ALBERT

Beschreibung: by Peng Zhao, Xue-mei Zhou, Li-yao Zhang, Wei Wang, Li-gang Ma, Li-bo Yang, Xiong-bo Peng, Peter V. Bozhkov, Meng-xiang Sun Plant zygote divides asymmetrically into an apical cell that develops into the embryo proper and a basal cell that generates the suspensor, a vital organ functioning as a conduit of nutrients and growth factors to the embryo proper. After the suspensor has fulfilled its function, it is removed by programmed cell death (PCD) at the late stages of embryogenesis. The molecular trigger of this PCD is unknown. Here we use tobacco ( Nicotiana tabacum ) embryogenesis as a model system to demonstrate that the mechanism triggering suspensor PCD is based on the antagonistic action of two proteins: a protease inhibitor, cystatin NtCYS, and its target, cathepsin H-like protease NtCP14. NtCYS is expressed in the basal cell of the proembryo, where encoded cystatin binds to and inhibits NtCP14, thereby preventing precocious onset of PCD. The anti-cell death effect of NtCYS is transcriptionally regulated and is repressed at the 32-celled embryo stage, leading to increased NtCP14 activity and initiation of PCD. Silencing of NtCYS or overexpression of NtCP14 induces precocious cell death in the basal cell lineage causing embryonic arrest and seed abortion. Conversely, overexpression of NtCYS or silencing of NtCP14 leads to profound delay of suspensor PCD. Our results demonstrate that NtCYS-mediated inhibition of NtCP14 protease acts as a bipartite molecular module to control initiation of PCD in the basal cell lineage of plant embryos.

Beschreibung: by William Henry Gilbert

Beschreibung: by Kaheina Aizel, Valérie Biou, Jorge Navaza, Lionel V. Duarte, Valérie Campanacci, Jacqueline Cherfils, Mahel Zeghouf The mechanisms whereby guanine nucleotide exchange factors (GEFs) coordinate their subcellular targeting to their activation of small GTPases remain poorly understood. Here we analyzed how membranes control the efficiency of human BRAG2, an ArfGEF involved in receptor endocytosis, Wnt signaling, and tumor invasion. The crystal structure of an Arf1–BRAG2 complex that mimics a membrane-bound intermediate revealed an atypical PH domain that is constitutively anchored to the catalytic Sec7 domain and interacts with Arf. Combined with the quantitative analysis of BRAG2 exchange activity reconstituted on membranes, we find that this PH domain potentiates nucleotide exchange by about 2,000-fold by cumulative conformational and membrane-targeting contributions. Furthermore, it restricts BRAG2 activity to negatively charged membranes without phosphoinositide specificity, using a positively charged surface peripheral to but excluding the canonical lipid-binding pocket. This suggests a model of BRAG2 regulation along the early endosomal pathway that expands the repertoire of GEF regulatory mechanisms. Notably, it departs from the auto-inhibitory and feedback loop paradigm emerging from studies of SOS and cytohesins. It also uncovers a novel mechanism of unspecific lipid-sensing by PH domains that may allow sustained binding to maturating membranes.

Beschreibung: by Tobias Eckle, Kelley Brodsky, Megan Bonney, Thomas Packard, Jun Han, Christoph H. Borchers, Thomas J. Mariani, Douglas J. Kominsky, Michel Mittelbronn, Holger K. Eltzschig Background While acute lung injury (ALI) contributes significantly to critical illness, it resolves spontaneously in many instances. The majority of patients experiencing ALI require mechanical ventilation. Therefore, we hypothesized that mechanical ventilation and concomitant stretch-exposure of pulmonary epithelia could activate endogenous pathways important in lung protection. Methods and Findings To examine transcriptional responses during ALI, we exposed pulmonary epithelia to cyclic mechanical stretch conditions—an in vitro model resembling mechanical ventilation. A genome-wide screen revealed a transcriptional response similar to hypoxia signaling. Surprisingly, we found that stabilization of hypoxia-inducible factor 1A (HIF1A) during stretch conditions in vitro or during ventilator-induced ALI in vivo occurs under normoxic conditions. Extension of these findings identified a functional role for stretch-induced inhibition of succinate dehydrogenase (SDH) in mediating normoxic HIF1A stabilization, concomitant increases in glycolytic capacity, and improved tricarboxylic acid (TCA) cycle function. Pharmacologic studies with HIF activator or inhibitor treatment implicated HIF1A-stabilization in attenuating pulmonary edema and lung inflammation during ALI in vivo. Systematic deletion of HIF1A in the lungs, endothelia, myeloid cells, or pulmonary epithelia linked these findings to alveolar-epithelial HIF1A. In vivo analysis of 13 C-glucose metabolites utilizing liquid-chromatography tandem mass-spectrometry demonstrated that increases in glycolytic capacity, improvement of mitochondrial respiration, and concomitant attenuation of lung inflammation during ALI were specific for alveolar-epithelial expressed HIF1A. Conclusions These studies reveal a surprising role for HIF1A in lung protection during ALI, where normoxic HIF1A stabilization and HIF-dependent control of alveolar-epithelial glucose metabolism function as an endogenous feedback loop to dampen lung inflammation.

Beschreibung: by Fred Opperdoes

Beschreibung: by Xiaojing Yang, Kai-Yeung Lau, Volkan Sevim, Chao Tang A hallmark of the G1/S transition in budding yeast cell cycle is the proteolytic degradation of the B-type cyclin-Cdk stoichiometric inhibitor Sic1. Deleting SIC1 or altering Sic1 degradation dynamics increases genomic instability. Certain key facts about the parts of the G1/S circuitry are established: phosphorylation of Sic1 on multiple sites is necessary for its destruction, and both the upstream kinase Cln1/2-Cdk1 and the downstream kinase Clb5/6-Cdk1 can phosphorylate Sic1 in vitro with varied specificity, cooperativity, and processivity. However, how the system works as a whole is still controversial due to discrepancies between in vitro , in vivo , and theoretical studies. Here, by monitoring Sic1 destruction in real time in individual cells under various perturbations to the system, we provide a clear picture of how the circuitry functions as a switch in vivo . We show that Cln1/2-Cdk1 sets the proper timing of Sic1 destruction, but does not contribute to its destruction speed; thus, it acts only as a trigger. Sic1's inhibition target Clb5/6-Cdk1 controls the speed of Sic1 destruction through a double-negative feedback loop, ensuring a robust all-or-none transition for Clb5/6-Cdk1 activity. Furthermore, we demonstrate that the degradation of a single-phosphosite mutant of Sic1 is rapid and switch-like, just as the wild-type form. Our mathematical model confirms our understanding of the circuit and demonstrates that the substrate sharing between the two kinases is not a redundancy but a part of the design to overcome the trade-off between the timing and sharpness of Sic1 degradation. Our study provides direct mechanistic insight into the design features underlying the yeast G1/S switch.

Beschreibung: by Cyrille Mionnet, Isabelle Mondor, Audrey Jorquera, Marie Loosveld, Julien Maurizio, Marie-Laure Arcangeli, Nancy H. Ruddle, Jonathan Nowak, Michel Aurrand-Lions, Hervé Luche, Marc Bajénoff Lymph node (LN) stromal cells provide survival signals and adhesive substrata to lymphocytes. During an immune response, B cell follicles enlarge, questioning how LN stromal cells manage these cellular demands. Herein, we used a murine fate mapping system to describe a new stromal cell type that resides in the T cell zone of resting LNs. We demonstrated that upon inflammation, B cell follicles progressively trespassed into the adjacent T cell zone and surrounded and converted these stromal cells into CXCL13 secreting cells that in return delineated the new boundaries of the growing follicle. Acute B cell ablation in inflamed LNs abolished CXCL13 secretion in these cells, while LT-β deficiency in B cells drastically affected this conversion. Altogether, we reveal the existence of a dormant stromal cell subset that can be functionally awakened by B cells to delineate the transient boundaries of their expanding territories upon inflammation.

Beschreibung: by Jinzhong Lin, Jing Lu, Yingang Feng, Mengyi Sun, Keqiong Ye A multitude of proteins and small nucleolar RNAs transiently associate with eukaryotic ribosomal RNAs to direct their modification and processing and the assembly of ribosomal proteins. Utp22 and Rrp7, two interacting proteins with no recognizable domain, are components of the 90S preribosome or the small subunit processome that conducts early processing of 18S rRNA. Here, we determine the cocrystal structure of Utp22 and Rrp7 complex at 1.97 Å resolution and the NMR structure of a C-terminal fragment of Rrp7, which is not visible in the crystal structure. The structure reveals that Utp22 surprisingly resembles a dimeric class I tRNA CCA-adding enzyme yet with degenerate active sites, raising an interesting evolutionary connection between tRNA and rRNA processing machineries. Rrp7 binds extensively to Utp22 using a deviant RNA recognition motif and an extended linker. Functional sites on the two proteins were identified by structure-based mutagenesis in yeast. We show that Rrp7 contains a flexible RNA-binding C-terminal tail that is essential for association with preribosomes. RNA–protein crosslinking shows that Rrp7 binds at the central domain of 18S rRNA and shares a neighborhood with two processing H/ACA snoRNAs snR30 and snR10. Depletion of snR30 prevents the stable assembly of Rrp7 into preribosomes. Our results provide insight into the evolutionary origin and functional context of Utp22 and Rrp7.

Beschreibung: by Karl S. Matlin

Beschreibung: by Brigitte Ritter, Sebastian Murphy, Hatem Dokainish, Martine Girard, Manasa V. Gudheti, Guennadi Kozlov, Marilene Halin, Jacynthe Philie, Erik M. Jorgensen, Kalle Gehring, Peter S. McPherson AP-2 is the core-organizing element in clathrin-mediated endocytosis. During the formation of clathrin-coated vesicles, clathrin and endocytic accessory proteins interact with AP-2 in a temporally and spatially controlled manner, yet it remains elusive as to how these interactions are regulated. Here, we demonstrate that the endocytic protein NECAP 1, which binds to the α-ear of AP-2 through a C-terminal WxxF motif, uses an N-terminal PH-like domain to compete with clathrin for access to the AP-2 β2-linker, revealing a means to allow AP-2–mediated coordination of accessory protein recruitment and clathrin polymerization at sites of vesicle formation. Knockdown and functional rescue studies demonstrate that through these interactions, NECAP 1 and AP-2 cooperate to increase the probability of clathrin-coated vesicle formation and to control the number, size, and cargo content of the vesicles. Together, our data demonstrate that NECAP 1 modulates the AP-2 interactome and reveal a new layer of organizational control within the endocytic machinery.

Beschreibung: by Richard Robinson

Beschreibung: by Caitlin Sedwick

Beschreibung: by Caitlin Sedwick

Beschreibung: by Artur A. Indzhykulian, Ruben Stepanyan, Anastasiia Nelina, Kateri J. Spinelli, Zubair M. Ahmed, Inna A. Belyantseva, Thomas B. Friedman, Peter G. Barr-Gillespie, Gregory I. Frolenkov Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism's life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca 2+ -dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery.

Beschreibung: by Richard Robinson

Beschreibung: by Ruth M. Fischer, Bruno M. Fontinha, Stephan Kirchmaier, Julia Steger, Susanne Bloch, Daigo Inoue, Satchidananda Panda, Simon Rumpel, Kristin Tessmar-Raible The functional principle of the vertebrate brain is often paralleled to a computer: information collected by dedicated devices is processed and integrated by interneuron circuits and leads to output. However, inter- and motorneurons present in today's vertebrate brains are thought to derive from neurons that combined sensory, integration, and motor function. Consistently, sensory inter­motorneurons have been found in the simple nerve nets of cnidarians, animals at the base of the evolutionary lineage. We show that light-sensory motorneurons and light-sensory interneurons are also present in the brains of vertebrates, challenging the paradigm that information processing and output circuitry in the central brain is shielded from direct environmental influences. We investigated two groups of nonvisual photopigments, VAL- and TMT-Opsins, in zebrafish and medaka fish; two teleost species from distinct habitats separated by over 300 million years of evolution. TMT-Opsin subclasses are specifically expressed not only in hypothalamic and thalamic deep brain photoreceptors, but also in interneurons and motorneurons with no known photoreceptive function, such as the typeXIV interneurons of the fish optic tectum. We further show that TMT-Opsins and Encephalopsin render neuronal cells light-sensitive. TMT-Opsins preferentially respond to blue light relative to rhodopsin, with subclass-specific response kinetics. We discovered that tmt-opsins co-express with val-opsins , known green light receptors, in distinct inter- and motorneurons. Finally, we show by electrophysiological recordings on isolated adult tectal slices that interneurons in the position of typeXIV neurons respond to light. Our work supports “sensory-inter-motorneurons” as ancient units for brain evolution. It also reveals that vertebrate inter- and motorneurons are endowed with an evolutionarily ancient, complex light-sensory ability that could be used to detect changes in ambient light spectra, possibly providing the endogenous equivalent to an optogenetic machinery.

Beschreibung: by Jonathan Chase

Beschreibung: by Steven A. Frank Gene expression varies widely in cells with the same genotype and environment [1],[2]. Predicting the patterns of stochastic cellular fluctuations remains an unsolved challenge. I propose that the degree to which varying cellular components combine to determine robust phenotypes may predict the amount of variability. Microbes provide excellent experimental models to analyze the relations between robust phenotypes and stochastic variability.

Beschreibung: by Smarajit Polley, De-Bin Huang, Arthur V. Hauenstein, Amanda J. Fusco, Xiangyang Zhong, Don Vu, Bärbel Schröfelbauer, Youngchang Kim, Alexander Hoffmann, Inder M. Verma, Gourisankar Ghosh, Tom Huxford Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of Xenopus IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote trans auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via trans auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent trans auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase.

Beschreibung: by Jennifer A. Dunne, Kevin D. Lafferty, Andrew P. Dobson, Ryan F. Hechinger, Armand M. Kuris, Neo D. Martinez, John P. McLaughlin, Kim N. Mouritsen, Robert Poulin, Karsten Reise, Daniel B. Stouffer, David W. Thieltges, Richard J. Williams, Claus Dieter Zander Comparative research on food web structure has revealed generalities in trophic organization, produced simple models, and allowed assessment of robustness to species loss. These studies have mostly focused on free-living species. Recent research has suggested that inclusion of parasites alters structure. We assess whether such changes in network structure result from unique roles and traits of parasites or from changes to diversity and complexity. We analyzed seven highly resolved food webs that include metazoan parasite data. Our analyses show that adding parasites usually increases link density and connectance (simple measures of complexity), particularly when including concomitant links (links from predators to parasites of their prey). However, we clarify prior claims that parasites “dominate” food web links. Although parasites can be involved in a majority of links, in most cases classic predation links outnumber classic parasitism links. Regarding network structure, observed changes in degree distributions, 14 commonly studied metrics, and link probabilities are consistent with scale-dependent changes in structure associated with changes in diversity and complexity. Parasite and free-living species thus have similar effects on these aspects of structure. However, two changes point to unique roles of parasites. First, adding parasites and concomitant links strongly alters the frequency of most motifs of interactions among three taxa, reflecting parasites' roles as resources for predators of their hosts, driven by trophic intimacy with their hosts. Second, compared to free-living consumers, many parasites' feeding niches appear broader and less contiguous, which may reflect complex life cycles and small body sizes. This study provides new insights about generic versus unique impacts of parasites on food web structure, extends the generality of food web theory, gives a more rigorous framework for assessing the impact of any species on trophic organization, identifies limitations of current food web models, and provides direction for future structural and dynamical models.

Beschreibung: by Rocio Sancho, Sophia M. Blake, Christian Tendeng, Bruce E. Clurman, Julian Lewis, Axel Behrens FBW7 is a crucial component of an SCF-type E3 ubiquitin ligase, which mediates degradation of an array of different target proteins. The Fbw7 locus comprises three different isoforms, each with its own promoter and each suspected to have a distinct set of substrates. Most FBW7 targets have important functions in developmental processes and oncogenesis, including Notch proteins, which are functionally important substrates of SCF(Fbw7). Notch signalling controls a plethora of cell differentiation decisions in a wide range of species. A prominent role of this signalling pathway is that of mediating lateral inhibition, a process where exchange of signals that repress Notch ligand production amplifies initial differences in Notch activation levels between neighbouring cells, resulting in unequal cell differentiation decisions. Here we show that the downstream Notch signalling effector HES5 directly represses transcription of the E3 ligase Fbw7β , thereby directly bearing on the process of lateral inhibition. Fbw7 Δ/+ heterozygous mice showed haploinsufficiency for Notch degradation causing impaired intestinal progenitor cell and neural stem cell differentiation. Notably, concomitant inactivation of Hes5 rescued both phenotypes and restored normal stem cell differentiation potential. In silico modelling suggests that the NICD/HES5/FBW7β positive feedback loop underlies Fbw7 haploinsufficiency. Thus repression of Fbw7β transcription by Notch signalling is an essential mechanism that is coupled to and required for the correct specification of cell fates induced by lateral inhibition.

Beschreibung: by Ryo Suzuki, Heather A. Ferris, Melissa J. Chee, Eleftheria Maratos-Flier, C. Ronald Kahn The sterol sensor SCAP is a key regulator of SREBP-2, the major transcription factor controlling cholesterol synthesis. Recently, we showed that there is a global down-regulation of cholesterol synthetic genes, as well as SREBP-2, in the brains of diabetic mice, leading to a reduction of cholesterol synthesis. We now show that in mouse models of type 1 and type 2 diabetes, this is, in part, the result of a decrease of SCAP. Homozygous disruption of the Scap gene in the brains of mice causes perinatal lethality associated with microcephaly and gliosis. Mice with haploinsufficiency of Scap in the brain show a 60% reduction of SCAP protein and ∼30% reduction in brain cholesterol synthesis, similar to what is observed in diabetic mice. This results in impaired synaptic transmission, as measured by decreased paired pulse facilitation and long-term potentiation, and is associated with behavioral and cognitive changes. Thus, reduction of SCAP and the consequent suppression of cholesterol synthesis in the brain may play an important role in the increased rates of cognitive decline and Alzheimer disease observed in diabetic states.

Beschreibung: by Janelle Weaver

Beschreibung: by Takashi Nobusawa, Yoko Okushima, Noriko Nagata, Mikiko Kojima, Hitoshi Sakakibara, Masaaki Umeda Plant organ growth is controlled by inter-cell-layer communication, which thus determines the overall size of the organism. The epidermal layer interfaces with the environment and participates in both driving and restricting growth via inter-cell-layer communication. However, it remains unknown whether the epidermis can send signals to internal tissue to limit cell proliferation in determinate growth. Very-long-chain fatty acids (VLCFAs) are synthesized in the epidermis and used in the formation of cuticular wax. Here we found that VLCFA synthesis in the epidermis is essential for proper development of Arabidopsis thaliana . Wild-type plants treated with a VLCFA synthesis inhibitor and pasticcino mutants with defects in VLCFA synthesis exhibited overproliferation of cells in the vasculature or in the rib zone of shoot apices. The decrease of VLCFA content increased the expression of IPT3 , a key determinant of cytokinin biosynthesis in the vasculature, and, indeed, elevated cytokinin levels. These phenotypes were suppressed in ipt3;5;7 triple mutants, and also by vasculature-specific expression of cytokinin oxidase, which degrades active forms of cytokinin. Our results imply that VLCFA synthesis in the epidermis is required to suppress cytokinin biosynthesis in the vasculature, thus fine-tuning cell division activity in internal tissue, and therefore that shoot growth is controlled by the interaction between the surface (epidermis) and the axis (vasculature) of the plant body.

Beschreibung: by Lucas B. Carey, David van Dijk, Peter M. A. Sloot, Jaap A. Kaandorp, Eran Segal The ability of cells to accurately control gene expression levels in response to extracellular cues is limited by the inherently stochastic nature of transcriptional regulation. A change in transcription factor (TF) activity results in changes in the expression of its targets, but the way in which cell-to-cell variability in expression (noise) changes as a function of TF activity, and whether targets of the same TF behave similarly, is not known. Here, we measure expression and noise as a function of TF activity for 16 native targets of the transcription factor Zap1 that are regulated by it through diverse mechanisms. For most activated and repressed Zap1 targets, noise decreases as expression increases. Kinetic modeling suggests that this is due to two distinct Zap1-mediated mechanisms that both change the frequency of transcriptional bursts. Notably, we found that another mechanism of repression by Zap1, which is encoded in the promoter DNA, likely decreases the size of transcriptional bursts, producing a unique transcriptional state characterized by low expression and low noise. In addition, we find that further reduction in noise is achieved when a single TF both activates and represses a single target gene. Our results suggest a global principle whereby at low TF concentrations, the dominant source of differences in expression between promoters stems from differences in burst frequency, whereas at high TF concentrations differences in burst size dominate. Taken together, we show that the precise amount by which noise changes with expression is specific to the regulatory mechanism of transcription and translation that acts at each gene.

Beschreibung: by Roland G. Roberts

Beschreibung: by Jamie L. Donnelly, Christopher M. Clark, Andrew M. Leifer, Jennifer K. Pirri, Marian Haburcak, Michael M. Francis, Aravinthan D. T. Samuel, Mark J. Alkema Monoamines provide chemical codes of behavioral states. However, the neural mechanisms of monoaminergic orchestration of behavior are poorly understood. Touch elicits an escape response in Caenorhabditis elegans where the animal moves backward and turns to change its direction of locomotion. We show that the tyramine receptor SER-2 acts through a Gα o pathway to inhibit neurotransmitter release from GABAergic motor neurons that synapse onto ventral body wall muscles. Extrasynaptic activation of SER-2 facilitates ventral body wall muscle contraction, contributing to the tight ventral turn that allows the animal to navigate away from a threatening stimulus. Tyramine temporally coordinates the different phases of the escape response through the synaptic activation of the fast-acting ionotropic receptor, LGC-55, and extrasynaptic activation of the slow-acting metabotropic receptor, SER-2. Our studies show, at the level of single cells, how a sensory input recruits the action of a monoamine to change neural circuit properties and orchestrate a compound motor sequence.

Beschreibung: by Thomas L. Rodgers, Philip D. Townsend, David Burnell, Matthew L. Jones, Shane A. Richards, Tom C. B. McLeish, Ehmke Pohl, Mark R. Wilson, Martin J. Cann Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distinct site. There is growing evidence that allosteric cooperativity can be communicated by modulation of protein dynamics without conformational change. The mechanisms, however, for communicating dynamic fluctuations between sites are debated. We provide a foundational theory for how allostery can occur as a function of low-frequency dynamics without a change in structure. We have generated coarse-grained models that describe the protein backbone motions of the CRP/FNR family transcription factors, CAP of Escherichia coli and GlxR of Corynebacterium glutamicum . The latter we demonstrate as a new exemplar for allostery without conformation change. We observe that binding the first molecule of cAMP ligand is correlated with modulation of the global normal modes and negative cooperativity for binding the second cAMP ligand without a change in mean structure. The theory makes key experimental predictions that are tested through an analysis of variant proteins by structural biology and isothermal calorimetry. Quantifying allostery as a free energy landscape revealed a protein “design space” that identified the inter- and intramolecular regulatory parameters that frame CRP/FNR family allostery. Furthermore, through analyzing CAP variants from diverse species, we demonstrate an evolutionary selection pressure to conserve residues crucial for allosteric control. This finding provides a link between the position of CRP/FNR transcription factors within the allosteric free energy landscapes and evolutionary selection pressures. Our study therefore reveals significant features of the mechanistic basis for allostery. Changes in low-frequency dynamics correlate with allosteric effects on ligand binding without the requirement for a defined spatial pathway. In addition to evolving suitable three-dimensional structures, CRP/FNR family transcription factors have been selected to occupy a dynamic space that fine-tunes biological activity and thus establishes the means to engineer allosteric mechanisms driven by low-frequency dynamics.

Beschreibung: by Min Ni, Marianna Feretzaki, Wenjun Li, Anna Floyd-Averette, Piotr Mieczkowski, Fred S. Dietrich, Joseph Heitman Aneuploidy is known to be deleterious and underlies several common human diseases, including cancer and genetic disorders such as trisomy 21 in Down's syndrome. In contrast, aneuploidy can also be advantageous and in fungi confers antifungal drug resistance and enables rapid adaptive evolution. We report here that sexual reproduction generates phenotypic and genotypic diversity in the human pathogenic yeast Cryptococcus neoformans , which is globally distributed and commonly infects individuals with compromised immunity, such as HIV/AIDS patients, causing life-threatening meningoencephalitis. C. neoformans has a defined a-α opposite sexual cycle; however, 〉99% of isolates are of the α mating type. Interestingly, α cells can undergo α-α unisexual reproduction, even involving genotypically identical cells. A central question is: Why would cells mate with themselves given that sex is costly and typically serves to admix preexisting genetic diversity from genetically divergent parents? In this study, we demonstrate that α-α unisexual reproduction frequently generates phenotypic diversity, and the majority of these variant progeny are aneuploid. Aneuploidy is responsible for the observed phenotypic changes, as chromosome loss restoring euploidy results in a wild-type phenotype. Other genetic changes, including diploidization, chromosome length polymorphisms, SNPs, and indels, were also generated. Phenotypic/genotypic changes were not observed following asexual mitotic reproduction. Aneuploidy was also detected in progeny from a-α opposite-sex congenic mating; thus, both homothallic and heterothallic sexual reproduction can generate phenotypic diversity de novo . Our study suggests that the ability to undergo unisexual reproduction may be an evolutionary strategy for eukaryotic microbial pathogens, enabling de novo genotypic and phenotypic plasticity and facilitating rapid adaptation to novel environments.

Beschreibung: by Charles Q. Choi

Beschreibung: by Jack Jing Lin Wong, Song Li, Edwin Kok Hao Lim, Yan Wang, Cheng Wang, Heng Zhang, Daniel Kirilly, Chunlai Wu, Yih-Cherng Liou, Hongyan Wang, Fengwei Yu Pruning that selectively eliminates unnecessary axons/dendrites is crucial for sculpting the nervous system during development. During Drosophila metamorphosis, dendrite arborization neurons, ddaCs, selectively prune their larval dendrites in response to the steroid hormone ecdysone, whereas mushroom body γ neurons specifically eliminate their axon branches within dorsal and medial lobes. However, it is unknown which E3 ligase directs these two modes of pruning. Here, we identified a conserved SCF E3 ubiquitin ligase that plays a critical role in pruning of both ddaC dendrites and mushroom body γ axons. The SCF E3 ligase consists of four core components Cullin1/Roc1a/SkpA/Slimb and promotes ddaC dendrite pruning downstream of EcR-B1 and Sox14, but independently of Mical. Moreover, we demonstrate that the Cullin1-based E3 ligase facilitates ddaC dendrite pruning primarily through inactivation of the InR/PI3K/TOR pathway. We show that the F-box protein Slimb forms a complex with Akt, an activator of the InR/PI3K/TOR pathway, and promotes Akt ubiquitination. Activation of the InR/PI3K/TOR pathway is sufficient to inhibit ddaC dendrite pruning. Thus, our findings provide a novel link between the E3 ligase and the InR/PI3K/TOR pathway during dendrite pruning.

Beschreibung: by Roland G. Roberts

Beschreibung: by Nicolas Rochereau, Daniel Drocourt, Eric Perouzel, Vincent Pavot, Pierre Redelinghuys, Gordon D. Brown, Gerard Tiraby, Xavier Roblin, Bernard Verrier, Christian Genin, Blaise Corthésy, Stéphane Paul Intestinal microfold (M) cells possess a high transcytosis capacity and are able to transport a broad range of materials including particulate antigens, soluble macromolecules, and pathogens from the intestinal lumen to inductive sites of the mucosal immune system. M cells are also the primary pathway for delivery of secretory IgA (SIgA) to the gut-associated lymphoid tissue. However, although the consequences of SIgA uptake by M cells are now well known and described, the mechanisms whereby SIgA is selectively bound and taken up remain poorly understood. Here we first demonstrate that both the Cα1 region and glycosylation, more particularly sialic acid residues, are involved in M cell–mediated reverse transcytosis. Second, we found that SIgA is taken up by M cells via the Dectin-1 receptor, with the possible involvement of Siglec-5 acting as a co-receptor. Third, we establish that transcytosed SIgA is taken up by mucosal CX3CR1 + dendritic cells (DCs) via the DC-SIGN receptor. Fourth, we show that mucosal and systemic antibody responses against the HIV p24-SIgA complexes administered orally is strictly dependent on the expression of Dectin-1. Having deciphered the mechanisms leading to specific targeting of SIgA-based Ag complexes paves the way to the use of such a vehicle for mucosal vaccination against various infectious diseases.

Beschreibung: by Christopher S. Carlson, Tara C. Matise, Kari E. North, Christopher A. Haiman, Megan D. Fesinmeyer, Steven Buyske, Fredrick R. Schumacher, Ulrike Peters, Nora Franceschini, Marylyn D. Ritchie, David J. Duggan, Kylee L. Spencer, Logan Dumitrescu, Charles B. Eaton, Fridtjof Thomas, Alicia Young, Cara Carty, Gerardo Heiss, Loic Le Marchand, Dana C. Crawford, Lucia A. Hindorff, Charles L. Kooperberg, for the PAGE Consortium The vast majority of genome-wide association study (GWAS) findings reported to date are from populations with European Ancestry (EA), and it is not yet clear how broadly the genetic associations described will generalize to populations of diverse ancestry. The Population Architecture Using Genomics and Epidemiology (PAGE) study is a consortium of multi-ancestry, population-based studies formed with the objective of refining our understanding of the genetic architecture of common traits emerging from GWAS. In the present analysis of five common diseases and traits, including body mass index, type 2 diabetes, and lipid levels, we compare direction and magnitude of effects for GWAS-identified variants in multiple non-EA populations against EA findings. We demonstrate that, in all populations analyzed, a significant majority of GWAS-identified variants have allelic associations in the same direction as in EA, with none showing a statistically significant effect in the opposite direction, after adjustment for multiple testing. However, 25% of tagSNPs identified in EA GWAS have significantly different effect sizes in at least one non-EA population, and these differential effects were most frequent in African Americans where all differential effects were diluted toward the null. We demonstrate that differential LD between tagSNPs and functional variants within populations contributes significantly to dilute effect sizes in this population. Although most variants identified from GWAS in EA populations generalize to all non-EA populations assessed, genetic models derived from GWAS findings in EA may generate spurious results in non-EA populations due to differential effect sizes. Regardless of the origin of the differential effects, caution should be exercised in applying any genetic risk prediction model based on tagSNPs outside of the ancestry group in which it was derived. Models based directly on functional variation may generalize more robustly, but the identification of functional variants remains challenging.

Beschreibung: by Arthur L. Caplan

Beschreibung: by Ross A. Breckenridge, Izabela Piotrowska, Keat-Eng Ng, Timothy J. Ragan, James A. West, Surendra Kotecha, Norma Towers, Michael Bennett, Petra C. Kienesberger, Ryszard T. Smolenski, Hillary K. Siddall, John L. Offer, Mihaela M. Mocanu, Derek M. Yelon, Jason R. B. Dyck, Jules L. Griffin, Andrey Y. Abramov, Alex P. Gould, Timothy J. Mohun Cardiomyocytes are vulnerable to hypoxia in the adult, but adapted to hypoxia in utero . Current understanding of endogenous cardiac oxygen sensing pathways is limited. Myocardial oxygen consumption is determined by regulation of energy metabolism, which shifts from glycolysis to lipid oxidation soon after birth, and is reversed in failing adult hearts, accompanying re-expression of several “fetal” genes whose role in disease phenotypes remains unknown. Here we show that hypoxia-controlled expression of the transcription factor Hand1 determines oxygen consumption by inhibition of lipid metabolism in the fetal and adult cardiomyocyte, leading to downregulation of mitochondrial energy generation. Hand1 is under direct transcriptional control by HIF1α. Transgenic mice prolonging cardiac Hand1 expression die immediately following birth, failing to activate the neonatal lipid metabolising gene expression programme. Deletion of Hand1 in embryonic cardiomyocytes results in premature expression of these genes. Using metabolic flux analysis, we show that Hand1 expression controls cardiomyocyte oxygen consumption by direct transcriptional repression of lipid metabolising genes. This leads, in turn, to increased production of lactate from glucose, decreased lipid oxidation, reduced inner mitochondrial membrane potential, and mitochondrial ATP generation. We found that this pathway is active in adult cardiomyocytes. Up-regulation of Hand1 is protective in a mouse model of myocardial ischaemia. We propose that Hand1 is part of a novel regulatory pathway linking cardiac oxygen levels with oxygen consumption. Understanding hypoxia adaptation in the fetal heart may allow development of strategies to protect cardiomyocytes vulnerable to ischaemia, for example during cardiac ischaemia or surgery.

Beschreibung: by Bibiana Peralta, David Gil-Carton, Daniel Castaño-Díez, Aurelie Bertin, Claire Boulogne, Hanna M. Oksanen, Dennis H. Bamford, Nicola G. A. Abrescia In internal membrane-containing viruses, a lipid vesicle enclosed by the icosahedral capsid protects the genome. It has been postulated that this internal membrane is the genome delivery device of the virus. Viruses built with this architectural principle infect hosts in all three domains of cellular life. Here, using a combination of electron microscopy techniques, we investigate bacteriophage PRD1, the best understood model for such viruses, to unveil the mechanism behind the genome translocation across the cell envelope. To deliver its double-stranded DNA, the icosahedral protein-rich virus membrane transforms into a tubular structure protruding from one of the 12 vertices of the capsid. We suggest that this viral nanotube exits from the same vertex used for DNA packaging, which is biochemically distinct from the other 11. The tube crosses the capsid through an aperture corresponding to the loss of the peripentonal P3 major capsid protein trimers, penton protein P31 and membrane protein P16. The remodeling of the internal viral membrane is nucleated by changes in osmolarity and loss of capsid-membrane interactions as consequence of the de-capping of the vertices. This engages the polymerization of the tail tube, which is structured by membrane-associated proteins. We have observed that the proteo-lipidic tube in vivo can pierce the gram-negative bacterial cell envelope allowing the viral genome to be shuttled to the host cell. The internal diameter of the tube allows one double-stranded DNA chain to be translocated. We conclude that the assembly principles of the viral tunneling nanotube take advantage of proteo-lipid interactions that confer to the tail tube elastic, mechanical and functional properties employed also in other protein-membrane systems.

Beschreibung: by Jill X. O'Reilly, Saad Jbabdi, Matthew F. S. Rushworth, Timothy E. J. Behrens A computational approach to functional specialization suggests that brain systems can be characterized in terms of the types of computations they perform, rather than their sensory or behavioral domains. We contrasted the neural systems associated with two computationally distinct forms of predictive model: a reinforcement-learning model of the environment obtained through experience with discrete events, and continuous dynamic forward modeling. By manipulating the precision with which each type of prediction could be used, we caused participants to shift computational strategies within a single spatial prediction task. Hence (using fMRI) we showed that activity in two brain systems (typically associated with reward learning and motor control) could be dissociated in terms of the forms of computations that were performed there, even when both systems were used to make parallel predictions of the same event. A region in parietal cortex, which was sensitive to the divergence between the predictions of the models and anatomically connected to both computational networks, is proposed to mediate integration of the two predictive modes to produce a single behavioral output.

Beschreibung: by Ryan C. Range, Robert C. Angerer, Lynne M. Angerer Patterning the neuroectoderm along the anterior–posterior (AP) axis is a critical event in the early development of deuterostome embryos. However, the mechanisms that regulate the specification and patterning of the neuroectoderm are incompletely understood. Remarkably, the anterior neuroectoderm (ANE) of the deuterostome sea urchin embryo expresses many of the same transcription factors and secreted modulators of Wnt signaling, as does the early vertebrate ANE (forebrain/eye field). Moreover, as is the case in vertebrate embryos, confining the ANE to the anterior end of the embryo requires a Wnt/β-catenin-dependent signaling mechanism. Here we use morpholino- or dominant negative-mediated interference to demonstrate that the early sea urchin embryo integrates information not only from Wnt/β-catenin but also from Wnt/Fzl5/8-JNK and Fzl1/2/7-PKC pathways to provide precise spatiotemporal control of neuroectoderm patterning along its AP axis. Together, through the Wnt1 and Wnt8 ligands, they orchestrate a progressive posterior-to-anterior wave of re-specification that restricts the initial, ubiquitous, maternally specified, ANE regulatory state to the most anterior blastomeres. There, the Wnt receptor antagonist, Dkk1, protects this state through a negative feedback mechanism. Because these different Wnt pathways converge on the same cell fate specification process, our data suggest they may function as integrated components of an interactive Wnt signaling network. Our findings provide strong support for the idea that the sea urchin ANE regulatory state and the mechanisms that position and define its borders represent an ancient regulatory patterning system that was present in the common echinoderm/vertebrate ancestor.

Beschreibung: by Allen Rodrigo, Susan Alberts, Karen Cranston, Joel Kingsolver, Hilmar Lapp, Craig McClain, Robin Smith, Todd Vision, Jory Weintraub, Brian Wiegmann

Beschreibung: by Supratim Ray, Amy M. Ni, John H. R. Maunsell Neuronal assemblies often exhibit stimulus-induced rhythmic activity in the gamma range (30–80 Hz), whose magnitude depends on the attentional load. This has led to the suggestion that gamma rhythms form dynamic communication channels across cortical areas processing the features of behaviorally relevant stimuli. Recently, attention has been linked to a normalization mechanism, in which the response of a neuron is suppressed (normalized) by the overall activity of a large pool of neighboring neurons. In this model, attention increases the excitatory drive received by the neuron, which in turn also increases the strength of normalization, thereby changing the balance of excitation and inhibition. Recent studies have shown that gamma power also depends on such excitatory–inhibitory interactions. Could modulation in gamma power during an attention task be a reflection of the changes in the underlying excitation–inhibition interactions? By manipulating the normalization strength independent of attentional load in macaque monkeys, we show that gamma power increases with increasing normalization, even when the attentional load is fixed. Further, manipulations of attention that increase normalization increase gamma power, even when they decrease the firing rate. Thus, gamma rhythms could be a reflection of changes in the relative strengths of excitation and normalization rather than playing a functional role in communication or control.

Beschreibung: by Caitlin Sedwick

Beschreibung: by Oliver E. Farrance, Eleanore Hann, Renata Kaminska, Nicholas G. Housden, Sasha R. Derrington, Colin Kleanthous, Sheena E. Radford, David J. Brockwell Colicins are protein antibiotics synthesised by Escherichia coli strains to target and kill related bacteria. To prevent host suicide, colicins are inactivated by binding to immunity proteins. Despite their high avidity (K d ≈fM, lifetime ≈4 days), immunity protein release is a pre-requisite of colicin intoxication, which occurs on a timescale of minutes. Here, by measuring the dynamic force spectrum of the dissociation of the DNase domain of colicin E9 (E9) and immunity protein 9 (Im9) complex using an atomic force microscope we show that application of low forces (

Beschreibung: by Matthew D. Herron, Michael Doebeli The causes and mechanisms of evolutionary diversification are central issues in biology. Geographic isolation is the traditional explanation for diversification, but recent theoretical and empirical studies have shown that frequency-dependent selection can drive diversification without isolation and that adaptive diversification occurring in sympatry may be an important source of biological diversity. However, there are no empirical examples in which sympatric lineage splits have been understood at the genetic level, and it is unknown how predictable this process is—that is, whether similar ecological settings lead to parallel evolutionary dynamics of diversification. We documented the genetic basis and the evolutionary dynamics of adaptive diversification in three replicate evolution experiments, in which competition for two carbon sources caused initially isogenic populations of the bacterium Escherichia coli to diversify into two coexisting ecotypes representing different physiological adaptations in the central carbohydrate metabolism. Whole-genome sequencing of clones of each ecotype from different populations revealed many parallel and some unique genetic changes underlying the derived phenotypes, including changes to the same genes and sometimes to the same nucleotide. Timelines of allele frequencies extracted from the frozen “fossil” record of the three evolving populations suggest parallel evolutionary dynamics driven at least in part by a co-evolutionary process in which mutations causing one type of physiology changed the ecological environment, allowing the invasion of mutations causing an alternate physiology. This process closely corresponds to the evolutionary dynamics seen in mathematical models of adaptive diversification due to frequency-dependent ecological interactions. The parallel genetic changes underlying similar phenotypes in independently evolved lineages provide empirical evidence of adaptive diversification as a predictable evolutionary process.

Beschreibung: by Richard Robinson

Beschreibung: by Amandine Girousse, Geneviève Tavernier, Carine Valle, Cedric Moro, Niklas Mejhert, Anne-Laure Dinel, Marianne Houssier, Balbine Roussel, Aurèle Besse-Patin, Marion Combes, Lucile Mir, Laurent Monbrun, Véronic Bézaire, Bénédicte Prunet-Marcassus, Aurélie Waget, Isabelle Vila, Sylvie Caspar-Bauguil, Katie Louche, Marie-Adeline Marques, Aline Mairal, Marie-Laure Renoud, Jean Galitzky, Cecilia Holm, Etienne Mouisel, Claire Thalamas, Nathalie Viguerie, Thierry Sulpice, Rémy Burcelin, Peter Arner, Dominique Langin When energy is needed, white adipose tissue (WAT) provides fatty acids (FAs) for use in peripheral tissues via stimulation of fat cell lipolysis. FAs have been postulated to play a critical role in the development of obesity-induced insulin resistance, a major risk factor for diabetes and cardiovascular disease. However, whether and how chronic inhibition of fat mobilization from WAT modulates insulin sensitivity remains elusive. Hormone-sensitive lipase (HSL) participates in the breakdown of WAT triacylglycerol into FAs. HSL haploinsufficiency and treatment with a HSL inhibitor resulted in improvement of insulin tolerance without impact on body weight, fat mass, and WAT inflammation in high-fat-diet–fed mice. In vivo palmitate turnover analysis revealed that blunted lipolytic capacity is associated with diminution in FA uptake and storage in peripheral tissues of obese HSL haploinsufficient mice. The reduction in FA turnover was accompanied by an improvement of glucose metabolism with a shift in respiratory quotient, increase of glucose uptake in WAT and skeletal muscle, and enhancement of de novo lipogenesis and insulin signalling in liver. In human adipocytes, HSL gene silencing led to improved insulin-stimulated glucose uptake, resulting in increased de novo lipogenesis and activation of cognate gene expression. In clinical studies, WAT lipolytic rate was positively and negatively correlated with indexes of insulin resistance and WAT de novo lipogenesis gene expression, respectively. In obese individuals, chronic inhibition of lipolysis resulted in induction of WAT de novo lipogenesis gene expression. Thus, reduction in WAT lipolysis reshapes FA fluxes without increase of fat mass and improves glucose metabolism through cell-autonomous induction of fat cell de novo lipogenesis, which contributes to improved insulin sensitivity.

Beschreibung: by Christopher J. Marx Few areas of science have benefited more from the expansion in sequencing capability than the study of microbial communities. Can sequence data, besides providing hypotheses of the functions the members possess, detect the evolutionary and ecological processes that are occurring? For example, can we determine if a species is adapting to one niche, or if it is diversifying into multiple specialists that inhabit distinct niches? Fortunately, adaptation of populations in the laboratory can serve as a model to test our ability to make such inferences about evolution and ecology from sequencing. Even adaptation to a single niche can give rise to complex temporal dynamics due to the transient presence of multiple competing lineages. If there are multiple niches, this complexity is augmented by segmentation of the population into multiple specialists that can each continue to evolve within their own niche. For a known example of parallel diversification that occurred in the laboratory, sequencing data gave surprisingly few obvious, unambiguous signs of the ecological complexity present. Whereas experimental systems are open to direct experimentation to test hypotheses of selection or ecological interaction, the difficulty in “seeing ecology” from sequencing for even such a simple system suggests translation to communities like the human microbiome will be quite challenging. This will require both improved empirical methods to enhance the depth and time resolution for the relevant polymorphisms and novel statistical approaches to rigorously examine time-series data for signs of various evolutionary and ecological phenomena within and between species.

Beschreibung: by Chiara Sinigaglia, Henriette Busengdal, Lucas Leclère, Ulrich Technau, Fabian Rentzsch The origin of the bilaterian head is a fundamental question for the evolution of animal body plans. The head of bilaterians develops at the anterior end of their primary body axis and is the site where the brain is located. Cnidarians, the sister group to bilaterians, lack brain-like structures and it is not clear whether the oral, the aboral, or none of the ends of the cnidarian primary body axis corresponds to the anterior domain of bilaterians. In order to understand the evolutionary origin of head development, we analysed the function of conserved genetic regulators of bilaterian anterior development in the sea anemone Nematostella vectensis . We show that orthologs of the bilaterian anterior developmental genes six3/6 , foxQ2 , and irx have dynamic expression patterns in the aboral region of Nematostella . Functional analyses reveal that NvSix3/6 acts upstream of NvFoxQ2a as a key regulator of the development of a broad aboral territory in Nematostella . NvSix3/6 initiates an autoregulatory feedback loop involving positive and negative regulators of FGF signalling, which subsequently results in the downregulation of NvSix3/6 and NvFoxQ2a in a small domain at the aboral pole, from which the apical organ develops. We show that signalling by NvFGFa1 is specifically required for the development of the apical organ, whereas NvSix3/6 has an earlier and broader function in the specification of the aboral territory. Our functional and gene expression data suggest that the head-forming region of bilaterians is derived from the aboral domain of the cnidarian-bilaterian ancestor.

Beschreibung: by Robin Mejia

Beschreibung: by Daniel Gurnon, Julian Voss-Andreae, Jacob Stanley

Beschreibung: by Phing Chian Chai, Zhong Liu, William Chia, Yu Cai In Drosophila postembryonic neuroblasts, transition in gene expression programs of a cascade of transcription factors (also known as the temporal series) acts together with the asymmetric division machinery to generate diverse neurons with distinct identities and regulate the end of neuroblast proliferation. However, the underlying mechanism of how this “temporal series” acts during development remains unclear. Here, we show that Hh signaling in the postembryonic brain is temporally regulated; excess (earlier onset of) Hh signaling causes premature neuroblast cell cycle exit and under-proliferation, whereas loss of Hh signaling causes delayed cell cycle exit and excess proliferation. Moreover, the Hh pathway functions downstream of Castor but upstream of Grainyhead, two components of the temporal series, to schedule neuroblast cell cycle exit. Interestingly, hh is likely a target of Castor. Hence, Hh signaling provides a link between the temporal series and the asymmetric division machinery in scheduling the end of neurogenesis.

Beschreibung: by Andy Dobson

Beschreibung: by Cheng-Hai Zhang, Lawrence M. Lifshitz, Karl F. Uy, Mitsuo Ikebe, Kevin E. Fogarty, Ronghua ZhuGe Bronchodilators are a standard medicine for treating airway obstructive diseases, and β2 adrenergic receptor agonists have been the most commonly used bronchodilators since their discovery. Strikingly, activation of G-protein-coupled bitter taste receptors (TAS2Rs) in airway smooth muscle (ASM) causes a stronger bronchodilation in vitro and in vivo than β2 agonists, implying that new and better bronchodilators could be developed. A critical step towards realizing this potential is to understand the mechanisms underlying this bronchodilation, which remain ill-defined. An influential hypothesis argues that bitter tastants generate localized Ca 2+ signals, as revealed in cultured ASM cells, to activate large-conductance Ca 2+ -activated K + channels, which in turn hyperpolarize the membrane, leading to relaxation. Here we report that in mouse primary ASM cells bitter tastants neither evoke localized Ca 2+ events nor alter spontaneous local Ca 2+ transients. Interestingly, they increase global intracellular [Ca 2+ ] i , although to a much lower level than bronchoconstrictors. We show that these Ca 2+ changes in cells at rest are mediated via activation of the canonical bitter taste signaling cascade (i.e., TAS2R-gustducin-phospholipase Cβ [PLCβ]- inositol 1,4,5-triphosphate receptor [IP3R]), and are not sufficient to impact airway contractility. But activation of TAS2Rs fully reverses the increase in [Ca 2+ ] i induced by bronchoconstrictors, and this lowering of the [Ca 2+ ] i is necessary for bitter tastant-induced ASM cell relaxation. We further show that bitter tastants inhibit L-type voltage-dependent Ca 2+ channels (VDCCs), resulting in reversal in [Ca 2+ ] i , and this inhibition can be prevented by pertussis toxin and G-protein βγ subunit inhibitors, but not by the blockers of PLCβ and IP3R. Together, we suggest that TAS2R stimulation activates two opposing Ca 2+ signaling pathways via Gβγ to increase [Ca 2+ ] i at rest while blocking activated L-type VDCCs to induce bronchodilation of contracted ASM. We propose that the large decrease in [Ca 2+ ] i caused by effective tastant bronchodilators provides an efficient cell-based screening method for identifying potent dilators from among the many thousands of available bitter tastants.

Beschreibung: by Kaare M. Nielsen

Beschreibung: by Uma Karthika Rajarajacholan, Subhash Thalappilly, Karl Riabowol The INhibitor of Growth (ING) proteins act as type II tumor suppressors and epigenetic regulators, being stoichiometric members of histone acetyltransferase and histone deacetylase complexes. Expression of the alternatively spliced ING1a tumor suppressor increases 〉10-fold during replicative senescence. ING1a overexpression inhibits growth; induces a large flattened cell morphology and the expression of senescence-associated β-galactosidase; increases Rb, p16, and cyclin D1 levels; and results in the accumulation of senescence-associated heterochromatic foci. Here we identify ING1a-regulated genes and find that ING1a induces the expression of a disproportionate number of genes whose products encode proteins involved in endocytosis. Intersectin 2 (ITSN2) is most affected by ING1a, being rapidly induced 〉25-fold. Overexpression of ITSN2 independently induces expression of the p16 and p57 KIP2 cyclin-dependent kinase inhibitors, which act to block Rb inactivation, acting as downstream effectors of ING1a. ITSN2 is also induced in normally senescing cells, consistent with elevated levels of ING1a inducing ITSN2 as part of a normal senescence program. Inhibition of endocytosis or altering the stoichiometry of endosome components such as Rab family members similarly induces senescence. Knockdown of ITSN2 also blocks the ability of ING1a to induce a senescent phenotype, confirming that ITSN2 is a major transducer of ING1a-induced senescence signaling. These data identify a pathway by which ING1a induces senescence and indicate that altered endocytosis activates the Rb pathway, subsequently effecting a senescent phenotype.

Beschreibung: by Janelle Weaver

Beschreibung: by Daven C. Presgraves The modern evolutionary synthesis codified the idea that species exist as distinct entities because intrinsic reproductive barriers prevent them from merging together. Understanding the origin of species therefore requires understanding the evolution and genetics of reproductive barriers between species. In most cases, speciation is an accident that happens as different populations adapt to different environments and, incidentally, come to differ in ways that render them reproductively incompatible. As with other reproductive barriers, the evolution and genetics of interspecific hybrid sterility and lethality were once also thought to evolve as pleiotripic side effects of adaptation. Recent work on the molecular genetics of speciation has raised an altogether different possibility—the genes that cause hybrid sterility and lethality often come to differ between species not because of adaptation to the external ecological environment but because of internal evolutionary arms races between selfish genetic elements and the genes of the host genome. Arguably one of the best examples supporting a role of ecological adaptation comes from a population of yellow monkey flowers, Mimulus guttatus , in Copperopolis, California, which recently evolved tolerance to soil contaminants from copper mines and simultaneously, as an incidental by-product, hybrid lethality in crosses with some off-mine populations. However, in new work, Wright and colleagues show that hybrid lethality is not a pleiotropic consequence of copper tolerance. Rather, the genetic factor causing hybrid lethality is tightly linked to copper tolerance and spread to fixation in Copperopolis by genetic hitchhiking.

Beschreibung: by Michael V. Airola, Nattakan Sukomon, Dipanjan Samanta, Peter P. Borbat, Jack H. Freed, Kylie J. Watts, Brian R. Crane HAMP domains are signal relay modules in 〉26,000 receptors of bacteria, eukaryotes, and archaea that mediate processes involved in chemotaxis, pathogenesis, and biofilm formation. We identify two HAMP conformations distinguished by a four- to two-helix packing transition at the C-termini that send opposing signals in bacterial chemoreceptors. Crystal structures of signal-locked mutants establish the observed structure-to-function relationships. Pulsed dipolar electron spin resonance spectroscopy of spin-labeled soluble receptors active in cells verify that the crystallographically defined HAMP conformers are maintained in the receptors and influence the structure and activity of downstream domains accordingly. Mutation of HR2, a key residue for setting the HAMP conformation and generating an inhibitory signal, shifts HAMP structure and receptor output to an activating state. Another HR2 variant displays an inverted response with respect to ligand and demonstrates the fine energetic balance between “on” and “off” conformers. A DExG motif found in membrane proximal HAMP domains is shown to be critical for responses to extracellular ligand. Our findings directly correlate in vivo signaling with HAMP structure, stability, and dynamics to establish a comprehensive model for HAMP-mediated signal relay that consolidates existing views on how conformational signals propagate in receptors. Moreover, we have developed a rational means to manipulate HAMP structure and function that may prove useful in the engineering of bacterial taxis responses.

Beschreibung: by Richard Robinson

Beschreibung: by Beverly S. Muhlhausler, Frank H. Bloomfield, Matthew W. Gillman

Beschreibung: by Cong Cao, Mengia S. Rioult-Pedotti, Paolo Migani, Crystal J. Yu, Rakesh Tiwari, Keykavous Parang, Mark R. Spaller, Dennis J. Goebel, John Marshall Angelman syndrome (AS) is a neurodevelopment disorder characterized by severe cognitive impairment and a high rate of autism. AS is caused by disrupted neuronal expression of the maternally inherited Ube3A ubiquitin protein ligase, required for the proteasomal degradation of proteins implicated in synaptic plasticity, such as the activity-regulated cytoskeletal-associated protein (Arc/Arg3.1). Mice deficient in maternal Ube3A express elevated levels of Arc in response to synaptic activity, which coincides with severely impaired long-term potentiation (LTP) in the hippocampus and deficits in learning behaviors. In this study, we sought to test whether elevated levels of Arc interfere with brain-derived neurotrophic factor (BDNF) TrkB receptor signaling, which is known to be essential for both the induction and maintenance of LTP. We report that TrkB signaling in the AS mouse is defective, and show that reduction of Arc expression to control levels rescues the signaling deficits. Moreover, the association of the postsynaptic density protein PSD-95 with TrkB is critical for intact BDNF signaling, and elevated levels of Arc were found to impede PSD-95/TrkB association. In Ube3A deficient mice, the BDNF-induced recruitment of PSD-95, as well as PLCγ and Grb2-associated binder 1 (Gab1) with TrkB receptors was attenuated, resulting in reduced activation of PLCγ-α-calcium/calmodulin-dependent protein kinase II (CaMKII) and PI3K-Akt, but leaving the extracellular signal-regulated kinase (Erk) pathway intact. A bridged cyclic peptide (CN2097), shown by nuclear magnetic resonance (NMR) studies to uniquely bind the PDZ1 domain of PSD-95 with high affinity, decreased the interaction of Arc with PSD-95 to restore BDNF-induced TrkB/PSD-95 complex formation, signaling, and facilitate the induction of LTP in AS mice. We propose that the failure of TrkB receptor signaling at synapses in AS is directly linked to elevated levels of Arc associated with PSD-95 and PSD-95 PDZ-ligands may represent a promising approach to reverse cognitive dysfunction.

Beschreibung: by Alexandra-Viola Bohne, Christian Schwarz, Marco Schottkowski, Michael Lidschreiber, Markus Piotrowski, William Zerges, Jörg Nickelsen Metabolic control of gene expression coordinates the levels of specific gene products to meet cellular demand for their activities. This control can be exerted by metabolites acting as regulatory signals and/or a class of metabolic enzymes with dual functions as regulators of gene expression. However, little is known about how metabolic signals affect the balance between enzymatic and regulatory roles of these dual functional proteins. We previously described the RNA binding activity of a 63 kDa chloroplast protein from Chlamydomonas reinhardtii , which has been implicated in expression of the psbA mRNA, encoding the D1 protein of photosystem II. Here, we identify this factor as dihydrolipoamide acetyltransferase (DLA2), a subunit of the chloroplast pyruvate dehydrogenase complex (cpPDC), which is known to provide acetyl-CoA for fatty acid synthesis. Analyses of RNAi lines revealed that DLA2 is involved in the synthesis of both D1 and acetyl-CoA. Gel filtration analyses demonstrated an RNP complex containing DLA2 and the chloroplast psbA mRNA specifically in cells metabolizing acetate. An intrinsic RNA binding activity of DLA2 was confirmed by in vitro RNA binding assays. Results of fluorescence microscopy and subcellular fractionation experiments support a role of DLA2 in acetate-dependent localization of the psbA mRNA to a translation zone within the chloroplast. Reciprocally, the activity of the cpPDC was specifically affected by binding of psbA mRNA. Beyond that, in silico analysis and in vitro RNA binding studies using recombinant proteins support the possibility that RNA binding is an ancient feature of dihydrolipoamide acetyltransferases. Our results suggest a regulatory function of DLA2 in response to growth on reduced carbon energy sources. This raises the intriguing possibility that this regulation functions to coordinate the synthesis of lipids and proteins for the biogenesis of photosynthetic membranes.

Beschreibung: by Mary Hoff

Beschreibung: by Mélodie Duval, Alexey Korepanov, Olivier Fuchsbauer, Pierre Fechter, Andrea Haller, Attilio Fabbretti, Laurence Choulier, Ronald Micura, Bruno P. Klaholz, Pascale Romby, Mathias Springer, Stefano Marzi Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5′ untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA–protein or mRNA–ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5′ ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features.

Beschreibung: by Richard Robinson

Beschreibung: by Jonathan Chase

Beschreibung: by Camilo Mora, Chih-Lin Wei, Audrey Rollo, Teresa Amaro, Amy R. Baco, David Billett, Laurent Bopp, Qi Chen, Mark Collier, Roberto Danovaro, Andrew J. Gooday, Benjamin M. Grupe, Paul R. Halloran, Jeroen Ingels, Daniel O. B. Jones, Lisa A. Levin, Hideyuki Nakano, Karl Norling, Eva Ramirez-Llodra, Michael Rex, Henry A. Ruhl, Craig R. Smith, Andrew K. Sweetman, Andrew R. Thurber, Jerry F. Tjiputra, Paolo Usseglio, Les Watling, Tongwen Wu, Moriaki Yasuhara Ongoing greenhouse gas emissions can modify climate processes and induce shifts in ocean temperature, pH, oxygen concentration, and productivity, which in turn could alter biological and social systems. Here, we provide a synoptic global assessment of the simultaneous changes in future ocean biogeochemical variables over marine biota and their broader implications for people. We analyzed modern Earth System Models forced by greenhouse gas concentration pathways until 2100 and showed that the entire world's ocean surface will be simultaneously impacted by varying intensities of ocean warming, acidification, oxygen depletion, or shortfalls in productivity. In contrast, only a small fraction of the world's ocean surface, mostly in polar regions, will experience increased oxygenation and productivity, while almost nowhere will there be ocean cooling or pH elevation. We compiled the global distribution of 32 marine habitats and biodiversity hotspots and found that they would all experience simultaneous exposure to changes in multiple biogeochemical variables. This superposition highlights the high risk for synergistic ecosystem responses, the suite of physiological adaptations needed to cope with future climate change, and the potential for reorganization of global biodiversity patterns. If co-occurring biogeochemical changes influence the delivery of ocean goods and services, then they could also have a considerable effect on human welfare. Approximately 470 to 870 million of the poorest people in the world rely heavily on the ocean for food, jobs, and revenues and live in countries that will be most affected by simultaneous changes in ocean biogeochemistry. These results highlight the high risk of degradation of marine ecosystems and associated human hardship expected in a future following current trends in anthropogenic greenhouse gas emissions.

Beschreibung: by Jonathan A. Eisen, Catriona J. MacCallum, Cameron Neylon

Beschreibung: by Adam Eyre-Walker, Nina Stoletzki The assessment of scientific publications is an integral part of the scientific process. Here we investigate three methods of assessing the merit of a scientific paper: subjective post-publication peer review, the number of citations gained by a paper, and the impact factor of the journal in which the article was published. We investigate these methods using two datasets in which subjective post-publication assessments of scientific publications have been made by experts. We find that there are moderate, but statistically significant, correlations between assessor scores, when two assessors have rated the same paper, and between assessor score and the number of citations a paper accrues. However, we show that assessor score depends strongly on the journal in which the paper is published, and that assessors tend to over-rate papers published in journals with high impact factors. If we control for this bias, we find that the correlation between assessor scores and between assessor score and the number of citations is weak, suggesting that scientists have little ability to judge either the intrinsic merit of a paper or its likely impact. We also show that the number of citations a paper receives is an extremely error-prone measure of scientific merit. Finally, we argue that the impact factor is likely to be a poor measure of merit, since it depends on subjective assessment. We conclude that the three measures of scientific merit considered here are poor; in particular subjective assessments are an error-prone, biased, and expensive method by which to assess merit. We argue that the impact factor may be the most satisfactory of the methods we have considered, since it is a form of pre-publication review. However, we emphasise that it is likely to be a very error-prone measure of merit that is qualitative, not quantitative.

Beschreibung: by Yu Hang Fong, Ho Chun Wong, Man Hon Yuen, Pak Ho Lau, Yu Wai Chen, Kam-Bo Wong Urease is a metalloenzyme essential for the survival of Helicobacter pylori in acidic gastric environment. Maturation of urease involves carbamylation of Lys219 and insertion of two nickel ions at its active site. This process requires GTP hydrolysis and the formation of a preactivation complex consisting of apo-urease and urease accessory proteins UreF, UreH, and UreG. UreF and UreH form a complex to recruit UreG, which is a SIMIBI class GTPase, to the preactivation complex. We report here the crystal structure of the UreG/UreF/UreH complex, which illustrates how UreF and UreH facilitate dimerization of UreG, and assembles its metal binding site by juxtaposing two invariant Cys66-Pro67-His68 metal binding motif at the interface to form the (UreG/UreF/UreH) 2 complex. Interaction studies revealed that addition of nickel and GTP to the UreG/UreF/UreH complex releases a UreG dimer that binds a nickel ion at the dimeric interface. Substitution of Cys66 and His68 with alanine abolishes the formation of the nickel-charged UreG dimer. This nickel-charged UreG dimer can activate urease in vitro in the presence of the UreF/UreH complex. Static light scattering and atomic absorption spectroscopy measurements demonstrated that the nickel-charged UreG dimer, upon GTP hydrolysis, reverts to its monomeric form and releases nickel to urease. Based on our results, we propose a mechanism on how urease accessory proteins facilitate maturation of urease.

Beschreibung: by Orli Yogev, Victoria C. Williams, Yaniv Hinits, Simon M. Hughes Muscle fiber size is activity-dependent and clinically important in ageing, bed-rest, and cachexia, where muscle weakening leads to disability, prolonged recovery times, and increased costs. Inactivity causes muscle wasting by triggering protein degradation and may simultaneously prevent protein synthesis. During development, muscle tissue grows by several mechanisms, including hypertrophy of existing fibers. As in other tissues, the TOR pathway plays a key role in promoting muscle protein synthesis by inhibition of eIF4EBPs (eukaryotic Initiation Factor 4E Binding Proteins), regulators of the translational initiation. Here, we tested the role of TOR-eIF4EBP in a novel zebrafish muscle inactivity model. Inactivity triggered up-regulation of eIF4EBP3L (a zebrafish homolog of eIF4EBP3) and diminished myosin and actin content, myofibrilogenesis, and fiber growth. The changes were accompanied by preferential reduction of the muscle transcription factor Mef2c, relative to Myod and Vinculin. Polysomal fractionation showed that Mef2c decrease was due to reduced translation of mef2ca mRNA. Loss of Mef2ca function reduced normal muscle growth and diminished the reduction in growth caused by inactivity. We identify eIF4EBP3L as a key regulator of Mef2c translation and protein level following inactivity; blocking eIF4EBP3L function increased Mef2ca translation. Such blockade also prevented the decline in mef2ca translation and level of Mef2c and slow myosin heavy chain proteins caused by inactivity. Conversely, overexpression of active eIF4EBP3L mimicked inactivity by decreasing the proportion of mef2ca mRNA in polysomes, the levels of Mef2c and slow myosin heavy chain, and myofibril content. Inhibiting the TOR pathway without the increase in eIF4EBP3L had a lesser effect on myofibrilogenesis and muscle size. These findings identify eIF4EBP3L as a key TOR-dependent regulator of muscle fiber size in response to activity. We suggest that by selectively inhibiting translational initiation of mef2ca and other mRNAs, eIF4EBP3L reprograms the translational profile of muscle, enabling it to adjust to new environmental conditions.

Beschreibung: by Hiruy S. Meharena, Philip Chang, Malik M. Keshwani, Krishnadev Oruganty, Aishwarya K. Nene, Natarajan Kannan, Susan S. Taylor, Alexandr P. Kornev Eukaryotic protein kinases (EPKs) regulate numerous signaling processes by phosphorylating targeted substrates through the highly conserved catalytic domain. Our previous computational studies proposed a model stating that a properly assembled nonlinear motif termed the Regulatory (R) spine is essential for catalytic activity of EPKs. Here we define the required intramolecular interactions and biochemical properties of the R-spine and the newly identified “Shell” that surrounds the R-spine using site-directed mutagenesis and various in vitro phosphoryl transfer assays using cyclic AMP-dependent protein kinase as a representative of the entire kinome. Analysis of the 172 available Apo EPK structures in the protein data bank (PDB) revealed four unique structural conformations of the R-spine that correspond with catalytic inactivation of various EPKs. Elucidating the molecular entities required for the catalytic activation of EPKs and the identification of these inactive conformations opens new avenues for the design of efficient therapeutic EPK inhibitors.

Beschreibung: by Zachary B. Gaber, Samantha J. Butler, Bennett G. Novitch Distinct classes of neurons and glial cells in the developing spinal cord arise at specific times and in specific quantities from spatially discrete neural progenitor domains. Thus, adjacent domains can exhibit marked differences in their proliferative potential and timing of differentiation. However, remarkably little is known about the mechanisms that account for this regional control. Here, we show that the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF) plays a critical role shaping patterns of neuronal differentiation by gating the expression of Fibroblast Growth Factor (FGF) Receptor 3 and responsiveness of progenitors to FGFs. PLZF elevation increases FGFR3 expression and STAT3 pathway activity, suppresses neurogenesis, and biases progenitors towards glial cell production. In contrast, PLZF loss reduces FGFR3 levels, leading to premature neuronal differentiation. Together, these findings reveal a novel transcriptional strategy for spatially tuning the responsiveness of distinct neural progenitor groups to broadly distributed mitogenic signals in the embryonic environment.

Beschreibung: by Anna Śledzińska, Saskia Hemmers, Florian Mair, Oliver Gorka, Jürgen Ruland, Lynsey Fairbairn, Anja Nissler, Werner Müller, Ari Waisman, Burkhard Becher, Thorsten Buch TGF-β is widely held to be critical for the maintenance and function of regulatory T (T reg ) cells and thus peripheral tolerance. This is highlighted by constitutive ablation of TGF-β receptor (TR) during thymic development in mice, which leads to a lethal autoimmune syndrome. Here we describe that TGF-β–driven peripheral tolerance is not regulated by TGF-β signalling on mature CD4 + T cells. Inducible TR2 ablation specifically on CD4 + T cells did not result in a lethal autoinflammation. Transfer of these TR2-deficient CD4 + T cells to lymphopenic recipients resulted in colitis, but not overt autoimmunity. In contrast, thymic ablation of TR2 in combination with lymphopenia led to lethal multi-organ inflammation. Interestingly, deletion of TR2 on mature CD4 + T cells does not result in the collapse of the T reg cell population as observed in constitutive models. Instead, a pronounced enlargement of both regulatory and effector memory T cell pools was observed. This expansion is cell-intrinsic and seems to be caused by increased T cell receptor sensitivity independently of common gamma chain-dependent cytokine signals. The expression of Foxp3 and other regulatory T cells markers was not dependent on TGF-β signalling and the TR2–deficient T reg cells retained their suppressive function both in vitro and in vivo . In summary, absence of TGF-β signalling on mature CD4 + T cells is not responsible for breakdown of peripheral tolerance, but rather controls homeostasis of mature T cells in adult mice.

Beschreibung: by Maël Lebreton, Maxime Bertoux, Claire Boutet, Stéphane Lehericy, Bruno Dubois, Philippe Fossati, Mathias Pessiglione Many choice situations require imagining potential outcomes, a capacity that was shown to involve memory brain regions such as the hippocampus. We reasoned that the quality of hippocampus-mediated simulation might therefore condition the subjective value assigned to imagined outcomes. We developed a novel paradigm to assess the impact of hippocampus structure and function on the propensity to favor imagined outcomes in the context of intertemporal choices. The ecological condition opposed immediate options presented as pictures (hence directly observable) to delayed options presented as texts (hence requiring mental stimulation). To avoid confounding simulation process with delay discounting, we compared this ecological condition to control conditions using the same temporal labels while keeping constant the presentation mode. Behavioral data showed that participants who imagined future options with greater details rated them as more likeable. Functional MRI data confirmed that hippocampus activity could account for subjects assigning higher values to simulated options. Structural MRI data suggested that grey matter density was a significant predictor of hippocampus activation, and therefore of the propensity to favor simulated options. Conversely, patients with hippocampus atrophy due to Alzheimer's disease, but not patients with Fronto-Temporal Dementia, were less inclined to favor options that required mental simulation. We conclude that hippocampus-mediated simulation plays a critical role in providing the motivation to pursue goals that are not present to our senses.

Beschreibung: by Emma Ganley

Beschreibung: by Cameron Neylon

Beschreibung: by Heather Joseph

Beschreibung: by Marie-Cécile Caillaud, Shuta Asai, Ghanasyam Rallapalli, Sophie Piquerez, Georgina Fabro, Jonathan D. G. Jones Plants are continually exposed to pathogen attack but usually remain healthy because they can activate defences upon perception of microbes. However, pathogens have evolved to overcome plant immunity by delivering effectors into the plant cell to attenuate defence, resulting in disease. Recent studies suggest that some effectors may manipulate host transcription, but the specific mechanisms by which such effectors promote susceptibility remain unclear. We study the oomycete downy mildew pathogen of Arabidopsis, Hyaloperonospora arabidopsidis ( Hpa ), and show here that the nuclear-localized effector HaRxL44 interacts with Mediator subunit 19a (MED19a), resulting in the degradation of MED19a in a proteasome-dependent manner. The Mediator complex of ∼25 proteins is broadly conserved in eukaryotes and mediates the interaction between transcriptional regulators and RNA polymerase II. We found MED19a to be a positive regulator of immunity against Hpa . Expression profiling experiments reveal transcriptional changes resembling jasmonic acid/ethylene (JA/ET) signalling in the presence of HaRxL44, and also 3 d after infection with Hpa . Elevated JA/ET signalling is associated with a decrease in salicylic acid (SA)–triggered immunity (SATI) in Arabidopsis plants expressing HaRxL44 and in med19a loss-of-function mutants, whereas SATI is elevated in plants overexpressing MED19a . Using a PR1 ::GUS reporter, we discovered that Hpa suppresses PR1 expression specifically in cells containing haustoria, into which RxLR effectors are delivered, but not in nonhaustoriated adjacent cells, which show high PR1 ::GUS expression levels. Thus, HaRxL44 interferes with Mediator function by degrading MED19, shifting the balance of defence transcription from SA-responsive defence to JA/ET-signalling, and enhancing susceptibility to biotrophs by attenuating SA-dependent gene expression.

Beschreibung: by Morgane Wartel, Adrien Ducret, Shashi Thutupalli, Fabian Czerwinski, Anne-Valérie Le Gall, Emilia M. F. Mauriello, Ptissam Bergam, Yves V. Brun, Joshua Shaevitz, Tâm Mignot Eukaryotic cells utilize an arsenal of processive transport systems to deliver macromolecules to specific subcellular sites. In prokaryotes, such transport mechanisms have only been shown to mediate gliding motility, a form of microbial surface translocation. Here, we show that the motility function of the Myxococcus xanthus Agl-Glt machinery results from the recent specialization of a versatile class of bacterial transporters. Specifically, we demonstrate that the Agl motility motor is modular and dissociates from the rest of the gliding machinery (the Glt complex) to bind the newly expressed Nfs complex, a close Glt paralogue, during sporulation. Following this association, the Agl system transports Nfs proteins directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by Agl-Nfs ensures its distribution around the spore. Thus, the Agl-Glt/Nfs machineries may constitute a novel class of directional bacterial surface transporters that can be diversified to specific tasks depending on the cognate cargo and machinery-specific accessories.

Beschreibung: by Thomas D. Pollard

Beschreibung: by Sarah E. Lester, Christopher Costello, Andrew Rassweiler, Steven D. Gaines, Robert Deacon

Beschreibung: by Esteban J. Beckwith, E. Axel Gorostiza, Jimena Berni, Carolina Rezával, Agustín Pérez-Santángelo, Alejandro D. Nadra, María Fernanda Ceriani Living organisms use biological clocks to maintain their internal temporal order and anticipate daily environmental changes. In Drosophila , circadian regulation of locomotor behavior is controlled by ∼150 neurons; among them, neurons expressing the PIGMENT DISPERSING FACTOR (PDF) set the period of locomotor behavior under free-running conditions. To date, it remains unclear how individual circadian clusters integrate their activity to assemble a distinctive behavioral output. Here we show that the BONE MORPHOGENETIC PROTEIN (BMP) signaling pathway plays a crucial role in setting the circadian period in PDF neurons in the adult brain. Acute deregulation of BMP signaling causes period lengthening through regulation of d Clock transcription, providing evidence for a novel function of this pathway in the adult brain. We propose that coherence in the circadian network arises from integration in PDF neurons of both the pace of the cell-autonomous molecular clock and information derived from circadian-relevant neurons through release of BMP ligands.

Beschreibung: by Yaron E. Antebi, Shlomit Reich-Zeliger, Yuval Hart, Avi Mayo, Inbal Eizenberg, Jacob Rimer, Prabhakar Putheti, Dana Pe'er, Nir Friedman Cell differentiation is typically directed by external signals that drive opposing regulatory pathways. Studying differentiation under polarizing conditions, with only one input signal provided, is limited in its ability to resolve the logic of interactions between opposing pathways. Dissection of this logic can be facilitated by mapping the system's response to mixtures of input signals, which are expected to occur in vivo , where cells are simultaneously exposed to various signals with potentially opposing effects. Here, we systematically map the response of naïve T cells to mixtures of signals driving differentiation into the Th1 and Th2 lineages. We characterize cell state at the single cell level by measuring levels of the two lineage-specific transcription factors (T-bet and GATA3) and two lineage characteristic cytokines (IFN-γ and IL-4) that are driven by these transcription regulators. We find a continuum of mixed phenotypes in which individual cells co-express the two lineage-specific master regulators at levels that gradually depend on levels of the two input signals. Using mathematical modeling we show that such tunable mixed phenotype arises if autoregulatory positive feedback loops in the gene network regulating this process are gradual and dominant over cross-pathway inhibition. We also find that expression of the lineage-specific cytokines follows two independent stochastic processes that are biased by expression levels of the master regulators. Thus, cytokine expression is highly heterogeneous under mixed conditions, with subpopulations of cells expressing only IFN-γ, only IL-4, both cytokines, or neither. The fraction of cells in each of these subpopulations changes gradually with input conditions, reproducing the continuous internal state at the cell population level. These results suggest a differentiation scheme in which cells reflect uncertainty through a continuously tuneable mixed phenotype combined with a biased stochastic decision rather than a binary phenotype with a deterministic decision.

Beschreibung: by Amy Coombs

Beschreibung: by Konstantinos K. Tsilidis, Orestis A. Panagiotou, Emily S. Sena, Eleni Aretouli, Evangelos Evangelou, David W. Howells, Rustam Al-Shahi Salman, Malcolm R. Macleod, John P. A. Ioannidis Animal studies generate valuable hypotheses that lead to the conduct of preventive or therapeutic clinical trials. We assessed whether there is evidence for excess statistical significance in results of animal studies on neurological disorders, suggesting biases. We used data from meta-analyses of interventions deposited in Collaborative Approach to Meta-Analysis and Review of Animal Data in Experimental Studies (CAMARADES). The number of observed studies with statistically significant results (O) was compared with the expected number (E), based on the statistical power of each study under different assumptions for the plausible effect size. We assessed 4,445 datasets synthesized in 160 meta-analyses on Alzheimer disease ( n  = 2), experimental autoimmune encephalomyelitis ( n  = 34), focal ischemia ( n  = 16), intracerebral hemorrhage ( n  = 61), Parkinson disease ( n  = 45), and spinal cord injury ( n  = 2). 112 meta-analyses (70%) found nominally ( p ≤0.05) statistically significant summary fixed effects. Assuming the effect size in the most precise study to be a plausible effect, 919 out of 4,445 nominally significant results were expected versus 1,719 observed ( p

Beschreibung: by Jacob E. Lazarus, Armen J. Moughamian, Mariko K. Tokito, Erika L. F. Holzbaur Regulation of microtubule dynamics in neurons is critical, as defects in the microtubule-based transport of axonal organelles lead to neurodegenerative disease. The microtubule motor cytoplasmic dynein and its partner complex dynactin drive retrograde transport from the distal axon. We have recently shown that the p150 Glued subunit of dynactin promotes the initiation of dynein-driven cargo motility from the microtubule plus-end. Because plus end-localized microtubule-associated proteins like p150 Glued may also modulate the dynamics of microtubules, we hypothesized that p150 Glued might promote cargo initiation by stabilizing the microtubule track. Here, we demonstrate in vitro using assembly assays and TIRF microscopy, and in primary neurons using live-cell imaging, that p150 Glued is a potent anti-catastrophe factor for microtubules. p150 Glued alters microtubule dynamics by binding both to microtubules and to tubulin dimers; both the N-terminal CAP-Gly and basic domains of p150 Glued are required in tandem for this activity. p150 Glued is alternatively spliced in vivo , with the full-length isoform including these two domains expressed primarily in neurons. Accordingly, we find that RNAi of p150 Glued in nonpolarized cells does not alter microtubule dynamics, while depletion of p150 Glued in neurons leads to a dramatic increase in microtubule catastrophe. Strikingly, a mutation in p150 Glued causal for the lethal neurodegenerative disorder Perry syndrome abrogates this anti-catastrophe activity. Thus, we find that dynactin has multiple functions in neurons, both activating dynein-mediated retrograde axonal transport and enhancing microtubule stability through a novel anti-catastrophe mechanism regulated by tissue-specific isoform expression; disruption of either or both of these functions may contribute to neurodegenerative disease.

Beschreibung: by Laura C. Wieland Brown, Cristina Penaranda, Purna C. Kashyap, Brianna B. Williams, Jon Clardy, Mitchell Kronenberg, Justin L. Sonnenburg, Laurie E. Comstock, Jeffrey A. Bluestone, Michael A. Fischbach While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise 〉50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of signaling molecules that play a key role in inducing apoptosis and modulating the host immune response. Although known for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined. Here, we use a combination of genetics and chemistry to identify the sphingolipids produced by Bacteroides fragilis NCTC 9343. We constructed a deletion mutant of BF2461, a putative serine palmitoyltransferase whose yeast homolog catalyzes the committed step in sphingolipid biosynthesis. We show that the Δ2461 mutant is sphingolipid deficient, enabling us to purify and solve the structures of three alkaline-stable lipids present in the wild-type strain but absent from the mutant. The first compound was the known sphingolipid ceramide phosphorylethanolamine, and the second was its corresponding dihydroceramide base. Unexpectedly, the third compound was the glycosphingolipid α-galactosylceramide (α-GalCer Bf ), which is structurally related to a sponge-derived sphingolipid (α-GalCer, KRN7000) that is the prototypical agonist of CD1d-restricted natural killer T (iNKT) cells. We demonstrate that α-GalCer Bf has similar immunological properties to KRN7000: it binds to CD1d and activates both mouse and human iNKT cells both in vitro and in vivo . Thus, our study reveals BF2461 as the first known member of the Bacteroides sphingolipid pathway, and it indicates that the committed steps of the Bacteroides and eukaryotic sphingolipid pathways are identical. Moreover, our data suggest that some Bacteroides sphingolipids might influence host immune homeostasis.

Beschreibung: by Alessia Soldano, Zeynep Okray, Pavlina Janovska, Kateřina Tmejová, Elodie Reynaud, Annelies Claeys, Jiekun Yan, Zeynep Kalender Atak, Bart De Strooper, Jean-Maurice Dura, Vítězslav Bryja, Bassem A. Hassan Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of Drosophila APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth.

Beschreibung: by Mauricio Menacho-Márquez, Ramón García-Escudero, Virginia Ojeda, Antonio Abad, Pilar Delgado, Clotilde Costa, Sergio Ruiz, Balbino Alarcón, Jesús M. Paramio, Xosé R. Bustelo The catalytic activity of GDP/GTP exchange factors (GEFs) is considered critical to maintain the typically high activity of Rho GTPases found in cancer cells. However, the large number of them has made it difficult to pinpoint those playing proactive, nonredundant roles in tumors. In this work, we have investigated whether GEFs of the Vav subfamily exert such specific roles in skin cancer. Using genetically engineered mice, we show here that Vav2 and Vav3 favor cooperatively the initiation and promotion phases of skin tumors. Transcriptomal profiling and signaling experiments indicate such function is linked to the engagement of, and subsequent participation in, keratinocyte-based autocrine/paracrine programs that promote epidermal proliferation and recruitment of pro-inflammatory cells. This is a pathology-restricted mechanism because the loss of Vav proteins does not cause alterations in epidermal homeostasis. These results reveal a previously unknown Rho GEF-dependent pro-tumorigenic mechanism that influences the biology of cancer cells and their microenvironment. They also suggest that anti-Vav therapies may be of potential interest in skin tumor prevention and/or treatment.

Beschreibung: by Robin Mejia

Beschreibung: by Fergal O'Farrell, Shenqiu Wang, Nadja Katheder, Tor Erik Rusten, Christos Samakovlis Body size in Drosophila larvae, like in other animals, is controlled by nutrition. Nutrient restriction leads to catabolic responses in the majority of tissues, but the Drosophila mitotic imaginal discs continue growing. The nature of these differential control mechanisms that spare distinct tissues from starvation are poorly understood. Here, we reveal that the Ret-like receptor tyrosine kinase (RTK), Stitcher (Stit), is required for cell growth and proliferation through the PI3K-I/TORC1 pathway in the Drosophila wing disc. Both Stit and insulin receptor (InR) signaling activate PI3K-I and drive cellular proliferation and tissue growth. However, whereas optimal growth requires signaling from both InR and Stit, catabolic changes manifested by autophagy only occur when both signaling pathways are compromised. The combined activities of Stit and InR in ectodermal epithelial tissues provide an RTK-mediated, two-tiered reaction threshold to varying nutritional conditions that promote epithelial organ growth even at low levels of InR signaling.

Beschreibung: by Sinem Beyhan, Matias Gutierrez, Mark Voorhies, Anita Sil Survival at host temperature is a critical trait for pathogenic microbes of humans. Thermally dimorphic fungal pathogens, including Histoplasma capsulatum , are soil fungi that undergo dramatic changes in cell shape and virulence gene expression in response to host temperature. How these organisms link changes in temperature to both morphologic development and expression of virulence traits is unknown. Here we elucidate a temperature-responsive transcriptional network in H. capsulatum , which switches from a filamentous form in the environment to a pathogenic yeast form at body temperature. The circuit is driven by three highly conserved factors, Ryp1, Ryp2, and Ryp3, that are required for yeast-phase growth at 37°C. Ryp factors belong to distinct families of proteins that control developmental transitions in fungi: Ryp1 is a member of the WOPR family of transcription factors, and Ryp2 and Ryp3 are both members of the Velvet family of proteins whose molecular function is unknown. Here we provide the first evidence that these WOPR and Velvet proteins interact, and that Velvet proteins associate with DNA to drive gene expression. Using genome-wide chromatin immunoprecipitation studies, we determine that Ryp1, Ryp2, and Ryp3 associate with a large common set of genomic loci that includes known virulence genes, indicating that the Ryp factors directly control genes required for pathogenicity in addition to their role in regulating cell morphology. We further dissect the Ryp regulatory circuit by determining that a fourth transcription factor, which we name Ryp4, is required for yeast-phase growth and gene expression, associates with DNA, and displays interdependent regulation with Ryp1, Ryp2, and Ryp3. Finally, we define cis -acting motifs that recruit the Ryp factors to their interwoven network of temperature-responsive target genes. Taken together, our results reveal a positive feedback circuit that directs a broad transcriptional switch between environmental and pathogenic states in response to temperature.

Beschreibung: by Cassandra Coburn, Erik Allman, Parag Mahanti, Alexandre Benedetto, Filipe Cabreiro, Zachary Pincus, Filip Matthijssens, Caroline Araiz, Abraham Mandel, Manolis Vlachos, Sally-Anne Edwards, Grahame Fischer, Alexander Davidson, Rosina E. Pryor, Ailsa Stevens, Frank J. Slack, Nektarios Tavernarakis, Bart P. Braeckman, Frank C. Schroeder, Keith Nehrke, David Gems For cells the passage from life to death can involve a regulated, programmed transition. In contrast to cell death, the mechanisms of systemic collapse underlying organismal death remain poorly understood. Here we present evidence of a cascade of cell death involving the calpain-cathepsin necrosis pathway that can drive organismal death in Caenorhabditis elegans . We report that organismal death is accompanied by a burst of intense blue fluorescence, generated within intestinal cells by the necrotic cell death pathway. Such death fluorescence marks an anterior to posterior wave of intestinal cell death that is accompanied by cytosolic acidosis. This wave is propagated via the innexin INX-16, likely by calcium influx. Notably, inhibition of systemic necrosis can delay stress-induced death. We also identify the source of the blue fluorescence, initially present in intestinal lysosome-related organelles (gut granules), as anthranilic acid glucosyl esters—not, as previously surmised, the damage product lipofuscin. Anthranilic acid is derived from tryptophan by action of the kynurenine pathway. These findings reveal a central mechanism of organismal death in C. elegans that is related to necrotic propagation in mammals—e.g., in excitotoxicity and ischemia-induced neurodegeneration. Endogenous anthranilate fluorescence renders visible the spatio-temporal dynamics of C. elegans organismal death.

Beschreibung: by Caitlin Sedwick

Beschreibung: by Logan D. Andrews, Tim D. Fenn, Daniel Herschlag Enzymes stabilize transition states of reactions while limiting binding to ground states, as is generally required for any catalyst. Alkaline Phosphatase (AP) and other nonspecific phosphatases are some of Nature's most impressive catalysts, achieving preferential transition state over ground state stabilization of more than 10 22 -fold while utilizing interactions with only the five atoms attached to the transferred phosphorus. We tested a model that AP achieves a portion of this preference by destabilizing ground state binding via charge repulsion between the anionic active site nucleophile, Ser102, and the negatively charged phosphate monoester substrate. Removal of the Ser102 alkoxide by mutation to glycine or alanine increases the observed P i affinity by orders of magnitude at pH 8.0. To allow precise and quantitative comparisons, the ionic form of bound P i was determined from pH dependencies of the binding of P i and tungstate, a P i analog lacking titratable protons over the pH range of 5–11, and from the 31 P chemical shift of bound P i . The results show that the P i trianion binds with an exceptionally strong femtomolar affinity in the absence of Ser102, show that its binding is destabilized by ≥10 8 -fold by the Ser102 alkoxide, and provide direct evidence for ground state destabilization. Comparisons of X-ray crystal structures of AP with and without Ser102 reveal the same active site and P i binding geometry upon removal of Ser102, suggesting that the destabilization does not result from a major structural rearrangement upon mutation of Ser102. Analogous P i binding measurements with a protein tyrosine phosphatase suggest the generality of this ground state destabilization mechanism. Our results have uncovered an important contribution of anionic nucleophiles to phosphoryl transfer catalysis via ground state electrostatic destabilization and an enormous capacity of the AP active site for specific and strong recognition of the phosphoryl group in the transition state.

Beschreibung: by Richard Robinson

Beschreibung: by Yasunobu Mano, Tetsuya J. Kobayashi, Jun-ichi Nakayama, Hiroyuki Uchida, Masaya Oki Differences in gene expression between individual cells can be mediated by epigenetic regulation; thus, methods that enable detailed analyses of single cells are crucial to understanding this phenomenon. In this study, genomic silencing regions of Saccharomyces cerevisiae that are subject to epigenetic regulation, including the HMR , HML , and telomere regions, were investigated using a newly developed single cell analysis method. This method uses fluorescently labeled proteins to track changes in gene expression over multiple generations of a single cell. Epigenetic control of gene expression differed depending on the specific silencing region at which the reporter gene was inserted. Correlations between gene expression at the HMR -left and HMR -right regions, as well as the HMR -right and HML -right regions, were observed in the single-cell level; however, no such correlations involving the telomere region were observed. Deletion of the histone acetyltransferase GCN5 gene from a yeast strain carrying a fluorescent reporter gene at the HMR -left region reduced the frequency of changes in gene expression over a generation. The results presented here suggest that epigenetic control within an individual cell is reversible and can be achieved via regulation of histone acetyltransferase activity.

Beschreibung: by Todd A. Gray, Janet A. Krywy, Jessica Harold, Michael J. Palumbo, Keith M. Derbyshire Horizontal gene transfer (HGT) in bacteria generates variation and drives evolution, and conjugation is considered a major contributor as it can mediate transfer of large segments of DNA between strains and species. We previously described a novel form of chromosomal conjugation in mycobacteria that does not conform to classic oriT -based conjugation models, and whose potential evolutionary significance has not been evaluated. Here, we determined the genome sequences of 22 F1-generation transconjugants, providing the first genome-wide view of conjugal HGT in bacteria at the nucleotide level. Remarkably, mycobacterial recipients acquired multiple, large, unlinked segments of donor DNA, far exceeding expectations for any bacterial HGT event. Consequently, conjugal DNA transfer created extensive genome-wide mosaicism within individual transconjugants, which generated large-scale sibling diversity approaching that seen in meiotic recombination. We exploited these attributes to perform genome-wide mapping and introgression analyses to map a locus that determines conjugal mating identity in M. smegmatis . Distributive conjugal transfer offers a plausible mechanism for the predicted HGT events that created the genome mosaicism observed among extant Mycobacterium tuberculosis and Mycobacterium canettii species. Mycobacterial distributive conjugal transfer permits innovative genetic approaches to map phenotypic traits and confers the evolutionary benefits of sexual reproduction in an asexual organism.

Beschreibung: by Kazuhiko Higashida, Sang Hyun Kim, Su Ryun Jung, Meiko Asaka, John O. Holloszy, Dong-Ho Han It has been reported that feeding mice resveratrol activates AMPK and SIRT1 in skeletal muscle leading to deacetylation and activation of PGC-1α, increased mitochondrial biogenesis, and improved running endurance. This study was done to further evaluate the effects of resveratrol, SIRT1, and PGC-1α deacetylation on mitochondrial biogenesis in muscle. Feeding rats or mice a diet containing 4 g resveratrol/kg diet had no effect on mitochondrial protein levels in muscle. High concentrations of resveratrol lowered ATP concentration and activated AMPK in C 2 C 12 myotubes, resulting in an increase in mitochondrial proteins. Knockdown of SIRT1, or suppression of SIRT1 activity with a dominant-negative (DN) SIRT1 construct, increased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C 2 C 12 cells. Expression of a DN SIRT1 in rat triceps muscle also induced an increase in mitochondrial proteins. Overexpression of SIRT1 decreased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C 2 C 12 myotubes. Overexpression of SIRT1 also resulted in a decrease in mitochondrial proteins in rat triceps muscle. We conclude that, contrary to some previous reports, the mechanism by which SIRT1 regulates mitochondrial biogenesis is by inhibiting PGC-1α coactivator activity, resulting in a decrease in mitochondria. We also conclude that feeding rodents resveratrol has no effect on mitochondrial biogenesis in muscle.