Development of a 3D atlas of the embryonic pancreas for topological and quantitative analysis of heterologous cell interactions.

Generating comprehensive image maps, while preserving spatial three-dimensional (3D) context, is essential in order to locate and assess quantitatively specific cellular features and cell-cell interactions during organ development. Despite recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on two-dimensional histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in three dimensions and map tissue interactions at key time points in the mouse embryo. We demonstrate the utility of the approach by providing volumetric data, 3D distribution of three main cellular components (epithelial, mesenchymal and endothelial cells) within the developing pancreas, and quantification of their relative cellular abundance within the tissue. Interestingly, our 3D images show that endocrine cells are constantly and increasingly in contact with endothelial cells forming small vessels, whereas the interactions with mesenchymal cells decrease over time. These findings suggest distinct cell-cell interaction requirements for early endocrine cell specification and late differentiation. Lastly, we combine our image data in an open-source online repository (referred to as the Pancreas Embryonic Cell Atlas).

The biomechanical basis of biased epithelial tube elongation in lung and kidney development.

During lung development, epithelial branches expand preferentially in a longitudinal direction. This bias in outgrowth has been linked to a bias in cell shape and in the cell division plane. How this bias arises is unknown. Here, we show that biased epithelial outgrowth occurs independent of the surrounding mesenchyme, of preferential turnover of the extracellular matrix at the bud tips and of FGF signalling. There is also no evidence for actin-rich filopodia at the bud tips. Rather, we find epithelial tubes to be collapsed during early lung and kidney development, and we observe fluid flow in the narrow tubes. By simulating the measured fluid flow inside segmented narrow epithelial tubes, we show that the shear stress levels on the apical surface are sufficient to explain the reported bias in cell shape and outgrowth. We use a cell-based vertex model to confirm that apical shear forces, unlike constricting forces, can give rise to both the observed bias in cell shapes and tube elongation. We conclude that shear stress may be a more general driver of biased tube elongation beyond its established role in angiogenesis. This article has an associated 'The people behind the papers' interview.

Impact of glutathione metabolism on zinc homeostasis in Saccharomyces cerevisiae.

Zinc is a crucial mineral for all organisms as it is an essential cofactor for the proper function of a plethora of proteins and depletion of zinc causes oxidative stress. Glutathione is the major redox buffering agent in the cell and therefore important for mitigation of the adverse effects of oxidative stress. In mammalian cells, zinc deficiency is accompanied by a glutathione depletion. In the yeast Saccharomyces cerevisiae, the opposite effect is observed: under low zinc conditions, an elevated glutathione concentration is found. The main regulator to overcome zinc deficiency is Zap1p. However, we show that Zap1p is not involved in this glutathione accumulation phenotype. Furthermore, we found that in glutathione-accumulating strains also the metal ion-binding phytochelatin-2, which is an oligomer of glutathione, is accumulated. This increased phytochelatin concentration correlates with a lower free zinc level in the vacuole. These results suggest that phytochelatin is important for zinc buffering in S. cerevisiae and thus explains how zinc homeostasis is connected with glutathione metabolism.

Impaired chromatin remodelling at STAT1-regulated promoters leads to global unresponsiveness of Toxoplasma gondii-infected macrophages to IFN-γ.

Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites' replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not ß-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors.

Overexpression of genes of the fatty acid biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strains with deregulated sterol and fatty acid biosynthesis pathways were analysed for sterol and fatty acid content and mRNA profiles, with the aim of identifying interactions between lipid biosynthesis pathways. Acetyl CoA carboxylase ACC1 and fatty acid synthases FAS1/FAS2 were overexpressed in wild-type and squalene-overproducing strains. ACC1 overexpression led to decreased fatty acid content in the squalene-overproducing strain (factor of 0.7), while sterols and squalene were increased (factor of 1.5). In the wild-type strain, ACC1 overexpression led to increased levels of both fatty acids and squalene/sterols (factors of 4.0 and 1.7, respectively). This parallel activation of the two pathways seems to be due to transcriptional co-regulation of ACC1 and HMG1. While FAS1 and FAS2 overexpression had no effect in the wild-type strain, FAS2 overexpression induced significant increase of sterols and squalene (factors of 7.2 and 1.3, respectively) and a concomitant decrease of both saturated and unsaturated fatty acids in the squalene-overproducing strain (factor of 0.6). The microarray expression profiles showed that genes upregulated in ACC1-overexpressing strains are FAS1, ERG11, ERG28, ERG5, ERG2 and ERG20, supporting the observed increase of zymosterol and saturated fatty acids. The high ACC1 expression level due to overexpression correlated with increased transcript levels of sphingolipid and sterol biosynthesis genes. The relationship between was shown using the Pathway Studio program.

Shifting the fermentative/oxidative balance in Saccharomyces cerevisiae by transcriptional deregulation of Snf1 via overexpression of the upstream activating kinase Sak1p.

With the aim to reduce fermentation by-products and to promote respiratory metabolism by shifting the fermentative/oxidative balance, we evaluated the constitutive overexpression of the SAK1 and HAP4 genes in Saccharomyces cerevisiae. Sak1p is one of three kinases responsible for the phosphorylation, and thereby the activation, of the Snf1p complex, while Hap4p is the activator subunit of the Hap2/3/4/5 transcriptional complex. We compared the physiology of a SAK1-overexpressing strain with that of a strain overexpressing the HAP4 gene in wild-type and sdh2 deletion (respiratory-deficient) backgrounds. Both SAK1 and HAP4 overexpressions led to the upregulation of glucose-repressed genes and to reduced by-product formation rates (ethanol and glycerol). SAK1 overexpression had a greater impact on growth rates than did HAP4 overexpression. Elevated transcript levels of SAK1, but not HAP4, resulted in increased biomass yields in batch cultures grown on glucose (aerobic and excess glucose) as well as on nonfermentable carbon sources. SAK1 overexpression, but not the combined overexpression of SAK1 and HAP4 or the overexpression of HAP4 alone, restored growth on ethanol in an sdh2 deletion strain. In glucose-grown shake flask cultures, the sdh2 deletion strain with SAK1 and HAP4 overexpression produced succinic acid at a titer of 8.5 g liter(-1) and a yield of 0.26 mol (mol glucose)(-1) within 216 h. We here report for the first time that a constitutively high level of expression of SAK1 alleviates glucose repression and shifts the fermentative/oxidative balance under both glucose-repressed and -derepressed conditions.

Protein trafficking, ergosterol biosynthesis and membrane physics impact recombinant protein secretion in Pichia pastoris.

BACKGROUND: The increasing availability of 'omics' databases provide important platforms for yeast engineering strategies since they offer a lot of information on the physiology of the cells under diverse growth conditions, including environmental stresses. Notably, only a few of these approaches have considered a performance under recombinant protein production conditions. Recently, we have identified a beneficial effect of low oxygen availability on the expression of a human Fab fragment in Pichia pastoris. Transcriptional analysis and data mining allowed for the selection of potential targets for strain improvement. A first selection of these candidates has been evaluated as recombinant protein secretion enhancers. RESULTS: Based on previous transcriptomics analyses, we selected 8 genes for co-expression in the P. pastoris strain already secreting a recombinant Fab fragment. Notably, WSC4 (which is involved in trafficking through the ER) has been identified as a novel potential target gene for strain improvement, with up to a 1.2-fold increase of product yield in shake flask cultures. A further transcriptomics-based strategy to modify the yeast secretion system was focused on the ergosterol pathway, an aerobic process strongly affected by oxygen depletion. By specifically partially inhibiting ergosterol synthesis with the antifungal agent fluconazole (inhibiting Erg11p), we tried to mimic the hypoxic conditions, in which the cellular ergosterol content was significantly decreased. This strategy led to an improved Fab yield (2-fold) without impairing cellular growth. Since ergosterol shortage provokes alterations in the plasma membrane composition, an important role of this cellular structure in protein secretion is suggested. This hypothesis was additionally supported by the fact that the addition of non-ionic surfactants also enhanced Fab secretion. CONCLUSIONS: The current study presents a systems biotechnology-based strategy for the engineering of the industrially important yeast P. pastoris combining the use of host specific DNA microarray technologies and physiological studies under well defined environmental conditions. Such studies allowed for the identification of novel targets related with protein trafficking and ergosterol biosynthesis for improved recombinant protein production. Nevertheless, further studies will be required to elucidate the precise mechanisms whereby membrane biogenesis and composition impact on protein secretion in P. pastoris.

Organ-Specific Branching Morphogenesis.

A common developmental process, called branching morphogenesis, generates the epithelial trees in a variety of organs, including the lungs, kidneys, and glands. How branching morphogenesis can create epithelial architectures of very different shapes and functions remains elusive. In this review, we compare branching morphogenesis and its regulation in lungs and kidneys and discuss the role of signaling pathways, the mesenchyme, the extracellular matrix, and the cytoskeleton as potential organ-specific determinants of branch position, orientation, and shape. Identifying the determinants of branch and organ shape and their adaptation in different organs may reveal how a highly conserved developmental process can be adapted to different structural and functional frameworks and should provide important insights into epithelial morphogenesis and developmental disorders.

The human c-fos and TNFalpha AU-rich elements show different effects on mRNA abundance and protein expression depending on the reporter in the yeast Pichia pastoris.

AU-rich elements (AREs) are located in the 3' untranslated region (3' UTR) of their host genes and tightly regulate mRNA degradation and expression. Examples for this kind of regulation are the human proto-oncogene c-fos and the cytokine TNFalpha. Despite large effort in this field, the exact mechanism of ARE-mediated mRNA turnover remains unclear. In this work we analysed the effects of c-fos- and TNFalpha AREs on mRNA abundance and protein expression of selected human cDNAs in the yeast Pichia pastoris. This yeast is exceedingly well known for its excellent protein production capacity; however, ARE-like mechanisms have not been studied in this yeast to date. Interestingly, we observed both stabilizing and destabilizing effects of the c-fos ARE, whereas the TNFalpha ARE has a destabilizing or expression-reducing function in all tested cDNAs. Based on this observation, we introduced a number of single-point mutations upstream of the introduced c-fos ARE into the 3' UTR of a single cDNA in order to demonstrate the importance of ARE-flanking sequences for their own regulation. In conclusion, we illustrate that the analysis of ARE-mediated effects on mRNA abundance and protein expression of a reporter depends on the sequence of the reporter itself as well as the ARE-surrounding sequences within the 3' UTR. For this reason, we question whether already established reporter constructs in other cellular systems display the true type of regulation of the tested AREs for its original host gene. Finally, we propose that AREs should be analysed in their native sequence context.

Metabolic engineering of Saccharomyces cerevisiae for the biotechnological production of succinic acid.

The production of bio-based succinic acid is receiving great attention, and several predominantly prokaryotic organisms have been evaluated for this purpose. In this study we report on the suitability of the highly acid- and osmotolerant yeast Saccharomyces cerevisiae as a succinic acid production host. We implemented a metabolic engineering strategy for the oxidative production of succinic acid in yeast by deletion of the genes SDH1, SDH2, IDH1 and IDP1. The engineered strains harbor a TCA cycle that is completely interrupted after the intermediates isocitrate and succinate. The strains show no serious growth constraints on glucose. In glucose-grown shake flask cultures, the quadruple deletion strain Δsdh1Δsdh2Δidh1Δidp1 produces succinic acid at a titer of 3.62 g L(-1) (factor 4.8 compared to wild-type) at a yield of 0.11 mol (mol glucose)(-1). Succinic acid is not accumulated intracellularly. This makes the yeast S. cerevisiae a suitable and promising candidate for the biotechnological production of succinic acid on an industrial scale.

Endocytic uptake of fluorescence labelled DNA in yeast.

After dispiriting results using viral vectors in gene therapy, by which a number of patients acquired cancer as a result of the use of retroviral vector constructs, the percentage of non-viral approaches has increased over recent years. To elucidate potential bottlenecks in the non-viral transfection process we here introduce a novel method to directly visualize endocytic non-viral DNA uptake in a transfection approach. This novel method allows for the first time to monitor the location of DNA which is taken up by endocytosis in yeast (Saccharomyces cerevisiae) wild type and mutant strains. More specifically it enables drawing conclusions about conditions favouring non-viral gene transfection.

Association of weight at enlistment with enrollment in the Army Weight Control Program and subsequent attrition in the Assessment of Recruit Motivation and Strength Study.

The ongoing obesity epidemic has made recruiting qualified Army applicants increasingly difficult. A cohort of 10,213 Army enlisted subjects was enrolled in the Assessment of Recruit Motivation and Strength (ARMS) study from February 2005 through September 2006. Overweight recruits obtained a waiver for enlistment (n = 990) if they passed a screening physical fitness test. Recruits were evaluated for enrollment into the Army Weight Control Program (AWCP) and discharged during the 15 months following enlistment. Enrollment was higher among overweight recruits than recruits who met entrance standards (men: adjusted OR = 13.3 [95% CI: 10.3, 17.2]; women: adjusted OR = 3.6 [3.3, 3.9]). Although the discharge frequency was higher in the waiver group than in those who met standards (25.4% versus 19.9%, p < 0.001), there were only 10 (0.5% of total) discharges directly attributed to weight. Granting overweight waivers through the ARMS program increases enrollment to the AWCP but has little effect on weight-related attrition.

Defects in intracellular trafficking and endocytic/vacuolar acidification determine the efficiency of endocytotic DNA uptake in yeast.

The yeast Saccharomyces cerevisiae is a standard model system to study endocytosis. Here we describe the examination of a representative subset of deletion mutants to identify and locate steps in endocytic transport, endosomal/lysosomal acidification and in intracellular transport of hydrolases in non-viral transfection processes. When transport in late endocytosis is inhibited, transfection efficiency is significantly enhanced. Similarly, transfection efficiency is enhanced when the pH-value of the endosomal/vacuolar system is modified. Transfection efficiency is furthermore elevated when the N+/K+ transport in the endosomal system is disturbed. Finally, we observe enhanced transfection efficiency in mutants disturbed in the CVT/autophagy pathway and in hydrolase transport to the vacuole. In summary, non-viral transfection efficiency can be significantly increased by either (i) inhibiting the transport of endocytosed material before it enters the vacuole, or (ii) inducing a non-natural pH-value of the endosomal/vacuolar system, or (iii) slowing down degradative processes by inhibiting vacuolar hydrolases or the transport between Golgi and late endosome/vacuole.

Image-based modeling of kidney branching morphogenesis reveals GDNF-RET based Turing-type mechanism and pattern-modulating WNT11 feedback.

Branching patterns and regulatory networks differ between branched organs. It has remained unclear whether a common regulatory mechanism exists and how organ-specific patterns can emerge. Of all previously proposed signalling-based mechanisms, only a ligand-receptor-based Turing mechanism based on FGF10 and SHH quantitatively recapitulates the lung branching patterns. We now show that a GDNF-dependent ligand-receptor-based Turing mechanism quantitatively recapitulates branching of cultured wildtype and mutant ureteric buds, and achieves similar branching patterns when directing domain outgrowth in silico. We further predict and confirm experimentally that the kidney-specific positive feedback between WNT11 and GDNF permits the dense packing of ureteric tips. We conclude that the ligand-receptor based Turing mechanism presents a common regulatory mechanism for lungs and kidneys, despite the differences in the molecular implementation. Given its flexibility and robustness, we expect that the ligand-receptor-based Turing mechanism constitutes a likely general mechanism to guide branching morphogenesis and other symmetry breaks during organogenesis.

Absence of See1p, a widely conserved Saccharomyces cerevisiae protein, confers both deficient heterologous protein production and endocytosis.

The uncharacterized Saccharomyces cerevisiae open reading frame (ORF) YIL064w is predicted to encode a cytoplasmic 28 kDa protein, recognized by sequence similarity as a putative S-adenosyl-L-methionine methyltransferase. A micro-scale screening performed in our laboratory with the EUROSCARF S. cerevisiae BY4741 deletion mutant collection identified YIL064w deletion as negatively affecting secretory production of reporter alpha-amylase. The work presented here corroborates the later observations of the yil064w mutant in a larger-scale assay and shows that Yil064p is necessary for the efficient secretory production of two reporter proteins, murine alpha-amylase and fungal polygalacturonase. Further, we analysed endocytosis in the yil064w mutant strain and observed defects at both very early and later stages of endocytic transport in cells in the late logarithmic phase. The defects at very early stages may decisively account for the low transfection (DNA uptake by endocytosis) efficiency that we also observed in the yil064w mutant. These are the first in vivo data reporting a functional role for the protein encoded by ORF YIL064w and identify Yil064p, named here secretion and early endocytosis 1 protein (See1p), as a novel component of intracellular transport.

Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview.

Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes.

Molecular mechanisms involved in the regulation of cytokine production by muramyl dipeptide.

MDP (muramyl dipeptide), a component of peptidoglycan, interacts with NOD2 (nucleotide-binding oligomerization domain 2) stimulating the NOD2-RIP2 (receptor-interacting protein 2) complex to activate signalling pathways important for antibacterial defence. Here we demonstrate that the protein kinase activity of RIP2 has two functions, namely to limit the strength of downstream signalling and to stabilize the active enzyme. Thus pharmacological inhibition of RIP2 kinase with either SB 203580 [a p38 MAPK (mitogen-activated protein kinase) inhibitor] or the Src family kinase inhibitor PP2 induces a rapid and drastic decrease in the level of the RIP2 protein, which may explain why these RIP2 inhibitors block MDP-stimulated downstream signalling and the production of IL-1beta (interleukin-1beta) and TNFalpha (tumour necrosis factor-alpha). We also show that RIP2 induces the activation of the protein kinase TAK1 (transforming-growth-factor-beta-activated kinase-1), that a dominant-negative mutant of TAK1 inhibits RIP2-induced activation of JNK (c-Jun N-terminal kinase) and p38alpha MAPK, and that signalling downstream of NOD2 or RIP2 is reduced by the TAK1 inhibitor (5Z)-7-oxozeaenol or in TAK1-deficient cells. We also show that MDP activates ERK1 (extracellular-signal-regulated kinase 1)/ERK2 and p38alpha MAPK in human peripheral-blood mononuclear cells and that the activity of both MAPKs and TAK1 are required for MDP-induced signalling and production of IL-1beta and TNFalpha in these cells. Taken together, our results indicate that the MDP-NOD2/RIP2 and LPS (lipopolysaccharide)-TLR4 (Toll-like receptor 4) signalling pathways converge at the level of TAK1 and that many subsequent events that lead to the production of pro-inflammatory cytokines are common to both pathways.

Mathematical Approaches of Branching Morphogenesis.

Many organs require a high surface to volume ratio to properly function. Lungs and kidneys, for example, achieve this by creating highly branched tubular structures during a developmental process called branching morphogenesis. The genes that control lung and kidney branching share a similar network structure that is based on ligand-receptor reciprocal signalling interactions between the epithelium and the surrounding mesenchyme. Nevertheless, the temporal and spatial development of the branched epithelial trees differs, resulting in organs of distinct shape and size. In the embryonic lung, branching morphogenesis highly depends on FGF10 signalling, whereas GDNF is the driving morphogen in the kidney. Knockout of Fgf10 and Gdnf leads to lung and kidney agenesis, respectively. However, FGF10 plays a significant role during kidney branching and both the FGF10 and GDNF pathway converge on the transcription factors ETV4/5. Although the involved signalling proteins have been defined, the underlying mechanism that controls lung and kidney branching morphogenesis is still elusive. A wide range of modelling approaches exists that differ not only in the mathematical framework (e.g., stochastic or deterministic) but also in the spatial scale (e.g., cell or tissue level). Due to advancing imaging techniques, image-based modelling approaches have proven to be a valuable method for investigating the control of branching events with respect to organ-specific properties. Here, we review several mathematical models on lung and kidney branching morphogenesis and suggest that a ligand-receptor-based Turing model represents a potential candidate for a general but also adaptive mechanism to control branching morphogenesis during development.