An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.

Desulfitobacterium strains have the ability to dechlorinate halogenated compounds under anaerobic conditions by dehalorespiration. The complete genome of the tetrachloroethene (PCE)-dechlorinating strain Desulfitobacterium hafniense Y51 is a 5,727,534-bp circular chromosome harboring 5,060 predicted protein coding sequences. This genome contains only two reductive dehalogenase genes, a lower number than reported in most other dehalorespiring strains. More than 50 members of the dimethyl sulfoxide reductase superfamily and 30 paralogs of the flavoprotein subunit of the fumarate reductase are encoded as well. A remarkable feature of the genome is the large number of O-demethylase paralogs, which allow utilization of lignin-derived phenyl methyl ethers as electron donors. The large genome reveals a more versatile microorganism that can utilize a larger set of specialized electron donors and acceptors than previously thought. This is in sharp contrast to the PCE-dechlorinating strain Dehalococcoides ethenogenes 195, which has a relatively small genome with a narrow metabolic repertoire. A genomic comparison of these two very different strains allowed us to narrow down the potential candidates implicated in the dechlorination process. Our results provide further impetus to the use of desulfitobacteria as tools for bioremediation.Halogenated organic compounds are released into the environment from natural and anthropogenic sources. Many anthropogenic halogenated chemicals, like chlorinated haloalkenes (7, 10, 46), benzenes (1), and dioxins (5), are of particular concern due to their toxicity to humans and other forms of life. This toxicity is often paired with high recalcitrance to degradation, especially in anaerobic environments, leading to persistent contamination.Anaerobic environments are frequently characterized by limited availability of electron acceptors. Theoretical calculations have shown that coupling the reduction of many halogenated organic compounds to the oxidation of suitable substrates is a way to harness energy (46). As determined two decades ago, this source of energy is utilized by the microbial community. The oxidation of available electron donors coupled to the reduction of halogenated organic compounds while energy is conserved is called dehalorespiration (7,10,46). Dehalorespiring strains have been isolated independently from contaminated sites around the world. The two most prominent genera resulting from these isolation efforts are Dehalococcoides (29) and Desulfitobacterium (51), and various strains of these genera are used as model systems to study dehalorespiration (8,11,51).Dehalococcoides ethenogenes 195 is one of the few strains isolated to date which can dechlorinate tetrachloroethene (PCE) to ethene (29). D. ethenogenes 195 can use only hydrogen as an electron donor and chlorinated compounds as electron acceptors (29).Desulfitobacterium strains are also known to dechlorinate a wide variety of substrates, including halophenolic compounds and chloroalkenes (7,10,46). Although several s...

A prerequisite for the systems biology analysis of tissues is an accurate digital three-dimensional reconstruction of tissue structure based on images of markers covering multiple scales. Here, we designed a flexible pipeline for the multi-scale reconstruction and quantitative morphological analysis of tissue architecture from microscopy images. Our pipeline includes newly developed algorithms that address specific challenges of thick dense tissue reconstruction. Our implementation allows for a flexible workflow, scalable to high-throughput analysis and applicable to various mammalian tissues. We applied it to the analysis of liver tissue and extracted quantitative parameters of sinusoids, bile canaliculi and cell shapes, recognizing different liver cell types with high accuracy. Using our platform, we uncovered an unexpected zonation pattern of hepatocytes with different size, nuclei and DNA content, thus revealing new features of liver tissue organization. The pipeline also proved effective to analyse lung and kidney tissue, demonstrating its generality and robustness.DOI: http://dx.doi.org/10.7554/eLife.11214.001

Oncostatin M (OSM) is a member of the IL-6 family of cytokines. Mice deficient in the OSM receptor (OSMR -/-) showed impaired liver regeneration with persistent parenchymal necrosis after carbon tetrachloride (CCl 4 ) exposure. The recovery of liver mass from partial hepatectomy was also significantly delayed in OSMR -/-mice. In contrast to wildtype mice, CCl 4 administration only marginally induced expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 genes in OSMR -/-mice, correlating with the increased gelatinase activity of matrix metalloproteinase (MMP)-9 and matrix degradation in injured livers. The activation of STAT3 and expression of immediate early genes and cyclins were decreased in OSMR -/-liver, indicating that OSM signaling is required for hepatocyte proliferation and tissue remodeling during liver regeneration. We also found that CCl 4 administration in IL-6 -/-mice failed to induce OSM expression and that OSM administration in IL-6 -/-mice after CCl 4 injection induced the expression of cyclin D1 and proliferating cell nuclear antigen, suggesting that OSM is a key mediator of IL-6 in liver regeneration. Consistent with these results, administration of OSM ameliorated liver injury in wildtype mice by preventing hepatocyte apoptosis as well as tissue destruction. In conclusion, OSM and its signaling pathway may provide a useful therapeutic target for liver regeneration.

Bile, the central metabolic product of the liver, is transported by the bile canaliculi network. The impairment of bile flow in cholestatic liver diseases has urged a demand for insights into its regulation. Here, we developed a predictive 3D multi-scale model that simulates fluid dynamic properties successively from the subcellular to the tissue level. The model integrates the structure of the bile canalicular network in the mouse liver lobule, as determined by high-resolution confocal and serial block-face scanning electron microscopy, with measurements of bile transport by intravital microscopy. The combined experiment-theory approach revealed spatial heterogeneities of biliary geometry and hepatocyte transport activity. Based on this, our model predicts gradients of bile velocity and pressure in the liver lobule. Validation of the model predictions by pharmacological inhibition of Rho kinase demonstrated a requirement of canaliculi contractility for bile flow in vivo. Our model can be applied to functionally characterize liver diseases and quantitatively estimate biliary transport upon drug-induced liver injury.

No abstract

A transcriptional profiling of the metabolism of Corynebacterium glutamicum under oxygen deprivation conditions is reported. It was observed that the glucose consumption rate per cell when C. glutamicum cells were incubated under oxygen deprivation conditions was higher than that achieved by cells incubated under aerobic growth conditions. Furthermore, DNA microarray and quantitative RT-PCR analyses revealed that the genes of several key enzymes of the glycolytic and organic acid production pathways, including gapA, pgk, tpi, ppc, ldhA and mdh, were significantly upregulated under oxygen deprivation conditions. The corresponding enzymic activities consistently correlated with the regulation patterns of the genetic expression observed at the transcriptional level. Studies of lacZ fusions with the gapA, ldhA and mdh genes indicated not only that these genes are strongly induced at the onset of the stationary phase under aerobic growth conditions, but also that high expression levels are maintained under oxygen deprivation conditions. These results indicate that the genetic expression of several key metabolic enzymes in C. glutamicum cells incubated under oxygen deprivation conditions is chiefly regulated at the transcriptional level. The physiological consequence of the observed increased transcription under oxygen deprivation conditions is an increased rate of carbon source consumption, which is accompanied by a concomitant increase in organic acid production.

An outstanding question is how receptor tyrosine kinases (RTKs) determine different cell-fate decisions despite sharing the same signalling cascades. Here, we uncovered an unexpected mechanism of RTK trafficking in this process. By quantitative high-resolution FRET microscopy, we found that phosphorylated epidermal growth factor receptor (p-EGFR) is not randomly distributed but packaged at constant mean amounts in endosomes. Cells respond to higher EGF concentrations by increasing the number of endosomes but keeping the mean p-EGFR content per endosome almost constant. By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output. Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation. We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response.DOI: http://dx.doi.org/10.7554/eLife.06156.001