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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.

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.

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.

Microtubules are dynamic cytoskeletal polymers, and their organization and stability are tightly regulated by numerous cellular factors. While regulatory proteins controlling 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 elongation and stability of ciliary axonemes and other microtubule-based structures.

Um 12.30 Uhr fand am Mittwoch, 25. November 2020, eine Online Veranstaltung der Universitätsbibliothek Hildesheim zum Forschungsdatenmanagement statt. Die Veranstaltung konnte über BBB verfolgt werden. Die Folien von Herrn Herrn Jan-Timo Zimmermann sind nach der Veranstaltung für alle Forscherinnen und Forscher sowie Interessierte öffentlich verfügbar. Referent: Jan-Timo Zimmermann (UB Hildesheim), Leitung Mediothek Thema: HilData Info: Die Nachhaltigkeit von Forschungsdaten, das Teilen von Forschungsdaten oder Publikationen von Datenbeständen gewinnen zunehmend an Bedeutung. Journals verlangen oft die Verfügbarmachung von Publikationen der zugrundeliegenden Daten eines Artikels. Universitäten, darunter auch die Universität Hildesheim, verabschieden Research Data Policies, und Forscherinnen und Forscher diskutieren sowohl die Chancen als auch die Risiken von "Open Data", die Herausforderungen des Datenschutzes und der Forschungsethik. Annette Strauch von der Universitätsbibliothek Hildesheim gibt einen kurzen Überblick zum Thema Forschungsdatenmanagement. Zusätzlich wird das Dienstleistungsspektrum der Uni-Bibliothek zu den E-Science Unterstützungsangeboten vorgestellt. &nbsp

Research question: The aim of this article is to analyse which objectives the national sport associations are pursuing with the introduction of league systems, which league constructs were chosen i...

HP1 is a structural component of heterochromatin. Mammalian HP1 isoforms HP1α, HP1β, and HP1γ play different roles in genome stability, but their precise role in heterochromatin structure is unclear. Analysis of Hp1α-/-, Hp1β-/-, 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, HP1β 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. This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2011-25860 and SAF2014-55964R to A.V.) and cofunded by FEDER funds/European Regional Development Fund (ERDF)-a way to build Europe, the Catalan Government Agency AGAUR (2009SGR-914 and 2014SGR-400 to A.V.), HGINJ (to L.S.), Deutsche Forschungsgemeinschaft (SFB1064 to G.S.), and the Canadian Institutes of Health Research (CIHR) (MOP-97878 to J.A.).

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.

Forster 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.

Aufbauend auf den Forschungserkenntnissen zur Steuerung von sportlichen Wettbewerben mittels Anreizen (Kapitel 2) sowie der Anreiz-Beitrags-Theorie (Kapitel 3) werden nachfolgend die Ergebnisse der Strukturanalyse von Ligen in Individualsportarten vorgestellt. Hierbei werden die Einrichtung und Ausgestaltung der Individualsportligen erforscht und damit grundlegende Informationen uber das Phanomen gewonnen. Ziel dessen ist es, zum einen zentrale Charakteristika und Besonderheiten der Individualsportligen zu identifizieren.

Um das Phanomen von Ligen in Individualsportarten zu verstehen und die Steuerung der Individualsportligen mittels Anreizen theoriegeleitet zu untersuchen, sind zunachst die theoretisch-konzeptionellen Grundlagen zu erarbeiten. Hierzu werden nachfolgend die vorliegenden Forschungserkenntnisse zur Steuerung von sportlichen Wettbewerben mit Fokus auf die Anreizsetzung als Steuerungsinstrument (Kap. 2) dargelegt. Ziel ist es dabei, zum einen Kenntnisse uber die Perspektiven und Ergebnisse der bisherigen Forschungsarbeiten zu dem Thema und dem Untersuchungsgegenstand zu gewinnen.

Aus den dargestellten Untersuchungsergebnissen zur Steuerung von Ligen in Individualsportarten mittels Anreizsetzung lassen sich Schlussfolgerungen fur die Praxis und Theorie ziehen. Im Hinblick auf die Praxis werden in Kapitel 11 Handlungsempfehlungen fur das Anreizmanagement der Ligaorganisatoren und der Vereine abgeleitet, um dieses zu optimieren. Auf dieser Basis kann die Existenz der Ligen gesichert und ihre Fortentwicklung initiiert werden.

Die vorangegangene Strukturanalyse der Ligen in Individualsportarten hat in Bezug auf die Anreiz-Beitrags-Dynamik gezeigt, dass der Ligawettbewerb aufgrund seiner Charakteristika und spezifischen Ausgestaltung uber Besonderheiten im Vergleich zu anderen Wettbewerbsformaten der Individualsportarten verfugt. Durch die Existenz des Ligawettbewerbs und die Art der Ausgestaltung des Ligasystems werden zugleich gegenuber den Vereinen und Athleten institutionelle, strukturelle, eigenwert-orientierte, interaktionelle, kompensatorische und replizierende Anreize zur Teilnahme an der Liga gesetzt. Die Wirkung dieser Anreize ist abhangig von dem Kontext der jeweiligen Individualsportart, sodass zwischen den Sportarten Unterschiede in der Ausgestaltung der Liga bestehen.

Aufbauend auf den im vorherigen Kapitel erlauterten theoretisch-konzeptionellen Grundlagen, wird nachfolgend die Forschungsmethodik fur die empirische Untersuchung der Steuerung von Ligen in Individualsportarten mittels Anreizen differenziert dargelegt. Der Studie liegt ein qualitatives Forschungsdesign zugrunde, bei dem deduktiv-induktiv vorgegangen wird.

The chapter focuses on golf tournaments, different special golf events and their spectators. First, the central organizational and economic aspects are presented for the most important women’s and men’s event series in golf. The primary focus is on the PGA Tour, which was the pioneer of tour systems, as well as its European counterpart, the European Tour. Two best-case examples of two different tournaments of the PGA and European Tour are presented. Subsequently, the focus is on special golf events, such as the Ryder Cup and the Solheim Cup as well as golf at the Olympics. In the third part of this chapter, the spectators at golf events are characterized, and how they differ from spectators at other sports is discussed. Finally, a short conclusion with an estimation of the future development of golf events and event series is provided.

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.

TANGO1 interacts with COPII components to generate a transport carrier for export of large cargo from the endoplasmic reticulum. Raote et al. show that TANGO1 molecules assemble to form a closed ribbon structure that encircles COPII components., 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.

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. CAPSULE: BACKGROUND: CEACAM3-mediated phagocytosis is an innate mechanism to eliminate CEACAM-binding pathogenic bacteria. RESULTS: Biochemical analyses and FRET-FLIM imaging reveal the direct binding of the adaptor protein Grb14 to the tyrosine-phosphorylated cytoplasmic domain of CEACAM3, and this interaction limits phagocytosis. CONCLUSION: Grb14 is a negative regulator of CEACAM3-mediated phagocytosis. SIGNIFICANCE: Deciphering CEACAM3 interaction partners illuminates the minimal set of cellular factors controlling phagocytosis.

SummaryHere we describe a lineage reprogramming system consisting of a B cell line with an estradiol-inducible form of C/EBPα where cells can be converted into macrophage-like cells at 100% efficiency within 2 to 3 days. The reprogrammed cells are larger, contain altered organelle and cytoskeletal structures, are phagocytic, and exhibit an inflammatory response. Time-lapse experiments showed that the cells acquire a macrophage morphology and increased migratory activity as early as 10 hr. During induction, thousands of genes become up- or downregulated, including several dozen transcription and chromatin-remodeling factors. Time-limited exposure of cells to the inducer showed that the reprogrammed cells become transgene independent within 1 to 2 days. The reprogramming can be inhibited, at least partially, by perturbation experiments with B cell and macrophage transcription factors. The tightness, robustness, and speed of the system described make it a versatile tool to study biochemical and biological aspects of lineage reprogramming.

Image stitching is the process of combining multiple images to produce a panorama or larger image. In many biomedical studies, including those of cancer and infection, the use of this approach is highly desirable in order to acquire large areas of certain structures or whole sections, while retaining microscopic resolution. In this study, we describe the application of Autostitch, viz. software that is normally used for the generation of panoramas in photography, in the seamless stitching of microscope images. First, we tested this software on image sets manually acquired by normal light microscopy and compared the performance with a manual stitching approach performed with Paint Shop Pro. Secondly, this software was applied to an image stack acquired by an automatic microscope. The stitching results were then compared with that generated by a self-programmed rectangular tiling macro integrated in Image J. Thirdly, this program was applied in the image stitching of images from electron microscopy. Thus, the automatic stitching program described here may find applications in convenient image stitching and virtual microscopy in the biomedical research.

Summary The COPII coat complex mediates the formation of transport carriers at specialized sites of the endoplasmic reticulum (ERES). It consists of the Sar1p GTPase and the Sec23/24p and the Sec13/31p subcomplexes [1–3]. Both stimulate the GTPase activity of Sar1p [4], which itself triggers coat disassembly. This built-in GAP activity makes the COPII complex in principle unstable and raises the question of how sufficient stability required for cargo capture and carrier formation is achieved. To address this, we analyzed COPII turnover at single ERES in living cells. The half times for Sar1p, Sec23p, and Sec24p turnover are 1.1, 3.7, and 3.9 s, respectively. Decreasing the amount of transport-competent cargo in the endoplasmic reticulum accelerates turnover of the Sec23/24p and slows down that of Sar1p. A mathematical model of COPII membrane turnover that reproduces the experimental in vivo FRAP kinetics and is consistent with existing in vitro data predicts that Sec23/24p remains membrane associated even after GTP hydrolysis by Sar1p for a duration that is strongly increased by the presence of cargo. We conclude that secretory cargo retains the COPII complex on membranes, after Sar1p release has occurred, and prevents premature disassembly of COPII during cargo sorting and transport carrier formation.

SummaryAnopheles gambiae uses effective immune responses, including phagocytosis, to fight microbial infection. We have developed a semiquantitative phagocytosis test and used it in conjunction with dsRNA gene silencing to test the in vivo roles of 71 candidate genes in phagocytosis of Escherichia coli and Staphylococcus aureus. Here, we show that inactivation of 26 genes changes the phagocytic activity by more than 45% and that two pathways similar to those that mediate apoptotic cell removal in Caenorhabditis elegans are used in A. gambiae for phagocytosis of microorganisms. Simultaneous inactivation of the identified regulators of phagocytosis and conserved components defining each signaling pathway permitted provisional assignment of the novel regulators to one or the other pathway. Pathway inactivation enhances at least three times the ability of E. coli and S. aureus to proliferate in the mosquito. Interestingly, mosquito survival is not compromised even if both pathways are perturbed simultaneously.

Invasion of the Anopheles mosquito midgut by the Plasmodium ookinete is a critical step in the malaria transmission cycle. We have generated a fluorescent P. berghei transgenic line that expresses GFP in the ookinete and oocyst stages, and used it to perform the first real-time analysis of midgut invasion in the living mosquito as well as in explanted intact midguts whose basolateral plasma membranes were vitally stained. These studies permitted detailed analysis of parasite motile behaviour in the midgut and cell biological analysis of the invasion process. Throughout its journey, the ookinete displays distinct modes of motility: stationary rotation, translocational spiralling and straight-segment motility. Spiralling is based on rotational motility combined with translocation steps and changes in direction, which are achieved by transient attachments of the ookinete's trailing end. As it moves from the apical to the basal side of the midgut epithelium, the ookinete uses a predominant intracellular route and appears to glide on the membrane in foldings of the basolateral domain. However, it traverses serially the cytoplasm of several midgut cells before entering and migrating through the basolateral intercellular space to access the basal lamina. The invaded cells commit apoptosis, and their expulsion from the epithelium invokes wound repair mechanisms including extensive lamellipodia crawling. A 'hood' of lamellipodial origin, provided by the invaded cell, covers the ookinete during its egress from the epithelium. The flexible ookinete undergoes shape changes and temporary constrictions associated with passage through the plasma membranes. Similar observations were made in both A. gambiae and A. stephensi, demonstrating the conservation of P. berghei interactions with these vectors.

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.

Purpur war in der Antike aufgrund der aufwendigen Herstellung eine kostbare Substanz. Obwohl der Farbstoff heute leicht herzustellen ist, ist er durch die leuchtenden synthetischen Farben fast v?llig in Vergessenheit geraten. In diesem Artikel wird ?ber die Geschichte, Chemie und Synthese des Farbstoffs berichtet und gezeigt, wie man echten „antiken” Purpur herstellen kann.

Light microscopy has been a tool for biological research for centuries and thus is one of the oldest methods used in biological and medical sciences. The inherent limitation of light microscope resolution to structures only slightly smaller than intracellular organelles has posed a severe limitation to this technology for more than a century and was one of the reasons why electron microscopy with its much higher resolution was developed. But instead of stagnating, the field of light microscopy has continued to provide exciting new ways of looking at biological samples by introducing epifluorescence imaging, improving its specific labelling, its detection sensitivity and alsoits resolution, first axially (in ‘z’) and recently also laterally (in ‘x’ and ‘y’).

Methods to present three-dimensional (3D) and time series of 3D datasets (4D) are demonstrated using the recent advances in confocal microscopy and computer visualization. The process of cell sorting during tip formation in the slime mould Dictyostelium discoideum is examined as an example by in vivo confocal microscopy of spectrally different green fluorescent protein (GFP) variants as reporters of cell-type specific gene expression. Also, cell sorting of the co-aggregating slime mould species D. discoideum and D. mucoroides is observed using a GFP variant and a spectrally distinguishable fluorescent vital stain. The confocal data are handled as 3D and 4D datasets, their processing and the advantages of different methods of visualization are discussed step by step. Selected sequences of the experiments can be viewed on the Internet, giving a much better impression of the complex cellular movements during Dictyostelium morphogenesis than printed photographs.

Using 3D reconstructions of serial ultrathin sections and extended-focus pictures of Golgi-impregnated and cobalt-stained visual fibres, we studied the branched short photoreceptor axons found in ancestral dipterans and in the scorpionfly Panorpa. In the 'phantom' midge Chaoborus (Nematocera), each cartridge of the lamina neuropil is innervated by collaterals of 24 photoreceptor axons from 18 neighbouring and next-but-one ommatidia, in a regular pattern of asymmetrical neural pooling. Comparison of axon morphologies in different groups (Tipulidae, Chironomidae, Culicidae) indicates that this pattern must represent an ancestral condition of the dipteran visual system and is thus a precursor of neural superposition found in flies (Brachycera) in which only one set of photoreceptors R1–R6 converges onto each cartridge instead of the four sets found in Chaoborus. It can be concluded that specific axonal input channels from the large array of innervating photoreceptors found in midges have been retained during the evolution of neural superposition.

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).

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.

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.

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.

Afferents of Tömösváry's organ innervate multiple synaptic sites within the ipsilateral protocerebrum, i.e. neuropil areas proximal to the 2nd optic neuropil, in the dorsolateral protocerebrum, and surrounding the pedunculus of the "mushroom bodies". Sensory input via Tömösváry's organ in Lithobius appears to follow a peculiar neuronal pathway bypassing the deutocerebrum and tritocerebrum which are innervated by many head sensilla in arthropods.

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+).

We present an in vitro system to analyze quantitatively the interactions of green fluorescent protein (GFP)-tagged recombinant proteins with microtubules. This method relies on photoconversion of GFP and time-lapse microscopy. Specific interactions can be detected and binding kinetics can be determined rapidly and accurately. This method provides an alternative to classical in vitro microtubule-binding assays to analyze microtubule-associated proteins binding to microtubules. It has the potential to be extended to study interactions of proteins or multi-protein complexes with different biopolymers like actin microfilaments or organelle membranes.