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Microbial metalloproteomes are largely uncharacterized

Sun, 07/18/2010 - 00:00

Microbial metalloproteomes are largely uncharacterized

Nature advance online publication 18 July 2010. doi:10.1038/nature09265

Authors: Aleksandar Cvetkovic, Angeli Lal Menon, Michael P. Thorgersen, Joseph W. Scott, Farris L. Poole II, Francis E. Jenney Jr, W. Andrew Lancaster, Jeremy L. Praissman, Saratchandra Shanmukh, Brian J. Vaccaro, Sunia A. Trauger, Ewa Kalisiak, Junefredo V. Apon, Gary Siuzdak, Steven M. Yannone, John A. Tainer & Michael W. W. Adams

Metal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability. Consequently, metalloproteins have key roles in most biological processes, including respiration (iron and copper), photosynthesis (manganese) and drug metabolism (iron). Yet, predicting from genome sequence the numbers and types of metal an organism assimilates from its environment or uses in its metalloproteome is currently impossible because metal coordination sites are diverse and poorly recognized. We present here a robust, metal-based approach to determine all metals an organism assimilates and identify its metalloproteins on a genome-wide scale. This shifts the focus from classical protein-based purification to metal-based identification and purification by liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) to characterize cytoplasmic metalloproteins from an exemplary microorganism (Pyrococcus furiosus). Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used (cobalt, iron, nickel, tungsten and zinc; 83 peaks) plus metals the organism was not thought to assimilate (lead, manganese, molybdenum, uranium and vanadium; 75 peaks). Purification of eight of 158 unexpected metal peaks yielded four novel nickel- and molybdenum-containing proteins, whereas four purified proteins contained sub-stoichiometric amounts of misincorporated lead and uranium. Analyses of two additional microorganisms (Escherichia coli and Sulfolobus solfataricus) revealed species-specific assimilation of yet more unexpected metals. Metalloproteomes are therefore much more extensive and diverse than previously recognized, and promise to provide key insights for cell biology, microbial growth and toxicity mechanisms.

Regulation of myeloid leukaemia by the cell-fate determinant Musashi

Sun, 07/18/2010 - 00:00

Regulation of myeloid leukaemia by the cell-fate determinant Musashi

Nature advance online publication 18 July 2010. doi:10.1038/nature09171

Authors: Takahiro Ito, Hyog Young Kwon, Bryan Zimdahl, Kendra L. Congdon, Jordan Blum, William E. Lento, Chen Zhao, Anand Lagoo, Gareth Gerrard, Letizia Foroni, John Goldman, Harriet Goh, Soo-Hyun Kim, Dong-Wook Kim, Charles Chuah, Vivian G. Oehler, Jerald P. Radich, Craig T. Jordan & Tannishtha Reya

Chronic myelogenous leukaemia (CML) can progress from a slow growing chronic phase to an aggressive blast crisis phase, but the molecular basis of this transition remains poorly understood. Here we have used mouse models of CML to show that disease progression is regulated by the Musashi–Numb signalling axis. Specifically, we find that the chronic phase is marked by high levels of Numb expression whereas the blast crisis phase has low levels of Numb expression, and that ectopic expression of Numb promotes differentiation and impairs advanced-phase disease in vivo. As a possible explanation for the decreased levels of Numb in the blast crisis phase, we show that NUP98–HOXA9, an oncogene associated with blast crisis CML, can trigger expression of the RNA-binding protein Musashi2 (Msi2), which in turn represses Numb. Notably, loss of Msi2 restores Numb expression and significantly impairs the development and propagation of blast crisis CML in vitro and in vivo. Finally we show that Msi2 expression is not only highly upregulated during human CML progression but is also an early indicator of poorer prognosis. These data show that the Musashi–Numb pathway can control the differentiation of CML cells, and raise the possibility that targeting this pathway may provide a new strategy for the therapy of aggressive leukaemias.

Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification

Sun, 07/18/2010 - 00:00

Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification

Nature advance online publication 18 July 2010. doi:10.1038/nature09303

Authors: Shinsuke Ito, Ana C. D’Alessio, Olena V. Taranova, Kwonho Hong, Lawrence C. Sowers & Yi Zhang

DNA methylation is one of the best-characterized epigenetic modifications. Although the enzymes that catalyse DNA methylation have been characterized, enzymes responsible for demethylation have been elusive. A recent study indicates that the human TET1 protein could catalyse the conversion of 5-methylcytosine (5mC) of DNA to 5-hydroxymethylcytosine (5hmC), raising the possibility that DNA demethylation may be a Tet1-mediated process. Here we extend this study by demonstrating that all three mouse Tet proteins (Tet1, Tet2 and Tet3) can also catalyse a similar reaction. Tet1 has an important role in mouse embryonic stem (ES) cell maintenance through maintaining the expression of Nanog in ES cells. Downregulation of Nanog via Tet1 knockdown correlates with methylation of the Nanog promoter, supporting a role for Tet1 in regulating DNA methylation status. Furthermore, knockdown of Tet1 in pre-implantation embryos results in a bias towards trophectoderm differentiation. Thus, our studies not only uncover the enzymatic activity of the Tet proteins, but also demonstrate a role for Tet1 in ES cell maintenance and inner cell mass cell specification.

Cross-species genomics matches driver mutations and cell compartments to model ependymoma

Sun, 07/18/2010 - 00:00

Cross-species genomics matches driver mutations and cell compartments to model ependymoma

Nature advance online publication 18 July 2010. doi:10.1038/nature09173

Authors: Robert A. Johnson, Karen D. Wright, Helen Poppleton, Kumarasamypet M. Mohankumar, David Finkelstein, Stanley B. Pounds, Vikki Rand, Sarah E. S. Leary, Elsie White, Christopher Eden, Twala Hogg, Paul Northcott, Stephen Mack, Geoffrey Neale, Yong-Dong Wang, Beth Coyle, Jennifer Atkinson, Mariko DeWire, Tanya A. Kranenburg, Yancey Gillespie, Jeffrey C. Allen, Thomas Merchant, Fredrick A. Boop, Robert. A. Sanford, Amar Gajjar, David W. Ellison, Michael D. Taylor, Richard G. Grundy & Richard J. Gilbertson

Understanding the biology that underlies histologically similar but molecularly distinct subgroups of cancer has proven difficult because their defining genetic alterations are often numerous, and the cellular origins of most cancers remain unknown. We sought to decipher this heterogeneity by integrating matched genetic alterations and candidate cells of origin to generate accurate disease models. First, we identified subgroups of human ependymoma, a form of neural tumour that arises throughout the central nervous system (CNS). Subgroup-specific alterations included amplifications and homozygous deletions of genes not yet implicated in ependymoma. To select cellular compartments most likely to give rise to subgroups of ependymoma, we matched the transcriptomes of human tumours to those of mouse neural stem cells (NSCs), isolated from different regions of the CNS at different developmental stages, with an intact or deleted Ink4a/Arf locus (that encodes Cdkn2a and b). The transcriptome of human supratentorial ependymomas with amplified EPHB2 and deleted INK4A/ARF matched only that of embryonic cerebral Ink4a/Arf−/− NSCs. Notably, activation of Ephb2 signalling in these, but not other, NSCs generated the first mouse model of ependymoma, which is highly penetrant and accurately models the histology and transcriptome of one subgroup of human supratentorial tumour. Further, comparative analysis of matched mouse and human tumours revealed selective deregulation in the expression and copy number of genes that control synaptogenesis, pinpointing disruption of this pathway as a critical event in the production of this ependymoma subgroup. Our data demonstrate the power of cross-species genomics to meticulously match subgroup-specific driver mutations with cellular compartments to model and interrogate cancer subgroups.

Regulation of heterochromatic DNA replication by histone H3 lysine 27 methyltransferases

Wed, 07/14/2010 - 00:00

Regulation of heterochromatic DNA replication by histone H3 lysine 27 methyltransferases

Nature advance online publication 14 July 2010. doi:10.1038/nature09290

Authors: Yannick Jacob, Hume Stroud, Chantal LeBlanc, Suhua Feng, Luting Zhou, Elena Caro, Christiane Hassel, Crisanto Gutierrez, Scott D. Michaels & Steven E. Jacobsen

Multiple pathways prevent DNA replication from occurring more than once per cell cycle. These pathways block re-replication by strictly controlling the activity of pre-replication complexes, which assemble at specific sites in the genome called origins. Here we show that mutations in the homologous histone 3 lysine 27 (H3K27) monomethyltransferases, ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6, lead to re-replication of specific genomic locations. Most of these locations correspond to transposons and other repetitive and silent elements of the Arabidopsis genome. These sites also correspond to high levels of H3K27 monomethylation, and mutation of the catalytic SET domain is sufficient to cause the re-replication defect. Mutation of ATXR5 and ATXR6 also causes upregulation of transposon expression and has pleiotropic effects on plant development. These results uncover a novel pathway that prevents over-replication of heterochromatin in Arabidopsis.

Microtubule nucleating γ-TuSC assembles structures with 13-fold microtubule-like symmetry

Wed, 07/14/2010 - 00:00

Microtubule nucleating γ-TuSC assembles structures with 13-fold microtubule-like symmetry

Nature advance online publication 14 July 2010. doi:10.1038/nature09207

Authors: Justin M. Kollman, Jessica K. Polka, Alex Zelter, Trisha N. Davis & David A. Agard

Microtubules are nucleated in vivo by γ-tubulin complexes. The 300-kDa γ-tubulin small complex (γ-TuSC), consisting of two molecules of γ-tubulin and one copy each of the accessory proteins Spc97 and Spc98, is the conserved, essential core of the microtubule nucleating machinery. In metazoa multiple γ-TuSCs assemble with other proteins into γ-tubulin ring complexes (γ-TuRCs). The structure of γ-TuRC indicated that it functions as a microtubule template. Because each γ-TuSC contains two molecules of γ-tubulin, it was assumed that the γ-TuRC-specific proteins are required to organize γ-TuSCs to match 13-fold microtubule symmetry. Here we show that Saccharomycescerevisiae γ-TuSC forms rings even in the absence of other γ-TuRC components. The yeast adaptor protein Spc110 stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8-Å cryo-electron microscopic reconstruction of the filament reveals 13 γ-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the γ-TuRC-specific proteins. The γ-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

Social learning promotes institutions for governing the commons

Wed, 07/14/2010 - 00:00

Social learning promotes institutions for governing the commons

Nature advance online publication 14 July 2010. doi:10.1038/nature09203

Authors: Karl Sigmund, Hannelore De Silva, Arne Traulsen & Christoph Hauert

Theoretical and empirical research highlights the role of punishment in promoting collaborative efforts. However, both the emergence and the stability of costly punishment are problematic issues. It is not clear how punishers can invade a society of defectors by social learning or natural selection, or how second-order free-riders (who contribute to the joint effort but not to the sanctions) can be prevented from drifting into a coercion-based regime and subverting cooperation. Here we compare the prevailing model of peer-punishment with pool-punishment, which consists in committing resources, before the collaborative effort, to prepare sanctions against free-riders. Pool-punishment facilitates the sanctioning of second-order free-riders, because these are exposed even if everyone contributes to the common good. In the absence of such second-order punishment, peer-punishers do better than pool-punishers; but with second-order punishment, the situation is reversed. Efficiency is traded for stability. Neither other-regarding tendencies or preferences for reciprocity and equity, nor group selection or prescriptions from higher authorities, are necessary for the emergence and stability of rudimentary forms of sanctioning institutions regulating common pool resources and enforcing collaborative efforts.

Convergent evolution of chicken Z and human X chromosomes by expansion and gene acquisition

Sun, 07/11/2010 - 00:00

Convergent evolution of chicken Z and human X chromosomes by expansion and gene acquisition

Nature advance online publication 11 July 2010. doi:10.1038/nature09172

Authors: Daniel W. Bellott, Helen Skaletsky, Tatyana Pyntikova, Elaine R. Mardis, Tina Graves, Colin Kremitzki, Laura G. Brown, Steve Rozen, Wesley C. Warren, Richard K. Wilson & David C. Page

In birds, as in mammals, one pair of chromosomes differs between the sexes. In birds, males are ZZ and females ZW. In mammals, males are XY and females XX. Like the mammalian XY pair, the avian ZW pair is believed to have evolved from autosomes, with most change occurring in the chromosomes found in only one sex—the W and Y chromosomes. By contrast, the sex chromosomes found in both sexes—the Z and X chromosomes—are assumed to have diverged little from their autosomal progenitors. Here we report findings that challenge this assumption for both the chicken Z chromosome and the human X chromosome. The chicken Z chromosome, which we sequenced essentially to completion, is less gene-dense than chicken autosomes but contains a massive tandem array containing hundreds of duplicated genes expressed in testes. A comprehensive comparison of the chicken Z chromosome with the finished sequence of the human X chromosome demonstrates that each evolved independently from different portions of the ancestral genome. Despite this independence, the chicken Z and human X chromosomes share features that distinguish them from autosomes: the acquisition and amplification of testis-expressed genes, and a low gene density resulting from an expansion of intergenic regions. These features were not present on the autosomes from which the Z and X chromosomes originated but were instead acquired during the evolution of Z and X as sex chromosomes. We conclude that the avian Z and mammalian X chromosomes followed convergent evolutionary trajectories, despite their evolving with opposite (female versus male) systems of heterogamety. More broadly, in birds and mammals, sex chromosome evolution involved not only gene loss in sex-specific chromosomes, but also marked expansion and gene acquisition in sex chromosomes common to males and females.

A novel pathway regulates memory and plasticity via SIRT1 and miR-134

Sun, 07/11/2010 - 00:00

A novel pathway regulates memory and plasticity via SIRT1 and miR-134

Nature advance online publication 11 July 2010. doi:10.1038/nature09271

Authors: Jun Gao, Wen-Yuan Wang, Ying-Wei Mao, Johannes Gräff, Ji-Song Guan, Ling Pan, Gloria Mak, Dohoon Kim, Susan C. Su & Li-Huei Tsai

The NAD-dependent deacetylase Sir2 was initially identified as a mediator of replicative lifespan in budding yeast and was subsequently shown to modulate longevity in worms and flies. Its mammalian homologue, SIRT1, seems to have evolved complex systemic roles in cardiac function, DNA repair and genomic stability. Recent studies suggest a functional relevance of SIRT1 in normal brain physiology and neurological disorders. However, it is unknown if SIRT1 has a role in higher-order brain functions. We report that SIRT1 modulates synaptic plasticity and memory formation via a microRNA-mediated mechanism. Activation of SIRT1 enhances, whereas its loss-of-function impairs, synaptic plasticity. Surprisingly, these effects were mediated via post-transcriptional regulation of cAMP response binding protein (CREB) expression by a brain-specific microRNA, miR-134. SIRT1 normally functions to limit expression of miR-134 via a repressor complex containing the transcription factor YY1, and unchecked miR-134 expression following SIRT1 deficiency results in the downregulated expression of CREB and brain-derived neurotrophic factor (BDNF), thereby impairing synaptic plasticity. These findings demonstrate a new role for SIRT1 in cognition and a previously unknown microRNA-based mechanism by which SIRT1 regulates these processes. Furthermore, these results describe a separate branch of SIRT1 signalling, in which SIRT1 has a direct role in regulating normal brain function in a manner that is disparate from its cell survival functions, demonstrating its value as a potential therapeutic target for the treatment of central nervous system disorders.

PHF8 mediates histone H4 lysine 20 demethylation events involved in cell cycle progression

Sun, 07/11/2010 - 00:00

PHF8 mediates histone H4 lysine 20 demethylation events involved in cell cycle progression

Nature advance online publication 11 July 2010. doi:10.1038/nature09272

Authors: Wen Liu, Bogdan Tanasa, Oksana V. Tyurina, Tian Yuan Zhou, Reto Gassmann, Wei Ting Liu, Kenneth A. Ohgi, Chris Benner, Ivan Garcia-Bassets, Aneel K. Aggarwal, Arshad Desai, Pieter C. Dorrestein, Christopher K. Glass & Michael G. Rosenfeld

While reversible histone modifications are linked to an ever-expanding range of biological functions, the demethylases for histone H4 lysine 20 and their potential regulatory roles remain unknown. Here we report that the PHD and Jumonji C (JmjC) domain-containing protein, PHF8, while using multiple substrates, including H3K9me1/2 and H3K27me2, also functions as an H4K20me1 demethylase. PHF8 is recruited to promoters by its PHD domain based on interaction with H3K4me2/3 and controls G1–S transition in conjunction with E2F1, HCF-1 (also known as HCFC1) and SET1A (also known as SETD1A), at least in part, by removing the repressive H4K20me1 mark from a subset of E2F1-regulated gene promoters. Phosphorylation-dependent PHF8 dismissal from chromatin in prophase is apparently required for the accumulation of H4K20me1 during early mitosis, which might represent a component of the condensin II loading process. Accordingly, the HEAT repeat clusters in two non-structural maintenance of chromosomes (SMC) condensin II subunits, N-CAPD3 and N-CAPG2 (also known as NCAPD3 and NCAPG2, respectively), are capable of recognizing H4K20me1, and ChIP-Seq analysis demonstrates a significant overlap of condensin II and H4K20me1 sites in mitotic HeLa cells. Thus, the identification and characterization of an H4K20me1 demethylase, PHF8, has revealed an intimate link between this enzyme and two distinct events in cell cycle progression.

Histone H4K20/H3K9 demethylase PHF8 regulates zebrafish brain and craniofacial development

Sun, 07/11/2010 - 00:00

Histone H4K20/H3K9 demethylase PHF8 regulates zebrafish brain and craniofacial development

Nature advance online publication 11 July 2010. doi:10.1038/nature09261

Authors: Hank H. Qi, Madathia Sarkissian, Gang-Qing Hu, Zhibin Wang, Arindam Bhattacharjee, D. Benjamin Gordon, Michelle Gonzales, Fei Lan, Pat P. Ongusaha, Maite Huarte, Nasser K. Yaghi, Huijun Lim, Benjamin A. Garcia, Leonardo Brizuela, Keji Zhao, Thomas M. Roberts & Yang Shi

X-linked mental retardation (XLMR) is a complex human disease that causes intellectual disability. Causal mutations have been found in approximately 90 X-linked genes; however, molecular and biological functions of many of these genetically defined XLMR genes remain unknown. PHF8 (PHD (plant homeo domain) finger protein 8) is a JmjC domain-containing protein and its mutations have been found in patients with XLMR and craniofacial deformities. Here we provide multiple lines of evidence establishing PHF8 as the first mono-methyl histone H4 lysine 20 (H4K20me1) demethylase, with additional activities towards histone H3K9me1 and me2. PHF8 is located around the transcription start sites (TSS) of ∼7,000 RefSeq genes and in gene bodies and intergenic regions (non-TSS). PHF8 depletion resulted in upregulation of H4K20me1 and H3K9me1 at the TSS and H3K9me2 in the non-TSS sites, respectively, demonstrating differential substrate specificities at different target locations. PHF8 positively regulates gene expression, which is dependent on its H3K4me3-binding PHD and catalytic domains. Importantly, patient mutations significantly compromised PHF8 catalytic function. PHF8 regulates cell survival in the zebrafish brain and jaw development, thus providing a potentially relevant biological context for understanding the clinical symptoms associated with PHF8 patients. Lastly, genetic and molecular evidence supports a model whereby PHF8 regulates zebrafish neuronal cell survival and jaw development in part by directly regulating the expression of the homeodomain transcription factor MSX1/MSXB, which functions downstream of multiple signalling and developmental pathways. Our findings indicate that an imbalance of histone methylation dynamics has a critical role in XLMR.

Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry

Wed, 07/07/2010 - 00:00

Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry

Nature advance online publication 07 July 2010. doi:10.1038/nature09159

Authors: Alexxai V. Kravitz, Benjamin S. Freeze, Philip R. L. Parker, Kenneth Kay, Myo T. Thwin, Karl Deisseroth & Anatol C. Kreitzer

Neural circuits of the basal ganglia are critical for motor planning and action selection. Two parallel basal ganglia pathways have been described, and have been proposed to exert opposing influences on motor function. According to this classical model, activation of the ‘direct’ pathway facilitates movement and activation of the ‘indirect’ pathway inhibits movement. However, more recent anatomical and functional evidence has called into question the validity of this hypothesis. Because this model has never been empirically tested, the specific function of these circuits in behaving animals remains unknown. Here we report direct activation of basal ganglia circuitry in vivo, using optogenetic control of direct- and indirect-pathway medium spiny projection neurons (MSNs), achieved through Cre-dependent viral expression of channelrhodopsin-2 in the striatum of bacterial artificial chromosome transgenic mice expressing Cre recombinase under control of regulatory elements for the dopamine D1 or D2 receptor. Bilateral excitation of indirect-pathway MSNs elicited a parkinsonian state, distinguished by increased freezing, bradykinesia and decreased locomotor initiations. In contrast, activation of direct-pathway MSNs reduced freezing and increased locomotion. In a mouse model of Parkinson’s disease, direct-pathway activation completely rescued deficits in freezing, bradykinesia and locomotor initiation. Taken together, our findings establish a critical role for basal ganglia circuitry in the bidirectional regulation of motor behaviour and indicate that modulation of direct-pathway circuitry may represent an effective therapeutic strategy for ameliorating parkinsonian motor deficits.

A new DAF-16 isoform regulates longevity

Wed, 07/07/2010 - 00:00

A new DAF-16 isoform regulates longevity

Nature advance online publication 07 July 2010. doi:10.1038/nature09184

Authors: Eun-Soo Kwon, Sri Devi Narasimhan, Kelvin Yen & Heidi A. Tissenbaum

The insulin/IGF-1 signalling (IIS) pathway has diverse roles from metabolism to longevity. In Caenorhabditis elegans, the single forkhead box O (FOXO) homologue, DAF-16, functions as the major target of the IIS pathway. One of two isoforms, DAF-16a, is known to regulate longevity, stress response and dauer diapause. However, it remains unclear how DAF-16 achieves its specificity in regulating these various biological processes. Here we identify a new isoform, DAF-16d/f, as an important isoform regulating longevity. We show that DAF-16 isoforms functionally cooperate to modulate IIS-mediated processes through differential tissue enrichment, preferential modulation by upstream kinases, and regulating distinct and overlapping target genes. Promoter-swapping experiments show both the promoter and the coding region of DAF-16 are important for its function. Importantly, in mammals, four FOXO genes have overlapping and different functions, and in C. elegans, a single FOXO/DAF-16 uses distinct isoforms to fine-tune the IIS-mediated processes in the context of a whole organism.

Subnanometre single-molecule localization, registration and distance measurements

Wed, 07/07/2010 - 00:00

Subnanometre single-molecule localization, registration and distance measurements

Nature advance online publication 07 July 2010. doi:10.1038/nature09163

Authors: Alexandros Pertsinidis, Yunxiang Zhang & Steven Chu

Remarkable progress in optical microscopy has been made in the measurement of nanometre distances. If diffraction blurs the image of a point object into an Airy disk with a root-mean-squared (r.m.s.) size of s =  0.44λ/2NA (∼90 nm for light with a wavelength of λ = 600 nm and an objective lens with a numerical aperture of NA = 1.49), limiting the resolution of the far-field microscope in use to d = 2.4s ≈ 200 nm, additional knowledge about the specimen can be used to great advantage. For example, if the source is known to be two spatially resolved fluorescent molecules, the distance between them is given by the separation of the centres of the two fluorescence images. In high-resolution microwave and optical spectroscopy, there are numerous examples where the line centre is determined with a precision of less than 10−6 of the linewidth. In contrast, in biological applications the brightest single fluorescent emitters can be detected with a signal-to-noise ratio of ∼100, limiting the centroid localization precision to sloc ≥ 1% (≥1 nm) of the r.m.s. size, s, of the microscope point spread function (PSF). Moreover, the error in co-localizing two or more single emitters is notably worse, remaining greater than 5–10% (5–10 nm) of the PSF size. Here we report a distance resolution of sreg = 0.50 nm (1σ) and an absolute accuracy of sdistance = 0.77 nm (1σ) in a measurement of the separation between differently coloured fluorescent molecules using conventional far-field fluorescence imaging in physiological buffer conditions. The statistical uncertainty in the mean for an ensemble of identical single-molecule samples is limited only by the total number of collected photons, to sloc ≈ 0.3 nm, which is ∼3 × 10−3 times the size of the optical PSF. Our method may also be used to improve the resolution of many subwavelength, far-field imaging methods such as those based on co-localization of molecules that are stochastically switched on in space. The improved resolution will allow the structure of large, multisubunit biological complexes in biologically relevant environments to be deciphered at the single-molecule level.

LRRC26 auxiliary protein allows BK channel activation at resting voltage without calcium

Wed, 07/07/2010 - 00:00

LRRC26 auxiliary protein allows BK channel activation at resting voltage without calcium

Nature advance online publication 07 July 2010. doi:10.1038/nature09162

Authors: Jiusheng Yan & Richard W. Aldrich

Large-conductance, voltage- and calcium-activated potassium (BK, or KCa1.1) channels are ubiquitously expressed in electrically excitable and non-excitable cells, either as α-subunit (BKα) tetramers or together with tissue specific auxiliary β-subunits (β1–β4). Activation of BK channels typically requires coincident membrane depolarization and elevation in free cytosolic Ca2+ concentration ([Ca2+]i), which are not physiological conditions for most non-excitable cells. Here we present evidence that in non-excitable LNCaP prostate cancer cells, BK channels can be activated at negative voltages without rises in [Ca2+]i through their complex with an auxiliary protein, leucine-rich repeat (LRR)-containing protein 26 (LRRC26). LRRC26 modulates the gating of a BK channel by enhancing the allosteric coupling between voltage-sensor activation and the channel’s closed–open transition. This finding reveals a novel auxiliary protein of a voltage-gated ion channel that gives an unprecedentedly large negative shift (∼−140 mV) in voltage dependence and provides a molecular basis for activation of BK channels at physiological voltages and calcium levels in non-excitable cells.

Sparse coding and high-order correlations in fine-scale cortical networks

Sun, 07/04/2010 - 00:00

Sparse coding and high-order correlations in fine-scale cortical networks

Nature advance online publication 04 July 2010. doi:10.1038/nature09178

Authors: Ifije E. Ohiorhenuan, Ferenc Mechler, Keith P. Purpura, Anita M. Schmid, Qin Hu & Jonathan D. Victor

Connectivity in the cortex is organized at multiple scales, suggesting that scale-dependent correlated activity is particularly important for understanding the behaviour of sensory cortices and their function in stimulus encoding. We analysed the scale-dependent structure of cortical interactions by using maximum entropy models to characterize multiple-tetrode recordings from primary visual cortex of anaesthetized macaque monkeys (Macaca mulatta). We compared the properties of firing patterns among local clusters of neurons (<300 μm apart) with those of neurons separated by larger distances (600–2,500 μm). Here we report that local firing patterns are distinctive: whereas multi-neuronal firing patterns at larger distances can be predicted by pairwise interactions, patterns within local clusters often show evidence of high-order correlations. Surprisingly, these local correlations are flexible and rapidly reorganized by visual input. Although they modestly reduce the amount of information that a cluster conveys, they also modify the format of this information, creating sparser codes by increasing the periods of total quiescence, and concentrating information into briefer periods of common activity. These results imply a hierarchical organization of neuronal correlations: simple pairwise correlations link neurons over scales of tens to hundreds of minicolumns, but on the scale of a few minicolumns, ensembles of neurons form complex subnetworks whose moment-to-moment effective connectivity is dynamically reorganized by the stimulus.

Co-option of the hormone-signalling module dafachronic acid–DAF-12 in nematode evolution

Wed, 06/30/2010 - 00:00

Co-option of the hormone-signalling module dafachronic acid–DAF-12 in nematode evolution

Nature advance online publication 30 June 2010. doi:10.1038/nature09164

Authors: Gilberto Bento, Akira Ogawa & Ralf J. Sommer

Morphological novelties are lineage-specific traits that serve new functions. Developmental polyphenisms have been proposed to be facilitators of phenotypic evolution, but little is known about the interplay between the associated genetic and environmental factors. Here, we study two alternative morphologies in the mouth of the nematode Pristionchus pacificus and the formation of teeth-like structures that are associated with bacteriovorous feeding and predatory behaviour on fungi and other worms. These teeth-like denticles represent an evolutionary novelty, which is restricted to some members of the nematode family Diplogastridae but is absent from Caenorhabditis elegans and related nematodes. We show that the mouth dimorphism is a polyphenism that is controlled by starvation and the co-option of an endocrine switch mechanism. Mutations in the nuclear hormone receptor DAF-12 and application of its ligand, the sterol hormone dafachronic acid, strongly influence this switch mechanism. The dafachronic acid–DAF-12 module has been shown to control the formation of arrested dauer larvae in both C. elegans and P. pacificus, as well as related life-history decisions in distantly related nematodes. The comparison of dauer formation and mouth morphology switch reveals that different thresholds of dafachronic acid signalling provide specificity. This study shows how hormonal signalling acts by coupling environmental change and genetic regulation and identifies dafachronic acid as a key hormone in nematode evolution.

Replacing underperforming protected areas achieves better conservation outcomes

Wed, 06/30/2010 - 00:00

Replacing underperforming protected areas achieves better conservation outcomes

Nature advance online publication 30 June 2010. doi:10.1038/nature09180

Authors: Richard A. Fuller, Eve McDonald-Madden, Kerrie A. Wilson, Josie Carwardine, Hedley S. Grantham, James E. M. Watson, Carissa J. Klein, David C. Green & Hugh P. Possingham

Protected areas vary enormously in their contribution to conserving biodiversity, and the inefficiency of protected area systems is widely acknowledged. However, conservation plans focus overwhelmingly on adding new sites to current protected area estates. Here we show that the conservation performance of a protected area system can be radically improved, without extra expenditure, by replacing a small number of protected areas with new ones that achieve more for conservation. Replacing the least cost-effective 1% of Australia’s 6,990 strictly protected areas could increase the number of vegetation types that have 15% or more of their original extent protected from 18 to 54, of a maximum possible of 58. Moreover, it increases markedly the area that can be protected, with no increase in overall spending. This new paradigm for protected area system expansion could yield huge improvements to global conservation at a time when competition for land is increasingly intense.

Allelic variation in a fatty-acyl reductase gene causes divergence in moth sex pheromones

Wed, 06/30/2010 - 00:00

Allelic variation in a fatty-acyl reductase gene causes divergence in moth sex pheromones

Nature advance online publication 30 June 2010. doi:10.1038/nature09058

Authors: Jean-Marc Lassance, Astrid T. Groot, Marjorie A. Liénard, Binu Antony, Christin Borgwardt, Fredrik Andersson, Erik Hedenström, David G. Heckel & Christer Löfstedt

Pheromone-based behaviours are crucial in animals from insects to mammals, and reproductive isolation is often based on pheromone differences. However, the genetic mechanisms by which pheromone signals change during the evolution of new species are largely unknown. In the sexual communication system of moths (Insecta: Lepidoptera), females emit a species-specific pheromone blend that attracts males over long distances. The European corn borer, Ostrinia nubilalis, consists of two sex pheromone races, Z and E, that use different ratios of the cis and trans isomers of acetate pheromone components. This subtle difference leads to strong reproductive isolation in the field between the two races, which could represent a first step in speciation. Female sex pheromone production and male behavioural response are under the control of different major genes, but the identity of these genes is unknown. Here we show that allelic variation in a fatty-acyl reductase gene essential for pheromone biosynthesis accounts for the phenotypic variation in female pheromone production, leading to race-specific signals. Both the cis and trans isomers of the pheromone precursors are produced by both races, but the precursors are differentially reduced to yield opposite ratios in the final pheromone blend as a result of the substrate specificity of the enzymes encoded by the Z and E alleles. This is the first functional characterization of a gene contributing to intraspecific behavioural reproductive isolation in moths, highlighting the importance of evolutionary diversification in a lepidopteran-specific family of reductases. Accumulation of substitutions in the coding region of a single biosynthetic enzyme can produce pheromone differences resulting in reproductive isolation, with speciation as a potential end result.

Blindsight depends on the lateral geniculate nucleus

Wed, 06/23/2010 - 00:00

Blindsight depends on the lateral geniculate nucleus

Nature advance online publication 23 June 2010. doi:10.1038/nature09179

Authors: Michael C. Schmid, Sylwia W. Mrowka, Janita Turchi, Richard C. Saunders, Melanie Wilke, Andrew J. Peters, Frank Q. Ye & David A. Leopold

Injury to the primary visual cortex (V1) leads to the loss of visual experience. Nonetheless, careful testing shows that certain visually guided behaviours can persist even in the absence of visual awareness. The neural circuits supporting this phenomenon, which is often termed blindsight, remain uncertain. Here we demonstrate that the thalamic lateral geniculate nucleus (LGN) has a causal role in V1-independent processing of visual information. By comparing functional magnetic resonance imaging (fMRI) and behavioural measures with and without temporary LGN inactivation, we assessed the contribution of the LGN to visual functions of macaque monkeys (Macaca mulatta) with chronic V1 lesions. Before LGN inactivation, high-contrast stimuli presented to the lesion-affected visual field (scotoma) produced significant V1-independent fMRI activation in the extrastriate cortical areas V2, V3, V4, V5/middle temporal (MT), fundus of the superior temporal sulcus (FST) and lateral intraparietal area (LIP) and the animals correctly located the stimuli in a detection task. However, following reversible inactivation of the LGN in the V1-lesioned hemisphere, fMRI responses and behavioural detection were abolished. These results demonstrate that direct LGN projections to the extrastriate cortex have a critical functional contribution to blindsight. They suggest a viable pathway to mediate fast detection during normal vision.