Microvesicle Shedding and Lysosomal Repair Fulfill Divergent Cellular Needs during the Repair of Streptolysin O-Induced Plasmalemmal Damage

Pathogenic bacteria secrete pore-forming toxins that permeabilize the plasma membrane of host cells. Nucleated cells possess protective mechanisms that repair toxin-damaged plasmalemma. Currently two putative repair scenarios are debated: either the isolation of the damaged membrane regions and their subsequent expulsion as microvesicles (shedding) or lysosome-dependent repair might allow the cell to rid itself of its toxic cargo and prevent lysis. Here we provide evidence that both mechanisms operate in tandem but fulfill diverse cellular needs. The prevalence of the repair strategy varies between cell types and is guided by the severity and the localization of the initial toxin-induced damage, by the morphology of a cell and, most important, by the incidence of the secondary mechanical damage. The surgically precise action of microvesicle shedding is best suited for the instant elimination of individual toxin pores, whereas lysosomal repair is indispensable for mending of self-inflicted mechanical injuries following initial plasmalemmal permeabilization by bacterial toxins. Our study provides new insights into the functioning of non-immune cellular defenses against bacterial pathogens.

THE MECHANISM OF RETINOIC ACID REGULATION OF TISSUE TRANSGLUTAMINASE GENE EXPRESSION

Retinoic acid regulates cellular growth and differentiation by altering the expression of specific sets of genes, but the molecular mechanism by which this is achieved is unknown. We have used the rapid induction of a specific enzyme, tissue transglutaminase in mouse macrophages, human leukemia cells and a variety of other cell types to study the regulation of gene expression by retinoic acid. Soluble retinoic acid binding proteins, such as cellular Retinoic Acid Binding Protein (cRABP), have been proposed as specific mediators of retinoic acid regulation of gene expression. This thesis demonstrates the lack of cRABP in a number of cell lines which are sensitive to retinoic acid regulation of tissue transglutaminase expression. These cells are also devoid of other soluble retinoic acid binding activity. The level of retinoic acid binding activity that could have been detected (6 fmol) is far below that of most cells and tissues which are sensitive to the effects of retinoic acid on growth and differentiation. A mouse melanoma cell line, S91-C2, was found to contain an unusual retinoic acid binding protein which has a lower affinity for retinoic acid than mouse tissue cRABP and also behaves differently on gel filtration HPLC chromatography.^ The induction of tissue transglutaminase by retinoic acid in macrophages is specifically inhibited by pertussis toxin. Pertussis toxin ADP-riblosylates membrane GTP-binding proteins such as N(,i) and interferes with signalling from plasma membrane receptors to regulatory enzymes. Pertussis toxin inhibition of transglutaminase induction is due to inhibition of tissue transglutaminase mRNA accumulation and is paralleled by the ADP-ribosylation of a 41,000 dalton macrophage membrane protein. It is concluded that soluble retinoic acid binding proteins are not essential for retinoic acid induction of tissue transglutaminase and that a membrane GTP-binding protein is closely linked to the sensitive response of macrophages to retinoic acid. ^

Loss of β1-integrin-deficient cells during the development of endoderm-derived epithelia

Beta1-integrins (beta1) represent cell surface receptors which mediate cell-matrix and cell-cell interactions. Fässler and Meyer described chimeric mice containing transgenic cells that express the LacZ gene instead of the beta1 gene. They observed beta1-negative cells in all germ layers at embryonic day E 8.5. Later in development, using a glucose phosphate isomerase assay of homogenized tissue samples, high levels of transgenic cells were found in skeletal muscle and gut, low levels in lung, heart, and kidney and none in the liver and spleen (Fässler and Meyer 1995). In order to study which cell types require beta1 during development of the primitive gut including its derivatives, chimeric fetuses containing 15 to 25% transgenic cells were obtained at days E 14.5 and E 15.5. They were LacZ (beta-galactosidase) stained "en bloc" and cross-sectioned head to tail. In esophagus, trachea, lung, stomach, hindgut, and the future urinary bladder, we observed various mesoderm-derived beta1-negative cells (e.g. fibroblasts, chondrocytes, endothelial cells, and smooth muscle cells) but no beta1-negative epithelial cells. Since the epithelia of lung, esophagus, trachea, stomach, hindgut, and urinary bladder are derived from the endodermal gut tube, we hypothesize that beta1 is essential for the development and/or survival of the epithelia of the fore- and hindgut and its derivatives.

Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2

Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1beta-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31 (downward arrow) AGD34 (downward arrow) A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase-mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor.

Characterization of the Effect of the Mitochondrial Protein Hint2 on Intracellular Ca2+ dynamics

Hint2, one of the five members of the superfamily of the histidine triad AMP-lysine hydrolase proteins, is expressed in mitochondria of various cell types. In human adrenocarcinoma cells, Hint2 modulates Ca2+ handling by mitochondria. As Hint2 is highly expressed in hepatocytes, we investigated if this protein affects Ca2+ dynamics in this cell type. We found that in hepatocytes isolated from Hint2−/− mice, the frequency of Ca2+ oscillations induced by 1 μM noradrenaline was 150% higher than in the wild-type. Using spectrophotometry, we analyzed the rates of Ca2+ pumping in suspensions of mitochondria prepared from hepatocytes of either wild-type or Hint2−/− mice; we found that Hint2 accelerates Ca2+ pumping into mitochondria. We then resorted to computational modeling to elucidate the possible molecular target of Hint2 that could explain both observations. On the basis of a detailed model for mitochondrial metabolism proposed in another study, we identified the respiratory chain as the most probable target of Hint2. We then used the model to predict that the absence of Hint2 leads to a premature opening of the mitochondrial permeability transition pore in response to repetitive additions of Ca2+ in suspensions of mitochondria. This prediction was then confirmed experimentally.

Simulating Cortical Development as a Self Constructing Process: A Novel Multi-Scale Approach Combining Molecular and Physical Aspects

Current models of embryological development focus on intracellular processes such as gene expression and protein networks, rather than on the complex relationship between subcellular processes and the collective cellular organization these processes support. We have explored this collective behavior in the context of neocortical development, by modeling the expansion of a small number of progenitor cells into a laminated cortex with layer and cell type specific projections. The developmental process is steered by a formal language analogous to genomic instructions, and takes place in a physically realistic three-dimensional environment. A common genome inserted into individual cells control their individual behaviors, and thereby gives rise to collective developmental sequences in a biologically plausible manner. The simulation begins with a single progenitor cell containing the artificial genome. This progenitor then gives rise through a lineage of offspring to distinct populations of neuronal precursors that migrate to form the cortical laminae. The precursors differentiate by extending dendrites and axons, which reproduce the experimentally determined branching patterns of a number of different neuronal cell types observed in the cat visual cortex. This result is the first comprehensive demonstration of the principles of self-construction whereby the cortical architecture develops. In addition, our model makes several testable predictions concerning cell migration and branching mechanisms.

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells.

Functional differentiation of spider hemocytes by light and transmission electron microscopy, and MALDI-MS-imaging

The most abundant cell types in the hemolymph of Cupiennius salei are plasmatocytes (70–80%) and granulocytes (20–30%). Both cells differ in shape, cytochemical and transmission electron microscopy staining of their cytoplasma and granules. According to MALDI-IMS (matrix-assisted laser desorption ionization mass spectrometry imaging), granulocytes exhibit ctenidin 1 (9510 Da) and ctenidin 3 (9568 Da), SIBD-1 (8675 Da), and unknown peptides with masses of 2207 and 6239 Da. Plasmatocytes exhibit mainly a mass of 6908 Da. Unknown peptides with masses of 1546 and 1960 Da were detected in plasmatocytes and granulocytes. Transmission electron microscopy confirms the presence of two compounds in one granule and cytochemical staining (light microscopy) tends to support this view. Two further hemocyte types (cyanocytes containing hemocyanin and prehemocytes as stem cells) are only rarely detected in the hemolymph. These four hemocyte types constitute the cellular part of the spider immune system and this is discussed in view of arachnid hemocyte evolution.

A next-generation tissue microarray (ngTMA) protocol for biomarker studies

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a 'donor' block into a 'recipient' block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 'recipient' blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.

Regulation of hematopoietic and leukemic stem cells by the immune system.

Hematopoietic stem cells (HSCs) are rare, multipotent cells that generate via progenitor and precursor cells of all blood lineages. Similar to normal hematopoiesis, leukemia is also hierarchically organized and a subpopulation of leukemic cells, the leukemic stem cells (LSCs), is responsible for disease initiation and maintenance and gives rise to more differentiated malignant cells. Although genetically abnormal, LSCs share many characteristics with normal HSCs, including quiescence, multipotency and self-renewal. Normal HSCs reside in a specialized microenvironment in the bone marrow (BM), the so-called HSC niche that crucially regulates HSC survival and function. Many cell types including osteoblastic, perivascular, endothelial and mesenchymal cells contribute to the HSC niche. In addition, the BM functions as primary and secondary lymphoid organ and hosts various mature immune cell types, including T and B cells, dendritic cells and macrophages that contribute to the HSC niche. Signals derived from the HSC niche are necessary to regulate demand-adapted responses of HSCs and progenitor cells after BM stress or during infection. LSCs occupy similar niches and depend on signals from the BM microenvironment. However, in addition to the cell types that constitute the HSC niche during homeostasis, in leukemia the BM is infiltrated by activated leukemia-specific immune cells. Leukemic cells express different antigens that are able to activate CD4(+) and CD8(+) T cells. It is well documented that activated T cells can contribute to the control of leukemic cells and it was hoped that these cells may be able to target and eliminate the therapy-resistant LSCs. However, the actual interaction of leukemia-specific T cells with LSCs remains ill-defined. Paradoxically, many immune mechanisms that evolved to activate emergency hematopoiesis during infection may actually contribute to the expansion and differentiation of LSCs, promoting leukemia progression. In this review, we summarize mechanisms by which the immune system regulates HSCs and LSCs.

Glycosylation of TRPM4 and TRPM5 channels: molecular determinants and functional aspects

The transient receptor potential channel, TRPM4, and its closest homolog, TRPM5, are non-selective cation channels that are activated by an increase in intracellular calcium. They are expressed in many cell types, including neurons and myocytes. Although the electrophysiological and pharmacological properties of these two channels have been previously studied, less is known about their regulation, in particular their post-translational modifications. We, and others, have reported that wild-type (WT) TRPM4 channels expressed in HEK293 cells, migrated on SDS-PAGE gel as doublets, similar to other ion channels and membrane proteins. In the present study, we provide evidence that TRPM4 and TRPM5 are each N-linked glycosylated at a unique residue, Asn(992) and Asn(932), respectively. N-linked glycosylated TRPM4 is also found in native cardiac cells. Biochemical experiments using HEK293 cells over-expressing WT TRPM4/5 or N992Q/N932Q mutants demonstrated that the abolishment of N-linked glycosylation did not alter the number of channels at the plasma membrane. In parallel, electrophysiological experiments demonstrated a decrease in the current density of both mutant channels, as compared to their respective controls, either due to the Asn to Gln mutations themselves or abolition of glycosylation. To discriminate between these possibilities, HEK293 cells expressing TRPM4 WT were treated with tunicamycin, an inhibitor of glycosylation. In contrast to N-glycosylation signal abolishment by mutagenesis, tunicamycin treatment led to an increase in the TRPM4-mediated current. Altogether, these results demonstrate that TRPM4 and TRPM5 are both N-linked glycosylated at a unique site and also suggest that TRPM4/5 glycosylation seems not to be involved in channel trafficking, but mainly in their functional regulation.

Interaction of small molecules with specific immune receptors: the p-i concept and its consequences

Drugs may stimulate the immune system by forming stable new antigenic complexes consisting of the drug or drug metabolite which is covalently bound to a protein or peptide (hapten-carrier complex). Both, B- and T-cell immunity may arise, the latter directed to hapten modified peptides presented by HLA molecules. Beside this immunological stimulation, drugs can also stimulate the immune system through binding by non-covalent bonds to proteins like immune receptors. This so-called “pharmacological interaction with immune receptors” concept (“p-i concept”) may occur with HLA or TCR molecules themselves (p-i HLA or p-i TCR), and not the immunogenic peptide. It is a type of “off-target” activity of the drug on immune receptors, but more complex as various cell types, cell interactions and functionally different T cells are involved. In this review the conditions which lead to activation of T cells by p-i are discussed: important factors for a functional consequence of drug binding is the location of binding (p-i HLA or p-i TCR); the exact site within these immune receptors; the affinity of binding and the finding that p-i HLA can stimulate the immune system like an allo-allele. The p-i concept is able to solve some puzzles of drug hypersensitivity reactions and are a basis to better treat and potentially avoid drug hypersensitivity reactions. Moreover, the p-i concept shows that in contrast to previous beliefs small molecules do interact with immune receptors with functional consequence. But these interactions are not based on “immune recognition”, are at odds with some immunological concepts, but may nevertheless open new possibilities to understand and even treat immune reactions

To sense or not to sense viral RNA--essentials of coronavirus innate immune evasion.

An essential function of innate immunity is to distinguish self from non-self and receptors have evolved to specifically recognize viral components and initiate the expression of antiviral proteins to restrict viral replication. Coronaviruses are RNA viruses that replicate in the host cytoplasm and evade innate immune sensing in most cell types, either passively by hiding their viral signatures and limiting exposure to sensors or actively, by encoding viral antagonists to counteract the effects of interferons. Since many cytoplasmic viruses exploit similar mechanisms of innate immune evasion, mechanistic insight into the direct interplay between viral RNA, viral RNA-processing enzymes, cellular sensors and antiviral proteins will be highly relevant to develop novel antiviral targets and to restrict important animal and human infections.

Primary human lung pericytes support and stabilize in-vitro perfusable microvessels.

The formation of blood vessels is a complex tissue-specific process that plays a pivotal role during developmental processes, in wound healing, cancer progression, fibrosis and other pathologies. To study vasculogenesis and vascular remodeling in the context of the lung, we developed an in-vitro microvascular model that closely mimics the human lung microvasculature in terms of 3D architecture, accessibility, functionality and cell types. Human pericytes from the distal airway were isolated and characterized using flow cytometry. To assess their role in the generation of normal microvessels, lung pericytes were mixed in fibrin gel and seeded into well-defined microcompartments together with primary endothelial cells (HUVEC). Patent microvessels covering an area of 3.1 mm2 formed within 3-5 days and were stable for up to 14 days. Soluble signals from the lung pericytes were necessary to establish perfusability, and pericytes migrated towards endothelial microvessels. Cell-cell communication in the form of adherens and tight junctions, as well as secretion of basement membrane was confirmed using transmission electron microscopy and immunocytochemistry on chip. Direct co-culture of pericytes with endothelial cells decreased the microvascular permeability by one order of magnitude from 17.8∙10-6 cm/s to 2.0∙10-6 cm/s and led to vessels with significantly smaller and less variable diameter. Upon phenylephrine administration, vasoconstriction was observed in microvessels lined with pericytes but not in endothelial microvessels only. Perfusable microvessels were also generated with human lung microvascular endothelial cells and lung pericytes. Human lung pericytes were thus shown to have a prominent influence on microvascular morphology, permeability, vasoconstriction and long-term stability in an in-vitro microvascular system. This biomimetic platform opens new possibilities to test functions and interactions of patient-derived cells in a physiologically relevant microvascular setting.

Modern techniques for the analysis of chromatin and nuclear organization in C. elegans.

In recent years, Caenorhabditis elegans has emerged as a new model to investigate the relationships between nuclear architecture, cellular differentiation, and organismal development. On one hand, C. elegans with its fixed lineage and transparent body is a great model organism to observe gene functions in vivo in specific cell types using microscopy. On the other hand, two different techniques have been applied in nematodes to identify binding sites for chromatin-associated proteins genome-wide: chromatin immunoprecipitation (ChIP), and Dam-mediated identification (DamID). We summarize here all three techniques together as they are complementary. We also highlight strengths and differences of the individual approaches.