PLoS Biology: New Articles

Multi-Cellular Rosettes in the Mouse Visceral Endoderm Facilitate the Ordered Migration of Anterior Visceral Endoderm Cells

2012-02-07T22:00:00Z

by Georgios Trichas, Aaron M. Smith, Natalia White, Vivienne Wilkins, Tomoko Watanabe, Abigail Moore, Bradley Joyce, Jacintha Sugnaseelan, Tristan A. Rodriguez, David Kay, Ruth E. Baker, Philip K. Maini, Shankar Srinivas

The visceral endoderm (VE) is a simple epithelium that forms the outer layer of the egg-cylinder stage mouse embryo. The anterior visceral endoderm (AVE), a specialised subset of VE cells, is responsible for specifying anterior pattern. AVE cells show a stereotypic migratory behaviour within the VE, which is responsible for correctly orientating the anterior-posterior axis. The epithelial integrity of the VE is maintained during the course of AVE migration, which takes place by intercalation of AVE and other VE cells. Though a continuous epithelial sheet, the VE is characterised by two regions of dramatically different behaviour, one showing robust cell movement and intercalation (in which the AVE migrates) and one that is static, with relatively little cell movement and mixing. Little is known about the cellular rearrangements that accommodate and influence the sustained directional movement of subsets of cells (such as the AVE) within epithelia like the VE. This study uses an interdisciplinary approach to further our understanding of cell movement in epithelia. Using both wild-type embryos as well as mutants in which AVE migration is abnormal or arrested, we show that AVE migration is specifically linked to changes in cell packing in the VE and an increase in multi-cellular rosette arrangements (five or more cells meeting at a point). To probe the role of rosettes during AVE migration, we develop a mathematical model of cell movement in the VE. To do this, we use a vertex-based model, implemented on an ellipsoidal surface to represent a realistic geometry for the mouse egg-cylinder. The potential for rosette formation is included, along with various junctional rearrangements. Simulations suggest that while rosettes are not essential for AVE migration, they are crucial for the orderliness of this migration observed in embryos. Our simulations are similar to results from transgenic embryos in which Planar Cell Polarity (PCP) signalling is disrupted. Such embryos have significantly reduced rosette numbers, altered epithelial packing, and show abnormalities in AVE migration. Our results show that the formation of multi-cellular rosettes in the mouse VE is dependent on normal PCP signalling. Taken together, our model and experimental observations suggest that rosettes in the VE epithelium do not form passively in response to AVE migration. Instead, they are a PCP-dependent arrangement of cells that acts to buffer the disequilibrium in cell packing generated in the VE by AVE migration, enabling AVE cells to migrate in an orderly manner.

Genome-Wide Analysis of the World's Sheep Breeds Reveals High Levels of Historic Mixture and Strong Recent Selection

2012-02-07T22:00:00Z

by James W. Kijas, Johannes A. Lenstra, Ben Hayes, Simon Boitard, Laercio R. Porto Neto, Magali San Cristobal, Bertrand Servin, Russell McCulloch, Vicki Whan, Kimberly Gietzen, Samuel Paiva, William Barendse, Elena Ciani, Herman Raadsma, John McEwan, Brian Dalrymple, other members of the International Sheep Genomics Consortium

Through their domestication and subsequent selection, sheep have been adapted to thrive in a diverse range of environments. To characterise the genetic consequence of both domestication and selection, we genotyped 49,034 SNP in 2,819 animals from a diverse collection of 74 sheep breeds. We find the majority of sheep populations contain high SNP diversity and have retained an effective population size much higher than most cattle or dog breeds, suggesting domestication occurred from a broad genetic base. Extensive haplotype sharing and generally low divergence time between breeds reveal frequent genetic exchange has occurred during the development of modern breeds. A scan of the genome for selection signals revealed 31 regions containing genes for coat pigmentation, skeletal morphology, body size, growth, and reproduction. We demonstrate the strongest selection signal has occurred in response to breeding for the absence of horns. The high density map of genetic variability provides an in-depth view of the genetic history for this important livestock species.

Development and Function of Invariant Natural Killer T Cells Producing TH2- and TH17-Cytokines

2012-02-07T22:00:00Z

by Hiroshi Watarai, Etsuko Sekine-Kondo, Tomokuni Shigeura, Yasutaka Motomura, Takuwa Yasuda, Rumi Satoh, Hisahiro Yoshida, Masato Kubo, Hiroshi Kawamoto, Haruhiko Koseki, Masaru Taniguchi

There is heterogeneity in invariant natural killer T (iNKT) cells based on the expression of CD4 and the IL-17 receptor B (IL-17RB), a receptor for IL-25 which is a key factor in T_H2 immunity. However, the development pathway and precise function of these iNKT cell subtypes remain unknown. IL-17RB⁺ iNKT cells are present in the thymic CD44^+/− NK1.1⁻ population and develop normally even in the absence of IL-15, which is required for maturation and homeostasis of IL-17RB⁻ iNKT cells producing IFN-γ. These results suggest that iNKT cells contain at least two subtypes, IL-17RB⁺ and IL-17RB⁻ subsets. The IL-17RB⁺ iNKT subtypes can be further divided into two subtypes on the basis of CD4 expression both in the thymus and in the periphery. CD4⁺ IL-17RB⁺ iNKT cells produce T_H2 (IL-13), T_H9 (IL-9 and IL-10), and T_H17 (IL-17A and IL-22) cytokines in response to IL-25 in an E4BP4-dependent fashion, whereas CD4⁻ IL-17RB⁺ iNKT cells are a retinoic acid receptor-related orphan receptor (ROR)γt⁺ subset producing T_H17 cytokines upon stimulation with IL-23 in an E4BP4-independent fashion. These IL-17RB⁺ iNKT cell subtypes are abundantly present in the lung in the steady state and mediate the pathogenesis in virus-induced airway hyperreactivity (AHR). In this study we demonstrated that the IL-17RB⁺ iNKT cell subsets develop distinct from classical iNKT cell developmental stages in the thymus and play important roles in the pathogenesis of airway diseases.

Desperately Seeking Stable 50-Year-Old Landscapes with Patches and Long, Wide Corridors

2012-01-31T22:00:00Z

by Paul Beier, Andrew J. Gregory

Built-in Timer Delays Differentiation

2012-01-31T22:00:00Z

by Liza Gross

Reconstructing Speech from Human Auditory Cortex

2012-01-31T22:00:00Z

by Brian N. Pasley, Stephen V. David, Nima Mesgarani, Adeen Flinker, Shihab A. Shamma, Nathan E. Crone, Robert T. Knight, Edward F. Chang

How the human auditory system extracts perceptually relevant acoustic features of speech is unknown. To address this question, we used intracranial recordings from nonprimary auditory cortex in the human superior temporal gyrus to determine what acoustic information in speech sounds can be reconstructed from population neural activity. We found that slow and intermediate temporal fluctuations, such as those corresponding to syllable rate, were accurately reconstructed using a linear model based on the auditory spectrogram. However, reconstruction of fast temporal fluctuations, such as syllable onsets and offsets, required a nonlinear sound representation based on temporal modulation energy. Reconstruction accuracy was highest within the range of spectro-temporal fluctuations that have been found to be critical for speech intelligibility. The decoded speech representations allowed readout and identification of individual words directly from brain activity during single trial sound presentations. These findings reveal neural encoding mechanisms of speech acoustic parameters in higher order human auditory cortex.

Pulsed Feedback Defers Cellular Differentiation

2012-01-31T22:00:00Z

by Joe H. Levine, Michelle E. Fontes, Jonathan Dworkin, Michael B. Elowitz

Environmental signals induce diverse cellular differentiation programs. In certain systems, cells defer differentiation for extended time periods after the signal appears, proliferating through multiple rounds of cell division before committing to a new fate. How can cells set a deferral time much longer than the cell cycle? Here we study Bacillus subtilis cells that respond to sudden nutrient limitation with multiple rounds of growth and division before differentiating into spores. A well-characterized genetic circuit controls the concentration and phosphorylation of the master regulator Spo0A, which rises to a critical concentration to initiate sporulation. However, it remains unclear how this circuit enables cells to defer sporulation for multiple cell cycles. Using quantitative time-lapse fluorescence microscopy of Spo0A dynamics in individual cells, we observed pulses of Spo0A phosphorylation at a characteristic cell cycle phase. Pulse amplitudes grew systematically and cell-autonomously over multiple cell cycles leading up to sporulation. This pulse growth required a key positive feedback loop involving the sporulation kinases, without which the deferral of sporulation became ultrasensitive to kinase expression. Thus, deferral is controlled by a pulsed positive feedback loop in which kinase expression is activated by pulses of Spo0A phosphorylation. This pulsed positive feedback architecture provides a more robust mechanism for setting deferral times than constitutive kinase expression. Finally, using mathematical modeling, we show how pulsing and time delays together enable “polyphasic” positive feedback, in which different parts of a feedback loop are active at different times. Polyphasic feedback can enable more accurate tuning of long deferral times. Together, these results suggest that Bacillus subtilis uses a pulsed positive feedback loop to implement a “timer” that operates over timescales much longer than a cell cycle.

Stochastic Expression of the Interferon-β Gene

2012-01-24T22:00:00Z

by Mingwei Zhao, Jiangwen Zhang, Hemali Phatnani, Stefanie Scheu, Tom Maniatis

Virus infection of mammalian cells induces the production of high levels of type I interferons (IFNα and β), cytokines that orchestrate antiviral innate and adaptive immunity. Previous studies have shown that only a fraction of the infected cells produce IFN. However, the mechanisms responsible for this stochastic expression are poorly understood. Here we report an in depth analysis of IFN-expressing and non-expressing mouse cells infected with Sendai virus. Mouse embryonic fibroblasts in which an internal ribosome entry site/yellow fluorescent protein gene was inserted downstream from the endogenous IFNβ gene were used to distinguish between the two cell types, and they were isolated from each other using fluorescence-activated cell sorting methods. Analysis of the separated cells revealed that stochastic IFNβ expression is a consequence of cell-to-cell variability in the levels and/or activities of limiting components at every level of the virus induction process, ranging from viral replication and expression, to the sensing of viral RNA by host factors, to activation of the signaling pathway, to the levels of activated transcription factors. We propose that this highly complex stochastic IFNβ gene expression evolved to optimize both the level and distribution of type I IFNs in response to virus infection.

Structural and Functional Loss in Restored Wetland Ecosystems

2012-01-24T22:00:00Z

by David Moreno-Mateos, Mary E. Power, Francisco A. Comín, Roxana Yockteng

Wetlands are among the most productive and economically valuable ecosystems in the world. However, because of human activities, over half of the wetland ecosystems existing in North America, Europe, Australia, and China in the early 20th century have been lost. Ecological restoration to recover critical ecosystem services has been widely attempted, but the degree of actual recovery of ecosystem functioning and structure from these efforts remains uncertain. Our results from a meta-analysis of 621 wetland sites from throughout the world show that even a century after restoration efforts, biological structure (driven mostly by plant assemblages), and biogeochemical functioning (driven primarily by the storage of carbon in wetland soils), remained on average 26% and 23% lower, respectively, than in reference sites. Either recovery has been very slow, or postdisturbance systems have moved towards alternative states that differ from reference conditions. We also found significant effects of environmental settings on the rate and degree of recovery. Large wetland areas (>100 ha) and wetlands restored in warm (temperate and tropical) climates recovered more rapidly than smaller wetlands and wetlands restored in cold climates. Also, wetlands experiencing more (riverine and tidal) hydrologic exchange recovered more rapidly than depressional wetlands. Restoration performance is limited: current restoration practice fails to recover original levels of wetland ecosystem functions, even after many decades. If restoration as currently practiced is used to justify further degradation, global loss of wetland ecosystem function and structure will spread.

Restoration of Ailing Wetlands

2012-01-24T22:00:00Z

by Oswald J. Schmitz

It is widely held that humankind's destructive tendencies when exploiting natural resources leads to irreparable harm to the environment. Yet, this thinking runs counter to evidence that many ecological systems damaged by severe natural environmental disturbances (e.g., hurricanes) can restore themselves via processes of natural recovery. The emerging field of restoration ecology is capitalizing on the natural restorative tendencies of ecological systems to build a science of repairing the harm inflicted by humans on natural environment. Evidence for this, for example, comes from a new meta-analysis of 124 studies that synthesizes recovery of impacted wetlands worldwide. While it may take up to two human generations to see full recovery, there is promise, given human will, to restore many damaged wetlands worldwide.

The Chromosomal Passenger Complex Activates Polo Kinase at Centromeres

2012-01-24T22:00:00Z

by Mar Carmena, Xavier Pinson, Melpi Platani, Zeina Salloum, Zhenjie Xu, Anthony Clark, Fiona MacIsaac, Hiromi Ogawa, Ulrike Eggert, David M. Glover, Vincent Archambault, William C. Earnshaw

The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules) requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. Our study concerns the coordination of the activities of these two kinases in vivo. We show that INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC), which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. We show here that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity) and is critical for Polo function at kinetochores. Our results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation.

Substrate Specificity within a Family of Outer Membrane Carboxylate Channels

2012-01-17T22:00:00Z

by Elif Eren, Jagamya Vijayaraghavan, Jiaming Liu, Belete R. Cheneke, Debra S. Touw, Bryan W. Lepore, Mridhu Indic, Liviu Movileanu, Bert van den Berg

Many Gram-negative bacteria, including human pathogens such as Pseudomonas aeruginosa, do not have large-channel porins. This results in an outer membrane (OM) that is highly impermeable to small polar molecules, making the bacteria intrinsically resistant towards many antibiotics. In such microorganisms, the majority of small molecules are taken up by members of the OprD outer membrane protein family. Here we show that OprD channels require a carboxyl group in the substrate for efficient transport, and based on this we have renamed the family Occ, for outer membrane carboxylate channels. We further show that Occ channels can be divided into two subfamilies, based on their very different substrate specificities. Our results rationalize how certain bacteria can efficiently take up a variety of substrates under nutrient-poor conditions without compromising membrane permeability. In addition, they explain how channel inactivation in response to antibiotics can cause resistance but does not lead to decreased fitness.

Sequential Analysis of Trans-SNARE Formation in Intracellular Membrane Fusion

2012-01-17T22:00:00Z

by Kannan Alpadi, Aditya Kulkarni, Veronique Comte, Monique Reinhardt, Andrea Schmidt, Sarita Namjoshi, Andreas Mayer, Christopher Peters

SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Q_a, Q_b, and Q_c) from target (t)-SNAREs and one (R) from the vesicular (v)-SNARE. NSF/Sec18 disrupts these cis-SNARE complexes, allowing reassembly of their subunits into trans-SNARE complexes and subsequent fusion. Studying these reactions in native yeast vacuoles, we found that NSF/Sec18 activates the vacuolar cis-SNARE complex by selectively displacing the vacuolar Q_a SNARE, leaving behind a Q_bcR subcomplex. This subcomplex serves as an acceptor for a Q_a SNARE from the opposite membrane, leading to Q_a-Q_bcR trans-complexes. Activity tests of vacuoles with diagnostic distributions of inactivating mutations over the two fusion partners confirm that this distribution accounts for a major share of the fusion activity. The persistence of the Q_bcR cis-complex and the formation of the Q_a-Q_bcR trans-complex are both sensitive to the Rab-GTPase inhibitor, GDI, and to mutations in the vacuolar tether complex, HOPS (HOmotypic fusion and vacuolar Protein Sorting complex). This suggests that the vacuolar Rab-GTPase, Ypt7, and HOPS restrict cis-SNARE disassembly and thereby bias trans-SNARE assembly into a preferred topology.

Rapid Evolution of Enormous, Multichromosomal Genomes in Flowering Plant Mitochondria with Exceptionally High Mutation Rates

2012-01-17T22:00:00Z

by Daniel B. Sloan, Andrew J. Alverson, John P. Chuckalovcak, Martin Wu, David E. McCauley, Jeffrey D. Palmer, Douglas R. Taylor

Genome size and complexity vary tremendously among eukaryotic species and their organelles. Comparisons across deeply divergent eukaryotic lineages have suggested that variation in mutation rates may explain this diversity, with increased mutational burdens favoring reduced genome size and complexity. The discovery that mitochondrial mutation rates can differ by orders of magnitude among closely related angiosperm species presents a unique opportunity to test this hypothesis. We sequenced the mitochondrial genomes from two species in the angiosperm genus Silene with recent and dramatic accelerations in their mitochondrial mutation rates. Contrary to theoretical predictions, these genomes have experienced a massive proliferation of noncoding content. At 6.7 and 11.3 Mb, they are by far the largest known mitochondrial genomes, larger than most bacterial genomes and even some nuclear genomes. In contrast, two slowly evolving Silene mitochondrial genomes are smaller than average for angiosperms. Consequently, this genus captures approximately 98% of known variation in organelle genome size. The expanded genomes reveal several architectural changes, including the evolution of complex multichromosomal structures (with 59 and 128 circular-mapping chromosomes, ranging in size from 44 to 192 kb). They also exhibit a substantial reduction in recombination and gene conversion activity as measured by the relative frequency of alternative genome conformations and the level of sequence divergence between repeat copies. The evolution of mutation rate, genome size, and chromosome structure can therefore be extremely rapid and interrelated in ways not predicted by current evolutionary theories. Our results raise the hypothesis that changes in recombinational processes, including gene conversion, may be a central force driving the evolution of both mutation rate and genome structure.

Two PI 3-Kinases and One PI 3-Phosphatase Together Establish the Cyclic Waves of Phagosomal PtdIns(3)P Critical for the Degradation of Apoptotic Cells

2012-01-17T22:00:00Z

by Nan Lu, Qian Shen, Timothy R. Mahoney, Lukas J. Neukomm, Ying Wang, Zheng Zhou

Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a signaling molecule important for many membrane trafficking events, including phagosome maturation. The level of PtdIns(3)P on phagosomes oscillates in two waves during phagosome maturation. However, the physiological significance of such oscillation remains unknown. Currently, the Class III PI 3-kinase (PI3K) Vps34 is regarded as the only kinase that produces PtdIns(3)P in phagosomal membranes. We report here that, in the nematode C. elegans, the Class II PI3K PIKI-1 plays a novel and crucial role in producing phagosomal PtdIns(3)P. PIKI-1 is recruited to extending pseudopods and nascent phagosomes prior to the appearance of PtdIns(3)P in a manner dependent on the large GTPase dynamin (DYN-1). PIKI-1 and VPS-34 act in sequence to provide overlapping pools of PtdIns(3)P on phagosomes. Inactivating both piki-1 and vps-34 completely abolishes the production of phagosomal PtdIns(3)P and disables phagosomes from recruiting multiple essential maturation factors, resulting in a complete arrest of apoptotic-cell degradation. We have further identified MTM-1, a PI 3-phosphatase that antagonizes the activities of PIKI-1 and VPS-34 by down-regulating PtdIns(3)P on phagosomes. Remarkably, persistent appearance of phagosomal PtdIns(3)P, as a result of inactivating mtm-1, blocks phagosome maturation. Our findings demonstrate that the proper oscillation pattern of PtdIns(3)P on phagosomes, programmed by the coordinated activities of two PI3Ks and one PI 3-phosphatase, is critical for phagosome maturation. They further shed light on how the temporally controlled reversible phosphorylation of phosphoinositides regulates the progression of multi-step cellular events.

Rise and Fall, and Rise Again: Phagosome Maturation Is Controlled by Two Kinases and One Phosphatase

2012-01-17T22:00:00Z

by Richard Robinson

Putting the Pieces Together: Integrative Modeling Platform Software for Structure Determination of Macromolecular Assemblies

2012-01-17T22:00:00Z

by Daniel Russel, Keren Lasker, Ben Webb, Javier Velázquez-Muriel, Elina Tjioe, Dina Schneidman-Duhovny, Bret Peterson, Andrej Sali

Functional Clustering Drives Encoding Improvement in a Developing Brain Network during Awake Visual Learning

2012-01-10T22:00:00Z

by Kaspar Podgorski, Derek Dunfield, Kurt Haas

Sensory experience drives dramatic structural and functional plasticity in developing neurons. However, for single-neuron plasticity to optimally improve whole-network encoding of sensory information, changes must be coordinated between neurons to ensure a full range of stimuli is efficiently represented. Using two-photon calcium imaging to monitor evoked activity in over 100 neurons simultaneously, we investigate network-level changes in the developing Xenopus laevis tectum during visual training with motion stimuli. Training causes stimulus-specific changes in neuronal responses and interactions, resulting in improved population encoding. This plasticity is spatially structured, increasing tuning curve similarity and interactions among nearby neurons, and decreasing interactions among distant neurons. Training does not improve encoding by single clusters of similarly responding neurons, but improves encoding across clusters, indicating coordinated plasticity across the network. NMDA receptor blockade prevents coordinated plasticity, reduces clustering, and abolishes whole-network encoding improvement. We conclude that NMDA receptors support experience-dependent network self-organization, allowing efficient population coding of a diverse range of stimuli.

New Signaling Chemicals Spur Worms to Seek Company

2012-01-10T22:00:00Z

by Janelle Weaver

A Modular Library of Small Molecule Signals Regulates Social Behaviors in Caenorhabditis elegans

2012-01-10T22:00:00Z

by Jagan Srinivasan, Stephan H. von Reuss, Neelanjan Bose, Alon Zaslaver, Parag Mahanti, Margaret C. Ho, Oran G. O'Doherty, Arthur S. Edison, Paul W. Sternberg, Frank C. Schroeder

The nematode C. elegans is an important model for the study of social behaviors. Recent investigations have shown that a family of small molecule signals, the ascarosides, controls population density sensing and mating behavior. However, despite extensive studies of C. elegans aggregation behaviors, no intraspecific signals promoting attraction or aggregation of wild-type hermaphrodites have been identified. Using comparative metabolomics, we show that the known ascarosides are accompanied by a series of derivatives featuring a tryptophan-derived indole moiety. Behavioral assays demonstrate that these indole ascarosides serve as potent intraspecific attraction and aggregation signals for hermaphrodites, in contrast to ascarosides lacking the indole group, which are repulsive. Hermaphrodite attraction to indole ascarosides depends on the ASK amphid sensory neurons. Downstream of the ASK sensory neuron, the interneuron AIA is required for mediating attraction to indole ascarosides instead of the RMG interneurons, which previous studies have shown to integrate attraction and aggregation signals from ASK and other sensory neurons. The role of the RMG interneuron in mediating aggregation and attraction is thought to depend on the neuropeptide Y-like receptor NPR-1, because solitary and social C. elegans strains are distinguished by different npr-1 variants. We show that indole ascarosides promote attraction and aggregation in both solitary and social C. elegans strains. The identification of indole ascarosides as aggregation signals reveals unexpected complexity of social signaling in C. elegans, which appears to be based on a modular library of ascarosides integrating building blocks derived from lipid β-oxidation and amino-acid metabolism. Variation of modules results in strongly altered signaling content, as addition of a tryptophan-derived indole unit to repellent ascarosides produces strongly attractive indole ascarosides. Our findings show that the library of ascarosides represents a highly developed chemical language integrating different neurophysiological pathways to mediate social communication in C. elegans.

Hedgehog-Regulated Ubiquitination Controls Smoothened Trafficking and Cell Surface Expression in Drosophila

2012-01-10T22:00:00Z

by Shuang Li, Yongbin Chen, Qing Shi, Tao Yue, Bing Wang, Jin Jiang

Hedgehog transduces signal by promoting cell surface expression of the seven-transmembrane protein Smoothened (Smo) in Drosophila, but the underlying mechanism remains unknown. Here we demonstrate that Smo is downregulated by ubiquitin-mediated endocytosis and degradation, and that Hh increases Smo cell surface expression by inhibiting its ubiquitination. We find that Smo is ubiquitinated at multiple Lysine residues including those in its autoinhibitory domain (SAID), leading to endocytosis and degradation of Smo by both lysosome- and proteasome-dependent mechanisms. Hh inhibits Smo ubiquitination via PKA/CK1-mediated phosphorylation of SAID, leading to Smo cell surface accumulation. Inactivation of the ubiquitin activating enzyme Uba1 or perturbation of multiple components of the endocytic machinery leads to Smo accumulation and Hh pathway activation. In addition, we find that the non-visual β-arrestin Kurtz (Krz) interacts with Smo and acts in parallel with ubiquitination to downregulate Smo. Finally, we show that Smo ubiquitination is counteracted by the deubiquitinating enzyme UBPY/USP8. Gain and loss of UBPY lead to reciprocal changes in Smo cell surface expression. Taken together, our results suggest that ubiquitination plays a key role in the downregulation of Smo to keep Hh pathway activity off in the absence of the ligand, and that Hh-induced phosphorylation promotes Smo cell surface accumulation by inhibiting its ubiquitination, which contributes to Hh pathway activation.

USP8 Promotes Smoothened Signaling by Preventing Its Ubiquitination and Changing Its Subcellular Localization

2012-01-10T22:00:00Z

by Ruohan Xia, Hongge Jia, Junkai Fan, Yajuan Liu, Jianhang Jia

The seven transmembrane protein Smoothened (Smo) is a critical component of the Hedgehog (Hh) signaling pathway and is regulated by phosphorylation, dimerization, and cell-surface accumulation upon Hh stimulation. However, it is not clear how Hh regulates Smo accumulation on the cell surface or how Hh regulates the intracellular trafficking of Smo. In addition, little is known about whether ubiquitination is involved in Smo regulation. In this study, we demonstrate that Smo is multi-monoubiquitinated and that Smo ubiquitination is inhibited by Hh and by phosphorylation. Using an in vivo RNAi screen, we identified ubiquitin-specific protease 8 (USP8) as a deubiquitinase that down-regulates Smo ubiquitination. Inactivation of USP8 increases Smo ubiquitination and attenuates Hh-induced Smo accumulation, leading to decreased Hh signaling activity. Moreover, overexpression of USP8 prevents Smo ubiquitination and elevates Smo accumulation, leading to increased Hh signaling activity. Mechanistically, we show that Hh promotes the interaction of USP8 with Smo aa625–753, which covers the three PKA and CK1 phosphorylation clusters. Finally, USP8 promotes the accumulation of Smo at the cell surface and prevents localization to the early endosomes, presumably by deubiquitinating Smo. Our studies identify USP8 as a positive regulator in Hh signaling by down-regulating Smo ubiquitination and thereby mediating Smo intracellular trafficking.

Differentiation of the Lateral Compartment of the Cochlea Requires a Temporally Restricted FGF20 Signal

2012-01-03T22:00:00Z

by Sung-Ho Huh, Jennifer Jones, Mark E. Warchol, David M. Ornitz

A large proportion of age-related hearing loss is caused by loss or damage to outer hair cells in the organ of Corti. The organ of Corti is the mechanosensory transducing apparatus in the inner ear and is composed of inner hair cells, outer hair cells, and highly specialized supporting cells. The mechanisms that regulate differentiation of inner and outer hair cells are not known. Here we report that fibroblast growth factor 20 (FGF20) is required for differentiation of cells in the lateral cochlear compartment (outer hair and supporting cells) within the organ of Corti during a specific developmental time. In the absence of FGF20, mice are deaf and lateral compartment cells remain undifferentiated, postmitotic, and unresponsive to Notch-dependent lateral inhibition. These studies identify developmentally distinct medial (inner hair and supporting cells) and lateral compartments in the developing organ of Corti. The viability and hearing loss in Fgf20 knockout mice suggest that FGF20 may also be a deafness-associated gene in humans.

Antarctic Marine Biodiversity and Deep-Sea Hydrothermal Vents

2012-01-03T22:00:00Z

by Steven L. Chown

The diversity of many marine benthic groups is unlike that of most other taxa. Rather than declining from the tropics to the poles, much of the benthos shows high diversity in the Southern Ocean. Moreover, many species are unique to the Antarctic region. Recent work has shown that this is also true of the communities of Antarctic deep-sea hydrothermal vents. Vent ecosystems have been documented from many sites across the globe, associated with the thermally and chemically variable habitats found around these, typically high temperature, streams that are rich in reduced compounds and polymetallic sulphides. The animal communities of the East Scotia Ridge vent ecosystems are very different to those elsewhere, though the microbiota, which form the basis of vent food webs, show less differentiation. Much of the biological significance of deep-sea hydrothermal vents lies in their biodiversity, the diverse biochemistry of their bacteria, the remarkable symbioses among many of the marine animals and these bacteria, and the prospects that investigations of these systems hold for understanding the conditions that may have led to the first appearance of life. The discovery of diverse and unusual Antarctic hydrothermal vent ecosystems provides opportunities for new understanding in these fields. Moreover, the Antarctic vents south of 60°S benefit from automatic conservation under the Convention on the Conservation of Antarctic Marine Living Resources and the Antarctic Treaty. Other deep-sea hydrothermal vents located in international waters are not protected and may be threatened by growing interests in deep-sea mining.

The Discovery of New Deep-Sea Hydrothermal Vent Communities in the Southern Ocean and Implications for Biogeography

2012-01-03T22:00:00Z

by Alex D. Rogers, Paul A. Tyler, Douglas P. Connelly, Jon T. Copley, Rachael James, Robert D. Larter, Katrin Linse, Rachel A. Mills, Alfredo Naveira Garabato, Richard D. Pancost, David A. Pearce, Nicholas V. C. Polunin, Christopher R. German, Timothy Shank, Philipp H. Boersch-Supan, Belinda J. Alker, Alfred Aquilina, Sarah A. Bennett, Andrew Clarke, Robert J. J. Dinley, Alastair G. C. Graham, Darryl R. H. Green, Jeffrey A. Hawkes, Laura Hepburn, Ana Hilario, Veerle A. I. Huvenne, Leigh Marsh, Eva Ramirez-Llodra, William D. K. Reid, Christopher N. Roterman, Christopher J. Sweeting, Sven Thatje, Katrin Zwirglmaier

Since the first discovery of deep-sea hydrothermal vents along the Galápagos Rift in 1977, numerous vent sites and endemic faunal assemblages have been found along mid-ocean ridges and back-arc basins at low to mid latitudes. These discoveries have suggested the existence of separate biogeographic provinces in the Atlantic and the North West Pacific, the existence of a province including the South West Pacific and Indian Ocean, and a separation of the North East Pacific, North East Pacific Rise, and South East Pacific Rise. The Southern Ocean is known to be a region of high deep-sea species diversity and centre of origin for the global deep-sea fauna. It has also been proposed as a gateway connecting hydrothermal vents in different oceans but is little explored because of extreme conditions. Since 2009 we have explored two segments of the East Scotia Ridge (ESR) in the Southern Ocean using a remotely operated vehicle. In each segment we located deep-sea hydrothermal vents hosting high-temperature black smokers up to 382.8°C and diffuse venting. The chemosynthetic ecosystems hosted by these vents are dominated by a new yeti crab (Kiwa n. sp.), stalked barnacles, limpets, peltospiroid gastropods, anemones, and a predatory sea star. Taxa abundant in vent ecosystems in other oceans, including polychaete worms (Siboglinidae), bathymodiolid mussels, and alvinocaridid shrimps, are absent from the ESR vents. These groups, except the Siboglinidae, possess planktotrophic larvae, rare in Antarctic marine invertebrates, suggesting that the environmental conditions of the Southern Ocean may act as a dispersal filter for vent taxa. Evidence from the distinctive fauna, the unique community structure, and multivariate analyses suggest that the Antarctic vent ecosystems represent a new vent biogeographic province. However, multivariate analyses of species present at the ESR and at other deep-sea hydrothermal vents globally indicate that vent biogeography is more complex than previously recognised.

Opening Up the Politics of Knowledge and Power in Bioscience

2012-01-03T22:00:00Z

by Andy Stirling

Alternative Splicing of RNA Triplets Is Often Regulated and Accelerates Proteome Evolution

2012-01-03T22:00:00Z

by Robert K. Bradley, Jason Merkin, Nicole J. Lambert, Christopher B. Burge

Thousands of human genes contain introns ending in NAGNAG (N any nucleotide), where both NAGs can function as 3′ splice sites, yielding isoforms that differ by inclusion/exclusion of three bases. However, few models exist for how such splicing might be regulated, and some studies have concluded that NAGNAG splicing is purely stochastic and nonfunctional. Here, we used deep RNA-Seq data from 16 human and eight mouse tissues to analyze the regulation and evolution of NAGNAG splicing. Using both biological and technical replicates to estimate false discovery rates, we estimate that at least 25% of alternatively spliced NAGNAGs undergo tissue-specific regulation in mammals, and alternative splicing of strongly tissue-specific NAGNAGs was 10 times as likely to be conserved between species as was splicing of non-tissue-specific events, implying selective maintenance. Preferential use of the distal NAG was associated with distinct sequence features, including a more distal location of the branch point and presence of a pyrimidine immediately before the first NAG, and alteration of these features in a splicing reporter shifted splicing away from the distal site. Strikingly, alignments of orthologous exons revealed a ∼15-fold increase in the frequency of three base pair gaps at 3′ splice sites relative to nearby exon positions in both mammals and in Drosophila. Alternative splicing of NAGNAGs in human was associated with dramatically increased frequency of exon length changes at orthologous exon boundaries in rodents, and a model involving point mutations that create, destroy, or alter NAGNAGs can explain both the increased frequency and biased codon composition of gained/lost sequence observed at the beginnings of exons. This study shows that NAGNAG alternative splicing generates widespread differences between the proteomes of mammalian tissues, and suggests that the evolutionary trajectories of mammalian proteins are strongly biased by the locations and phases of the introns that interrupt coding sequences.

Role of Pleiotropy in the Evolution of a Cryptic Developmental Variation in Caenorhabditis elegans

2012-01-03T22:00:00Z

by Fabien Duveau, Marie-Anne Félix

Robust biological systems are expected to accumulate cryptic genetic variation that does not affect the system output in standard conditions yet may play an evolutionary role once phenotypically expressed under a strong perturbation. Genetic variation that is cryptic relative to a robust trait may accumulate neutrally as it does not change the phenotype, yet it could also evolve under selection if it affects traits related to fitness in addition to its cryptic effect. Cryptic variation affecting the vulval intercellular signaling network was previously uncovered among wild isolates of Caenorhabditis elegans. Using a quantitative genetic approach, we identify a non-synonymous polymorphism of the previously uncharacterized nath-10 gene that affects the vulval phenotype when the system is sensitized with different mutations, but not in wild-type strains. nath-10 is an essential protein acetyltransferase gene and the homolog of human NAT10. The nath-10 polymorphism also presents non-cryptic effects on life history traits. The nath-10 allele carried by the N2 reference strain leads to a subtle increase in the egg laying rate and in the total number of sperm, a trait affecting the trade-off between fertility and minimal generation time in hermaphrodite individuals. We show that this allele appeared during early laboratory culture of N2, which allowed us to test whether it may have evolved under selection in this novel environment. The derived allele indeed strongly outcompetes the ancestral allele in laboratory conditions. In conclusion, we identified the molecular nature of a cryptic genetic variation and characterized its evolutionary history. These results show that cryptic genetic variation does not necessarily accumulate neutrally at the whole-organism level, but may evolve through selection for pleiotropic effects that alter fitness. In addition, cultivation in the laboratory has led to adaptive evolution of the reference strain N2 to the laboratory environment, which may modify other phenotypes of interest.

Mitotic Spindle Assembly around RCC1-Coated Beads in Xenopus Egg Extracts

2011-12-27T22:00:00Z

by David Halpin, Petr Kalab, Jay Wang, Karsten Weis, Rebecca Heald

During cell division the genetic material on chromosomes is distributed to daughter cells by a dynamic microtubule structure called the mitotic spindle. Here we establish a reconstitution system to assess the contribution of individual chromosome proteins to mitotic spindle formation around single 10 µm diameter porous glass beads in Xenopus egg extracts. We find that Regulator of Chromosome Condensation 1 (RCC1), the Guanine Nucleotide Exchange Factor (GEF) for the small GTPase Ran, can induce bipolar spindle formation. Remarkably, RCC1 beads oscillate within spindles from pole to pole, a behavior that could be converted to a more typical, stable association by the addition of a kinesin together with RCC1. These results identify two activities sufficient to mimic chromatin-mediated spindle assembly, and establish a foundation for future experiments to reconstitute spindle assembly entirely from purified components.

How the Brain Homes in on Valuable Objects

2011-12-27T22:00:00Z

by Janelle Weaver