Colon cancer cell-derived 12(S)-HETE induces the retraction of cancer-associated fibroblast via MLC2, RHO/ROCK and Ca<Superscript>2+</Superscript> signalling

Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca²⁺ levels were measured and pharmacological- or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca²⁺, Ca²⁺-calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca²⁺ release. Thus, Ca²⁺ signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour–stroma interaction.     

A novel sensor to map auxin response and distribution at high spatio-temporal resolution

Auxin is a key plant morphogenetic signal but tools to analyse dynamically its distribution and signalling during development are still limited. Auxin perception directly triggers the degradation of Aux/IAA repressor proteins. Here we describe a novel Aux/IAA-based auxin signalling sensor termed DII-VENUS that was engineered in the model plant Arabidopsis thaliana. The VENUS fast maturing form of yellow fluorescent protein was fused in-frame to the Aux/IAA auxin-interaction domain (termed domain II; DII) and expressed under a constitutive promoter. We initially show that DII-VENUS abundance is dependent on auxin, its TIR1/AFBs co-receptors and proteasome activities. Next, we demonstrate that DII-VENUS provides a map of relative auxin distribution at cellular resolution in different tissues. DII-VENUS is also rapidly degraded in response to auxin and we used it to visualize dynamic changes in cellular auxin distribution successfully during two developmental responses, the root gravitropic response and lateral organ production at the shoot apex. Our results illustrate the value of developing response input sensors such as DII-VENUS to provide high-resolution spatio-temporal information about hormone distribution and response during plant growth and development.     

Deregulated MYC expression induces dependence upon AMPK-related kinase 5

Deregulated expression of the MYC oncoprotein contributes to the genesis of many human tumours, yet strategies to exploit this for a rational tumour therapy are scarce. MYC promotes cell growth and proliferation, and alters cellular metabolism to enhance the provision of precursors for phospholipids and cellular macromolecules. Here we show in human and murine cell lines that oncogenic levels of MYC establish a dependence on AMPK-related kinase 5 (ARK5; also known as NUAK1) for maintaining metabolic homeostasis and for cell survival. ARK5 is an upstream regulator of AMPK and limits protein synthesis via inhibition of the mammalian target of rapamycin 1 (mTORC1) signalling pathway. ARK5 also maintains expression of mitochondrial respiratory chain complexes and respiratory capacity, which is required for efficient glutamine metabolism. Inhibition of ARK5 leads to a collapse of cellular ATP levels in cells expressing deregulated MYC, inducing multiple pro-apoptotic responses as a secondary consequence. Depletion of ARK5 prolongs survival in MYC-driven mouse models of hepatocellular carcinoma, demonstrating that targeting cellular energy homeostasis is a valid therapeutic strategy to eliminate tumour cells that express deregulated MYC.     

SbsB structure and lattice reconstruction unveil Ca2+ triggered S-layer assembly

S-layers are regular two-dimensional semipermeable protein layers that constitute a major cell-wall component in archaea and many bacteria. The nanoscale repeat structure of the S-layer lattices and their self-assembly from S-layer proteins (SLPs) have sparked interest in their use as patterning and display scaffolds for a range of nano-biotechnological applications. Despite their biological abundance and the technological interest in them, structural information about SLPs is limited to truncated and assembly-negative proteins. Here we report the X-ray structure of the SbsB SLP of Geobacillus stearothermophilus PV72/p2 by the use of nanobody-aided crystallization. SbsB consists of a seven-domain protein, formed by an amino-terminal cell-wall attachment domain and six consecutive immunoglobulin-like domains, that organize into a φ-shaped disk-like monomeric crystallization unit stabilized by interdomain Ca(2+) ion coordination. A Ca(2+)-dependent switch to the condensed SbsB quaternary structure pre-positions intermolecular contact zones and renders the protein competent for S-layer assembly. On the basis of crystal packing, chemical crosslinking data and cryo-electron microscopy projections, we present a model for the molecular organization of this SLP into a porous protein sheet inside the S-layer. The SbsB lattice represents a previously undescribed structural model for protein assemblies and may advance our understanding of SLP physiology and self-assembly, as well as the rational design of engineered higher-order structures for biotechnology.     

STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis

Synaptic transmission is mediated by neurotransmitters that are stored in synaptic vesicles and released by exocytosis upon activation. The vesicle membrane is then retrieved by endocytosis, and synaptic vesicles are regenerated and re-filled with neurotransmitter. Although many aspects of vesicle recycling are understood, the fate of the vesicles after fusion is still unclear. Do their components diffuse on the plasma membrane, or do they remain together? This question has been difficult to answer because synaptic vesicles are too small (approximately 40 nm in diameter) and too densely packed to be resolved by available fluorescence microscopes. Here we use stimulated emission depletion (STED) to reduce the focal spot area by about an order of magnitude below the diffraction limit, thereby resolving individual vesicles in the synapse. We show that synaptotagmin I, a protein resident in the vesicle membrane, remains clustered in isolated patches on the presynaptic membrane regardless of whether the nerve terminals are mildly active or intensely stimulated. This suggests that at least some vesicle constituents remain together during recycling. Our study also demonstrates that questions involving cellular structures with dimensions of a few tens of nanometres can be resolved with conventional far-field optics and visible light.     

Patterning organic single-crystal transistor arrays

Field-effect transistors made of organic single crystals are ideal for studying the charge transport characteristics of organic semiconductor materials. Their outstanding device performance, relative to that of transistors made of organic thin films, makes them also attractive candidates for electronic applications such as active matrix displays and sensor arrays. These applications require minimal cross-talk between neighbouring devices. In the case of thin film systems, simple patterning of the active semiconductor layer minimizes cross-talk. But when using organic single crystals, the only approach currently available for creating arrays of separate devices is manual selection and placing of individual crystals-a process prohibitive for producing devices at high density and with reasonable throughput. In contrast, inorganic crystals have been grown in extended arrays, and efficient and large-area fabrication of silicon crystalline islands with high mobilities for electronic applications has been reported. Here we describe a method for effectively fabricating large arrays of single crystals of a wide range of organic semiconductor materials directly onto transistor source-drain electrodes. We find that film domains of octadecyltriethoxysilane microcontact-printed onto either clean Si/SiO(2) surfaces or flexible plastic provide control over the nucleation of vapour-grown organic single crystals. This allows us to fabricate large arrays of high-performance organic single-crystal field-effect transistors with mobilities as high as 2.4 cm(2) V(-1) s(-1) and on/off ratios greater than 10(7), and devices on flexible substrates that retain their performance after significant bending. These results suggest that our fabrication approach constitutes a promising step that might ultimately allow us to utilize high-performance organic single-crystal field-effect transistors for large-area electronics applications.     

Arctic hydrology during global warming at the Palaeocene/Eocene thermal maximum

The Palaeocene/Eocene thermal maximum represents a period of rapid, extreme global warming 55 million years ago, superimposed on an already warm world. This warming is associated with a severe shoaling of the ocean calcite compensation depth and a >2.5 per mil negative carbon isotope excursion in marine and soil carbonates. Together these observations indicate a massive release of 13C-depleted carbon and greenhouse-gas-induced warming. Recently, sediments were recovered from the central Arctic Ocean, providing the first opportunity to evaluate the environmental response at the North Pole at this time. Here we present stable hydrogen and carbon isotope measurements of terrestrial-plant- and aquatic-derived n-alkanes that record changes in hydrology, including surface water salinity and precipitation, and the global carbon cycle. Hydrogen isotope records are interpreted as documenting decreased rainout during moisture transport from lower latitudes and increased moisture delivery to the Arctic at the onset of the Palaeocene/Eocene thermal maximum, consistent with predictions of poleward storm track migrations during global warming. The terrestrial-plant carbon isotope excursion (about -4.5 to -6 per mil) is substantially larger than those of marine carbonates. Previously, this offset was explained by the physiological response of plants to increases in surface humidity. But this mechanism is not an effective explanation in this wet Arctic setting, leading us to hypothesize that the true magnitude of the excursion--and associated carbon input--was greater than originally surmised. Greater carbon release and strong hydrological cycle feedbacks may help explain the maintenance of this unprecedented warmth.     

Pluripotency of spermatogonial stem cells from adult mouse testis

Embryonic germ cells as well as germline stem cells from neonatal mouse testis are pluripotent and have differentiation potential similar to embryonic stem cells, suggesting that the germline lineage may retain the ability to generate pluripotent cells. However, until now there has been no evidence for the pluripotency and plasticity of adult spermatogonial stem cells (SSCs), which are responsible for maintaining spermatogenesis throughout life in the male. Here we show the isolation of SSCs from adult mouse testis using genetic selection, with a success rate of 27%. These isolated SSCs respond to culture conditions and acquire embryonic stem cell properties. We name these cells multipotent adult germline stem cells (maGSCs). They are able to spontaneously differentiate into derivatives of the three embryonic germ layers in vitro and generate teratomas in immunodeficient mice. When injected into an early blastocyst, SSCs contribute to the development of various organs and show germline transmission. Thus, the capacity to form multipotent cells persists in adult mouse testis. Establishment of human maGSCs from testicular biopsies may allow individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells. Furthermore, these cells may provide new opportunities to study genetic diseases in various cell lineages.     

Evolution des prostates rudimentaires d'Ellobius lutescens (Microtinae) en culture organotypique; action des androgènes

Summary The structure of the rudimentary prostates ofEllobius lutescens is maintained intact after 6 days of organotypic culture inthe absence of male hormones. Comparion with controls even shows a noticeable increase in the size of the epithelial cells. Adding male hormones to the culture medium does not modify the morphology of adult prostates, while it induces a sharp stimulation of immature prostates. In accordance with our previous results, these experiments show that the prostates ofEllobius lutescens lose their sensivity to androgens after puberty

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Ce travil a pu être réalisé grâce au Fonds n^o3919072 attribué par le Fonds National Suisse de la Recherche Scientifique et à la contribution du Fonds Klaraz.