Chiral blastomere arrangement dictates zygotic left–right asymmetry pathway in snails

Nature advance online publication 25 November 2009. doi:10.1038/nature08597

Authors: Reiko Kuroda, Bunshiro Endo, Masanori Abe & Miho Shimizu

Most animals display internal and/or external left–right asymmetry. Several mechanisms for left–right asymmetry determination have been proposed for vertebrates and invertebrates but they are still not well characterized, particularly at the early developmental stage. The gastropods Lymnaeastagnalis and the closely related Lymnaea peregra have both the sinistral (recessive) and the dextral (dominant) snails within a species and the chirality is hereditary, determined by a single locus that functions maternally. Intriguingly, the handedness-determining gene(s) and the mechanisms are not yet identified. Here we show that in L. stagnalis, the chiral blastomere arrangement at the eight-cell stage (but not the two- or four-cell stage) determines the left–right asymmetry throughout the developmental programme, and acts upstream of the Nodal signalling pathway. Thus, we could demonstrate that mechanical micromanipulation of the third cleavage chirality (from the four- to the eight-cell stage) leads to reversal of embryonic handedness. These manipulated embryos grew to ‘dextralized’ sinistral and ‘sinistralized’ dextral snails—that is, normal healthy fertile organisms with all the usual left–right asymmetries reversed to that encoded by the mothers’ genetic information. Moreover, manipulation reversed the embryonic nodal expression patterns. Using backcrossed F7 congenic animals, we could demonstrate a strong genetic linkage between the handedness-determining gene(s) and the chiral cytoskeletal dynamics at the third cleavage that promotes the dominant-type blastomere arrangement. These results establish the crucial importance of the maternally determined blastomere arrangement at the eight-cell stage in dictating zygotic signalling pathways in the organismal chiromorphogenesis. Similar chiral blastomere configuration mechanisms may also operate upstream of the Nodal pathway in left–right patterning of deuterostomes/vertebrates.

Nature advance online publication 25 November 2009. doi:10.1038/nature08620

Authors: Natasha Yeung, Ying-Wu Lin, Yi-Gui Gao, Xuan Zhao, Brandy S. Russell, Lanyu Lei, Kyle D. Miner, Howard Robinson & Yi Lu

Protein design provides a rigorous test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. Whereas progress has been made in designing proteins that mimic native proteins structurally, it is more difficult to design functional proteins. In comparison to recent successes in designing non-metalloproteins, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes. This is because protein metal-binding sites are much more varied than non-metal-containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal-binding site properties in silico, as many of the parameters, such as force fields, are ill-defined. Therefore, the successful design of a structural and functional metalloprotein would greatly advance the field of protein design and our understanding of enzymes. Here we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a haem/non-haem FeB centre that is remarkably similar to that in the crystal structure. This designed protein also exhibits NO reduction activity, and so models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.

Nature advance online publication 22 November 2009. doi:10.1038/nature08578

Authors: M. Tavani, A. Bulgarelli, G. Piano, S. Sabatini, E. Striani, Y. Evangelista, A. Trois, G. Pooley, S. Trushkin, N. A. Nizhelskij, M. McCollough, K. I. I. Koljonen, G. Pucella, A. Giuliani, A. W. Chen, E. Costa, V. Vittorini, M. Trifoglio, F. Gianotti, A. Argan, G. Barbiellini, P. Caraveo, P. W. Cattaneo, V. Cocco, T. Contessi, F. D’Ammando, E. Del Monte, G. De Paris, G. Di Cocco, G. Di Persio, I. Donnarumma, M. Feroci, A. Ferrari, F. Fuschino, M. Galli, C. Labanti, I. Lapshov, F. Lazzarotto, P. Lipari, F. Longo, E. Mattaini, M. Marisaldi, M. Mastropietro, A. Mauri, S. Mereghetti, E. Morelli, A. Morselli, L. Pacciani, A. Pellizzoni, F. Perotti, P. Picozza, M. Pilia, M. Prest, M. Rapisarda, A. Rappoldi, E. Rossi, A. Rubini, E. Scalise, P. Soffitta, E. Vallazza, S. Vercellone, A. Zambra, D. Zanello, C. Pittori, F. Verrecchia, P. Giommi, S. Colafrancesco, P. Santolamazza, A. Antonelli & L. Salotti

Super-massive black holes in active galaxies can accelerate particles to relativistic energies, producing jets with associated γ-ray emission. Galactic ‘microquasars’, which are binary systems consisting of a neutron star or stellar-mass black hole accreting gas from a companion star, also produce relativistic jets, generally together with radio flares. Apart from an isolated event detected in Cygnus X-1, there has hitherto been no systematic evidence for the acceleration of particles to gigaelectronvolt or higher energies in a microquasar, with the consequence that we are as yet unsure about the mechanism of jet energization. Here we report four γ-ray flares with energies above 100 MeV from the microquasar Cygnus X-3 (an exceptional X-ray binary that sporadically produces radio jets). There is a clear pattern of temporal correlations between the γ-ray flares and transitional spectral states of the radio-frequency and X-ray emission. Particle acceleration occurred a few days before radio-jet ejections for two of the four flares, meaning that the process of jet formation implies the production of very energetic particles. In Cygnus X-3, particle energies during the flares can be thousands of times higher than during quiescent states.

Nature advance online publication 18 November 2009. doi:10.1038/nature08599

Authors: Hiroaki Fujii, Viswanathan Chinnusamy, Americo Rodrigues, Silvia Rubio, Regina Antoni, Sang-Youl Park, Sean R. Cutler, Jen Sheen, Pedro L. Rodriguez & Jian-Kang Zhu

The phytohormone abscisic acid (ABA) regulates the expression of many genes in plants; it has critical functions in stress resistance and in growth and development. Several proteins have been reported to function as ABA receptors, and many more are known to be involved in ABA signalling. However, the identities of ABA receptors remain controversial and the mechanism of signalling from perception to downstream gene expression is unclear. Here we show that by combining the recently identified ABA receptor PYR1 with the type 2C protein phosphatase (PP2C) ABI1, the serine/threonine protein kinase SnRK2.6/OST1 and the transcription factor ABF2/AREB1, we can reconstitute ABA-triggered phosphorylation of the transcription factor in vitro. Introduction of these four components into plant protoplasts results in ABA-responsive gene expression. Protoplast and test-tube reconstitution assays were used to test the function of various members of the receptor, protein phosphatase and kinase families. Our results suggest that the default state of the SnRK2 kinases is an autophosphorylated, active state and that the SnRK2 kinases are kept inactive by the PP2Cs through physical interaction and dephosphorylation. We found that in the presence of ABA, the PYR/PYL (pyrabactin resistance 1/PYR1-like) receptor proteins can disrupt the interaction between the SnRK2s and PP2Cs, thus preventing the PP2C-mediated dephosphorylation of the SnRK2s and resulting in the activation of the SnRK2 kinases. Our results reveal new insights into ABA signalling mechanisms and define a minimal set of core components of a complete major ABA signalling pathway.

Nature advance online publication 18 November 2009. doi:10.1038/nature08611

Authors: Manjula Pandey, Salman Syed, Ilker Donmez, Gayatri Patel, Taekjip Ha & Smita S. Patel

Genomic DNA is replicated by two DNA polymerase molecules, one of which works in close association with the helicase to copy the leading-strand template in a continuous manner while the second copies the already unwound lagging-strand template in a discontinuous manner through the synthesis of Okazaki fragments. Considering that the lagging-strand polymerase has to recycle after the completion of every Okazaki fragment through the slow steps of primer synthesis and hand-off to the polymerase, it is not understood how the two strands are synthesized with the same net rate. Here we show, using the T7 replication proteins, that RNA primers are made ‘on the fly’ during ongoing DNA synthesis and that the leading-strand T7 replisome does not pause during primer synthesis, contrary to previous reports. Instead, the leading-strand polymerase remains limited by the speed of the helicase; it therefore synthesizes DNA more slowly than the lagging-strand polymerase. We show that the primase–helicase T7 gp4 maintains contact with the priming sequence during ongoing DNA synthesis; the nascent lagging-strand template therefore organizes into a priming loop that keeps the primer in physical proximity to the replication complex. Our findings provide three synergistic mechanisms of coordination: first, primers are made concomitantly with DNA synthesis; second, the priming loop ensures efficient primer use and hand-off to the polymerase; and third, the lagging-strand polymerase copies DNA faster, which allows it to keep up with leading-strand DNA synthesis overall.

Nature advance online publication 15 November 2009. doi:10.1038/nature08505

Authors: Rebecca P. Seal, Xidao Wang, Yun Guan, Srinivasa N. Raja, C. Jeffery Woodbury, Allan I. Basbaum & Robert H. Edwards

Mechanical pain contributes to the morbidity associated with inflammation and trauma, but primary sensory neurons that convey the sensation of acute and persistent mechanical pain have not been identified. Dorsal root ganglion (DRG) neurons transmit sensory information to the spinal cord using the excitatory transmitter glutamate, a process that depends on glutamate transport into synaptic vesicles for regulated exocytotic release. Here we report that a small subset of cells in the DRG expresses the low abundance vesicular glutamate transporter VGLUT3 (also known as SLC17A8). In the dorsal horn of the spinal cord, these afferents project to lamina I and the innermost layer of lamina II, which has previously been implicated in persistent pain caused by injury. Because the different VGLUT isoforms generally have a non-redundant pattern of expression, we used Vglut3 knockout mice to assess the role of VGLUT3+ primary afferents in the behavioural response to somatosensory input. The loss of VGLUT3 specifically impairs mechanical pain sensation, and in particular the mechanical hypersensitivity to normally innocuous stimuli that accompanies inflammation, nerve injury and trauma. Direct recording from VGLUT3+ neurons in the DRG further identifies them as a poorly understood population of unmyelinated, low threshold mechanoreceptors (C-LTMRs). The analysis of Vglut3-/- mice now indicates a critical role for C-LTMRs in the mechanical hypersensitivity caused by injury.

Nature advance online publication 15 November 2009. doi:10.1038/nature08584

Authors: Gustavo S. Wiederhecker, Long Chen, Alexander Gondarenko & Michal Lipson

The use of optical forces to manipulate small objects is well known. Applications include the manipulation of living cells by optical tweezers and optical cooling in atomic physics. The miniaturization of optical systems (to the micro and nanoscale) has resulted in very compliant systems with masses of the order of nanograms, rendering them susceptible to optical forces. Optical forces have been exploited to demonstrate chaotic quivering of microcavities, optical cooling of mechanical modes, actuation of a tapered-fibre waveguide and excitation of the mechanical modes of silicon nano-beams. Despite recent progress in this field, it is challenging to manipulate the optical response of photonic structures using optical forces; this is because of the large forces that are required to induce appreciable changes in the geometry of the structure. Here we implement a resonant structure whose optical response can be efficiently statically controlled using relatively weak attractive and repulsive optical forces. We demonstrate a static mechanical deformation of up to 20 nanometres in a silicon nitride structure, using three milliwatts of continuous optical power. Because of the sensitivity of the optical response to this deformation, such optically induced static displacement introduces resonance shifts spanning 80 times the intrinsic resonance linewidth.

Nature advance online publication 08 November 2009. doi:10.1038/nature08591

Authors: Julia Santiago, Florine Dupeux, Adam Round, Regina Antoni, Sang-Youl Park, Marc Jamin, Sean R. Cutler, Pedro Luis Rodriguez & José Antonio Márquez

The plant hormone abscisic acid (ABA) has a central role in coordinating the adaptive response in situations of decreased water availability as well as the regulation of plant growth and development. Recently, a 14-member family of intracellular ABA receptors, named PYR/PYL/RCAR, has been identified. These proteins inhibit in an ABA-dependent manner the activity of a family of key negative regulators of the ABA signalling pathway: the group-A protein phosphatases type 2C (PP2Cs). Here we present the crystal structure of Arabidopsis thaliana PYR1, which consists of a dimer in which one of the subunits is bound to ABA. In the ligand-bound subunit, the loops surrounding the entry to the binding cavity fold over the ABA molecule, enclosing it inside, whereas in the empty subunit they form a channel leaving an open access to the cavity, indicating that conformational changes in these loops have a critical role in the stabilization of the hormone–receptor complex. By providing structural details on the ABA-binding pocket, this work paves the way for the development of new small molecules able to activate the plant stress response.

Nature advance online publication 01 November 2009. doi:10.1038/nature08557

Authors:

Although Galactic cosmic rays (protons and nuclei) are widely believed to be mainly accelerated by the winds and supernovae of massive stars, definitive evidence of this origin remains elusive nearly a century after their discovery. The active regions of starburst galaxies have exceptionally high rates of star formation, and their large size—more than 50 times the diameter of similar Galactic regions—uniquely enables reliable calorimetric measurements of their potentially high cosmic-ray density. The cosmic rays produced in the formation, life and death of massive stars in these regions are expected to produce diffuse γ-ray emission through interactions with interstellar gas and radiation. M82, the prototype small starburst galaxy, is predicted to be the brightest starburst galaxy in terms of γ-ray emission. Here we report the detection of >700-GeV γ-rays from M82. From these data we determine a cosmic-ray density of 250 eV cm-3 in the starburst core, which is about 500 times the average Galactic density. This links cosmic-ray acceleration to star formation activity, and suggests that supernovae and massive-star winds are the dominant accelerators.