediate compartment 53)-containing membranes to generate a mega-transport carrier for export of collagens and apolipoproteins from the ER. We now show that TANGO1, at the ER, assembles in a ring that encircles COPII components. The C-terminal, proline-rich domains of TANGO1 molecules in the ring are initially tilted onto COPII coats but appear to be pushed apart as the carrier grows. These findings lend support to our suggestion that growth of transport carriers for exporting bulky cargoes requires addition of membranes and not simply COPII-mediated accretion of a larger surface of ER. TANGO1 remains at the neck of the newly forming transport carrier, which grows in size by addition of ERGIC-53-containing membranes to generate a transport intermediate for the export of bulky collagens.

Molecular Interaction and Cellular Location of RecA and CheW Proteins in Salmonella enterica during SOS Response and Their Implication in Swarming.

In addition to its role in DNA damage repair and recombination, the RecA protein, through its interaction with CheW, is involved in swarming motility, a form of flagella-dependent movement across surfaces. In order to better understand how SOS response modulates swarming, in this work the location of RecA and CheW proteins within the swarming cells has been studied by using super-resolution microscopy. Further, and after in silico docking studies, the specific RecA and CheW regions associated with the RecA-CheW interaction have also been confirmed by site-directed mutagenesis and immunoprecipitation techniques. Our results point out that the CheW distribution changes, from the cell poles to foci distributed in a helical pattern along the cell axis when SOS response is activated or RecA protein is overexpressed. In this situation, the CheW presents the same subcellular location as that of RecA, pointing out that the previously described RecA storage structures may be modulators of swarming motility. Data reported herein not only confirmed that the RecA-CheW pair is essential for swarming motility but it is directly involved in the CheW distribution change associated to SOS response activation. A model explaining not only the mechanism by which DNA damage modulates swarming but also how both the lack and the excess of RecA protein impair this motility is proposed.

Compensatory Cell Movements Confer Robustness to Mechanical Deformation during Embryonic Development.

Embryonic development must proceed despite both internal molecular fluctuations and external perturbations. However, mechanisms that provide robustness to mechanical perturbation remain largely uncharacterized. Here, we use light-sheet microscopy, comprehensive single-cell tracking, and targeted cell ablation to study the response of Caenorhabditis elegans embryos to external compression. Compression changes the relative positions of many cells and causes severe distortions of the embryonic axes. A large-scale movement of cells then corrects this distortion. Only a few specific cells are required for these compensatory movements, and one cell, ABarppap, appears to generate force, dramatically changing as it moves to its correct local cellular environment. During these movements, we also observed "egressions", cells moving out onto the surface, and lineages that undergo both ingression and egression. In total, our work describes how the embryo responds to a major mechanical deformation that can occur during the early development in situ and puts forward a model to explain how the response is coordinated.

The evolution and development of neural superposition.

Visual systems have a rich history as model systems for the discovery and understanding of basic principles underlying neuronal connectivity. The compound eyes of insects consist of up to thousands of small unit eyes that are connected by photoreceptor axons to set up a visual map in the brain. The photoreceptor axon terminals thereby represent neighboring points seen in the environment in neighboring synaptic units in the brain. Neural superposition is a special case of such a wiring principle, where photoreceptors from different unit eyes that receive the same input converge upon the same synaptic units in the brain. This wiring principle is remarkable, because each photoreceptor in a single unit eye receives different input and each individual axon, among thousands others in the brain, must be sorted together with those few axons that have the same input. Key aspects of neural superposition have been described as early as 1907. Since then neuroscientists, evolutionary and developmental biologists have been fascinated by how such a complicated wiring principle could evolve, how it is genetically encoded, and how it is developmentally realized. In this review article, we will discuss current ideas about the evolutionary origin and developmental program of neural superposition. Our goal is to identify in what way the special case of neural superposition can help us answer more general questions about the evolution and development of genetically "hard-wired" synaptic connectivity in the brain.

Quantification of ErbB network proteins in three cell types using complementary approaches identifies cell-general and cell-type-specific signaling proteins.

Relating protein concentration to cell-type-specific responses is one of the remaining challenges for obtaining a quantitative systems level understanding of mammalian signaling. Here we used mass-spectrometry (MS)- and antibody-based quantitative proteomic approaches to measure protein abundances for 75% of a hand-curated reconstructed ErbB network of 198 proteins, in two established cell types (HEK293 and MCF-7) and in primary keratinocyte cells. Comparison with other quantitative studies allowed building a set of ErbB network proteins expressed in all cells and another which are cell-specific and could impart specific properties to the network. As a proof-of-concept of the importance of protein concentration, we generated a small simplified mathematical model encompassing ligand binding, followed by receptor dimerization, activation, and degradation. The model predicts ErbB phosphorylation in HEK293, MCF-7, and keratinocyte cells simply by incorporating cell-type-specific ErbB1, ErbB2, and caveolin-1 abundances but otherwise contains similar rate constants. Altogether, the data provide a resource for protein abundances and localization to be included in larger mathematical models, enabling the generation of cell-type-specific computational models. MS data have been deposited to the ProteomeXchange via PRIDE (with identifier PXD000623) and PASSEL (with identifier PASS00372).

Genetically encoded sender-receiver system in 3D mammalian cell culture.

Engineering spatial patterning in mammalian cells, employing entirely genetically encoded components, requires solving several problems. These include how to code secreted activator or inhibitor molecules and how to send concentration-dependent signals to neighboring cells, to control gene expression. The Madin-Darby Canine Kidney (MDCK) cell line is a potential engineering scaffold as it forms hollow spheres (cysts) in 3D culture and tubulates in response to extracellular hepatocyte growth factor (HGF). We first aimed to graft a synthetic patterning system onto single developing MDCK cysts. We therefore developed a new localized transfection method to engineer distinct sender and receiver regions. A stable reporter line enabled reversible EGFP activation by HGF and modulation by a secreted repressor (a truncated HGF variant, NK4). By expanding the scale to wide fields of cysts, we generated morphogen diffusion gradients, controlling reporter gene expression. Together, these components provide a toolkit for engineering cell-cell communication networks in 3D cell culture.

Clearing up the signal: spectral imaging and linear unmixing in fluorescence microscopy.

The ongoing progress in fluorescence labeling and in microscope instrumentation allows the generation and the imaging of complex biological samples that contain increasing numbers of fluorophores. For the correct quantitative analysis of datasets with multiple fluorescence channels, it is essential that the signals of the different fluorophores are reliably separated. Due to the width of fluorescence spectra, this cannot always be achieved using the fluorescence filters in the microscope. In such cases spectral imaging of the fluorescence data and subsequent linear unmixing allows the separation even of highly overlapping fluorophores into pure signals. In this chapter, the problems of fluorescence cross talk are defined, the concept of spectral imaging and separation by linear unmixing is described, and an overview of the microscope types suitable for spectral imaging are given.

Engineering of weak helper interactions for high-efficiency FRET probes.

Fluorescence resonance energy transfer (FRET)-based detection of protein interactions is limited by the very narrow range of FRET-permitting distances. We show two different strategies for the rational design of weak helper interactions that co-recruit donor and acceptor fluorophores for a more robust detection of bimolecular FRET: (i) in silico design of electrostatically driven encounter complexes and (ii) fusion of tunable domain-peptide interaction modules based on WW or SH3 domains. We tested each strategy for optimization of FRET between (m)Citrine and mCherry, which do not natively interact. Both approaches yielded comparable and large increases in FRET efficiencies with little or no background. Helper-interaction modules can be fused to any pair of fluorescent proteins and could, we found, enhance FRET between mTFP1 and mCherry as well as between mTurquoise2 and mCitrine. We applied enhanced helper-interaction FRET (hiFRET) probes to study the binding between full-length H-Ras and Raf1 as well as the drug-induced interaction between Raf1 and B-Raf.

Grb14 is a negative regulator of CEACAM3-mediated phagocytosis of pathogenic bacteria.

Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is a phagocytic receptor on human granulocytes, which mediates the opsonin-independent recognition and internalization of a restricted set of Gram-negative bacteria such as Neisseria gonorrhoeae. In an unbiased screen using a SH2 domain microarray we identified the SH2 domain of growth factor receptor-bound protein 14 (Grb14) as a novel binding partner of CEACAM3. Biochemical assays and microscopic studies demonstrated that the Grb14 SH2 domain promoted the rapid recruitment of this adaptor protein to the immunoreceptor-based activation motif (ITAM)-like sequence within the cytoplasmic domain of CEACAM3. Furthermore, FRET-FLIM analyses confirmed the direct association of Grb14 and CEACAM3 in intact cells at the sites of bacteria-host cell contact. Knockdown of endogenous Grb14 by RNA interference as well as Grb14 overexpression indicate an inhibitory role for this adapter protein in CEACAM3-mediated phagocytosis. Therefore, Grb14 is the first negative regulator of CEACAM3-initiated bacterial phagocytosis and might help to focus granulocyte responses to the subcellular sites of pathogen-host cell contact.

Quantifying the influence of yellow fluorescent protein photoconversion on acceptor photobleaching-based fluorescence resonance energy transfer measurements.

Fluorescence resonance energy transfer (FRET) efficiency measurements based on acceptor photobleaching of yellow fluorescent protein (YFP) are affected by the fact that bleaching of YFP produces a fluorescent species that is detectable in cyan fluorescent protein (CFP) image channels. The presented quantitative measurement of this conversion makes it possible to correct the obtained FRET signal to increase the accuracy of intensity based CFP/YFP FRET measurements. The described method can additionally be used to compare samples with very different fluorescence levels.

ADF/cofilin regulates secretory cargo sorting at the TGN via the Ca2+ ATPase SPCA1.

Actin-severing proteins ADF/cofilin are required for the sorting of secretory cargo at the trans-Golgi network (TGN) in mammalian cells. How do these cytoplasmic proteins interact with the cargoes in the lumen of the TGN? Put simply, how are these two sets of proteins connected across the TGN membrane? Mass spectrometry of cofilin1 immunoprecipitated from HeLa cells revealed the presence of actin and the Ca(2+) ATPase SPCA1. Moreover, cofilin1 was localized to the TGN and bound to SPCA1 via dynamic actin. SPCA1 knockdown, like ADF/cofilin1 knockdown, inhibited Ca(2+) uptake into the TGN and caused missorting of secretory cargo. These defects were rescued by the overexpression of the TGN-localized SPCA1. We propose that ADF/cofilin-dependent severing of actin filaments exposes and promotes the activation of SPCA1, which pumps Ca(2+) into the lumen of the TGN for the sorting of the class of secretory cargo that binds Ca(2+).