目次

Editorials

米国は、銃に関連した研究への公的助成の禁止を続けるべきではない。

Under the gun p.409

A ban on advocacy and promotion of gun control is keeping US agencies from conducting research that is sorely needed to inform policy on firearms and prevent shootings.

doi: 10.1038/495409a

情報の公開、共有、再利用は技術革新により進んできたが、科学者の受けとめ方は分野によって違いがある。

Disciplinary action p.409

How scientists share and reuse information is driven by technology but shaped by discipline.

doi: 10.1038/495409b

最新の民間研究船の就航により、海洋科学に新たな世界が開けるかもしれない。

Push the boat out p.410

The latest private research vessel to be launched could open up the world of marine science.

doi: 10.1038/495410a

News

プランク宇宙望遠鏡による高精度マイクロ波地図で、誕生直後の宇宙の姿が明らかに。

Planck snaps infant Universe p.417

Space telescope culls exotic creation theories with ultra-precise microwave map.

doi: 10.1038/495417a

イタリアの保健大臣が法的安全基準にのっとらない幹細胞治療を是認し、科学界から怒りの声が。

Stem-cell ruling riles researchers p.418

Italian health minister’s support for a controversial treatment appals the country’s scientists.

doi: 10.1038/495418a

ヨーロッパも、医薬品の臨床試験データを公開する方向へ。

Drug-company data vaults to be opened p.419

European agency will publish firms’ clinical-trial results.

doi: 10.1038/495419a

グーグル社の資金による研究船が、海洋研究に新たな変化を。

Private research ship makes waves p.420

Falkor plots a fresh course for ocean studies with Google cash.

doi: 10.1038/495420a

実在の論文誌の偽サイトを使って科学者から手数料をだまし取る、ネット詐欺事件が。

Sham journals scam authors p.421

Con artists are stealing the identities of real journals to cheat scientists out of publishing fees.

doi: 10.1038/495421a

凝縮物質系におけるヒッグス類似粒子が、ヒッグス粒子の理解に役立つ可能性も。

Higgs physics on the cheap p.422

Tabletop Higgs particles may illuminate cosmic cousin.

doi: 10.1038/495422a

News Features

新しいページ

The future of publishing: A new page p.425

この特集では、様変わりしていく科学論文発表の在り方を見つめる。

doi: 10.1038/495425a

科学出版の本当のコスト

Open access: The true cost of science publishing p.426

安価なオープンアクセス誌の増加で、論文出版にかかる費用や出版社による付加価値に疑問の目が向けられている。

doi: 10.1038/495426a

図書館の再起動

Publishing frontiers: The library reboot p.430

データ公開が推進される中で、図書館や研究者も遅れまいと努力している。

doi: 10.1038/495430a

出版の暗部

Investigating journals: The dark side of publishing p.433

オープンアクセス出版の急増で、運営のうさんくさいところも増えている。

doi: 10.1038/495433a

News & Views

国際保健:エイズ政策についての説得力のある証拠

Global health: Persuasive evidence on HIV policy p.452

抗レトロウイルス療法はHIV/エイズの世界的流行に対する闘いに大変革をもたらした。南アフリカ農村地帯における大規模集団の調査解析は、この治療法に対する持続的な支援が不可欠であることを示す説得力ある事例となっている。

doi: 10.1038/495452a

材料科学:お盆に乗せて差し出されたナノ粒子構造

Materials science: Nanoparticle structures served up on a tray p.453

一連のナノ粒子をイリジウム基板に支持されたグラフェンシート上に並べるなどの巧妙な方法により、ナノ粒子の原子構造の正確な測定が可能になった。

doi: 10.1038/495453a

地球科学:大洋が息を吐く仕組み

Earth science: How the ocean exhales p.454

亜熱帯大西洋における生物起源のオパール産生の記録は、最終氷期の終わり頃に海洋深層水からの二酸化炭素の放出を駆動したのは、風ではなく、大洋循環であったという説を裏付けている。

doi: 10.1038/495454a

X線結晶学:ほとんどのサイズに適応可能

X-ray crystallography: One size fits most p.456

結晶性の「スポンジ」を使えば、小型の分子も規則正しく並べることができるので、その構造をX線結晶学によって解明できる。この技術を使えば、ナノグラム程度の量で物質の解析が可能になる。

doi: 10.1038/495456a

細胞生物学:受容体からのシグナルは波のように押し寄せる

Cell biology: Receptor signals come in waves p.457

Gタンパク質共役受容体の活性は細胞表面に限られるものではない。顕微鏡観察によって、これらの受容体によって送り出される、時間的、空間的そしておそらくは機能的にも異なる3種類のシグナル伝達の波の存在を示す証拠が得られた。

doi: 10.1038/nature12086

古生物学:バージェス頁岩から得られた、管の中に棲む虫

Palaeontology: Tubular worms from the Burgess Shale p.458

5億年前の岩石から、管の中に棲んでいたギボシムシの化石が見つかったことは、脊椎動物の祖先が移動性のある虫に似た動物なのか、それとも固着性の群体の一員だったのかという議論に解決の光明を投じることになりそうだ。

doi: 10.1038/nature11963

Articles

化学:多孔性錯体を用いたナノグラムからマイクログラムスケールのX線解析

X-ray analysis on the nanogram to microgram scale using porous complexes p.461

Absorption of target molecules into a porous matrix permits single-crystal X-ray diffraction analysis of the ‘guest’ molecules, avoiding the need to obtain them in single-crystal form and making analysis possible using as little as 80 nanograms of sample.

doi: 10.1038/nature11990

細胞生物学:hnRNPA2B1とhnRNPA1のプリオン様ドメインに生じた変異が多系統プロテイノパチーとALSを引き起こす

Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS p.467

The identification of pathogenic mutations within prion-like domains (PrLDs) of the RNA-binding proteins hnRNPA2B1 and hnRNPA1 add to our understanding of how mutations in these proteins lead to degenerative disease, and highlight the potential importance of PrLDs in degenerative diseases of the nervous system, muscle and bone.

doi: 10.1038/nature11922

神経科学:CLP1はtRNA代謝を運動ニューロンの進行性消失に関連付ける

CLP1 links tRNA metabolism to progressive motor-neuron loss p.474

Inactivating the CLP1 RNA kinase in mice leads to a progressive loss of motor neurons, through a mechanism related to the accumulation of a novel set of small RNA fragments derived from aberrant processing of tyrosine pre-transfer RNA.

doi: 10.1038/nature11923

構造生物学:ヒトでの転写開始における重要なステップの構造的可視化

Structural visualization of key steps in human transcription initiation p.481

Cryo-electron microscopy structures of key intermediates during the sequential assembly of the pre-initiation complex are presented; structures of the closed and open-promoter complexes allow insights into the process of promoter melting.

doi: 10.1038/nature11991

Letters

宇宙:矮小不規則銀河 WLM の低金属量ガス雲における一酸化炭素

Carbon monoxide in clouds at low metallicity in the dwarf irregular galaxy WLM p.487

Carbon monoxide (CO) is the primary tracer for interstellar clouds where stars form, but it has never been detected in galaxies in which the oxygen abundance relative to hydrogen is less than 20 per cent of that of the Sun, even though such ‘low-metallicity’ galaxies often form stars. This raises the question of whether stars can form in dense gas without molecules, cooling to the required near-zero temperatures by atomic transitions and dust radiation rather than by molecular line emission; and it highlights uncertainties about star formation in the early Universe, when the metallicity was generally low. Here we report the detection of CO in two regions of a local dwarf irregular galaxy, WLM, where the metallicity is 13 per cent of the solar value. We use new submillimetre observations and archival far-infrared observations to estimate the cloud masses, which are both slightly greater than 100,000 solar masses. The clouds have produced stars at a rate per molecule equal to 10 per cent of that in the local Orion nebula cloud. The CO fraction of the molecular gas is also low, about 3 per cent of the Milky Way value. These results suggest that in small galaxies both star-forming cores and CO molecules become increasingly rare in molecular hydrogen clouds as the metallicity decreases.

doi: 10.1038/nature11933

物理:並進温度の低い分子の共同振動冷却を示す証拠

Evidence for sympathetic vibrational cooling of translationally cold molecules p.490

Compared with atoms, molecules have a rich internal structure that offers many opportunities for technological and scientific advancement. The study of this structure could yield critical insights into quantum chemistry, new methods for manipulating quantum information, and improved tests of discrete symmetry violation and fundamental constant variation. Harnessing this potential typically requires the preparation of cold molecules in their quantum rovibrational ground state. However, the molecular internal structure severely complicates efforts to produce such samples. Removal of energy stored in long-lived vibrational levels is particularly problematic because optical transitions between vibrational levels are not governed by strict selection rules, which makes laser cooling difficult. Additionally, traditional collisional, or sympathetic, cooling methods are inefficient at quenching molecular vibrational motion. Here we experimentally demonstrate that the vibrational motion of trapped BaCl+ molecules is quenched by collisions with ultracold calcium atoms at a rate comparable to the classical scattering, or Langevin, rate. This is over four orders of magnitude more efficient than traditional sympathetic cooling schemes. The high cooling rate, a consequence of a strong interaction potential (due to the high polarizability of calcium), along with the low collision energies involved, leads to molecular samples with a vibrational ground-state occupancy of at least 90 per cent. Our demonstration uses a novel thermometry technique that relies on relative photodissociation yields. Although the decrease in vibrational temperature is modest, with straightforward improvements it should be possible to produce molecular samples with a vibrational ground-state occupancy greater than 99 per cent in less than 100 milliseconds. Because sympathetic cooling of molecular rotational motion is much more efficient than vibrational cooling in traditional systems, we expect that the method also allows efficient cooling of the rotational motion of the molecules. Moreover, the technique should work for many different combinations of ultracold atoms and molecules.

doi: 10.1038/nature11937

気候:亜熱帯北大西洋への深海のケイ酸塩の、退氷による一時的な流入

Deglacial pulses of deep-ocean silicate into the subtropical North Atlantic Ocean p.495

Growing evidence suggests that the low atmospheric CO2 concentration of the ice ages resulted from enhanced storage of CO2 in the ocean interior, largely as a result of changes in the Southern Ocean. Early in the most recent deglaciation, a reduction in North Atlantic overturning circulation seems to have driven CO2 release from the Southern Ocean, but the mechanism connecting the North Atlantic and the Southern Ocean remains unclear. Biogenic opal export in the low-latitude ocean relies on silicate from the underlying thermocline, the concentration of which is affected by the circulation of the ocean interior. Here we report a record of biogenic opal export from a coastal upwelling system off the coast of northwest Africa that shows pronounced opal maxima during each glacial termination over the past 550,000 years. These opal peaks are consistent with a strong deglacial reduction in the formation of silicate-poor glacial North Atlantic intermediate water (GNAIW). The loss of GNAIW allowed mixing with underlying silicate-rich deep water to increase the silicate supply to the surface ocean. An increase in westerly-wind-driven upwelling in the Southern Ocean in response to the North Atlantic change has been proposed to drive the deglacial rise in atmospheric CO2 (refs 3, 4). However, such a circulation change would have accelerated the formation of Antarctic intermediate water and sub-Antarctic mode water, which today have as little silicate as North Atlantic Deep Water and would have thus maintained low silicate concentrations in the Atlantic thermocline. The deglacial opal maxima reported here suggest an alternative mechanism for the deglacial CO2 release. Just as the reduction in GNAIW led to upward silicate transport, it should also have allowed the downward mixing of warm, low-density surface water to reach into the deep ocean. The resulting decrease in the density of the deep Atlantic relative to the Southern Ocean surface promoted Antarctic overturning, which released CO2 to the atmosphere.

doi: 10.1038/nature12006

地球:東太平洋海膨北部下にある受動的なマントル上昇流の電気的画像

Electrical image of passive mantle upwelling beneath the northern East Pacific Rise p.499

Melt generated by mantle upwelling is fundamental to the production of new oceanic crust at mid-ocean ridges, yet the forces controlling this process are debated. Passive-flow models predict symmetric upwelling due to viscous drag from the diverging tectonic plates, but have been challenged by geophysical observations of asymmetric upwelling that suggest anomalous mantle pressure and temperature gradients, and by observations of concentrated upwelling centres consistent with active models where buoyancy forces give rise to focused convective flow. Here we use sea-floor magnetotelluric soundings at the fast-spreading northern East Pacific Rise to image mantle electrical structure to a depth of about 160 kilometres. Our data reveal a symmetric, high-conductivity region at depths of 20–90 kilometres that is consistent with partial melting of passively upwelling mantle. The triangular region of conductive partial melt matches passive-flow predictions, suggesting that melt focusing to the ridge occurs in the porous melting region rather than along the shallower base of the thermal lithosphere. A deeper conductor observed east of the ridge at a depth of more than 100 kilometres is explained by asymmetric upwelling due to viscous coupling across two nearby transform faults. Significant electrical anisotropy occurs only in the shallowest mantle east of the ridge axis, where high vertical conductivity at depths of 10–20 kilometres indicates localized porous conduits. This suggests that a coincident seismic-velocity anomaly is evidence of shallow magma transport channels rather than deeper off-axis upwelling. We interpret the mantle electrical structure as evidence that plate-driven passive upwelling dominates this ridge segment, with dynamic forces being negligible.

doi: 10.1038/nature11932

進化:カンブリア紀層で見つかった、棲管を形成する腸鰓類

Tubicolous enteropneusts from the Cambrian period p.503

Hemichordates are a marine group that, apart from one monospecific pelagic larval form, are represented by the vermiform enteropneusts and minute colonial tube-dwelling pterobranchs. Together with echinoderms, they comprise the clade Ambulacraria. Despite their restricted diversity, hemichordates provide important insights into early deuterostome evolution, notably because of their pharyngeal gill slits. Hemichordate phylogeny has long remained problematic, not least because the nature of any transitional form that might serve to link the anatomically disparate enteropneusts and pterobranchs is conjectural. Hence, inter-relationships have also remained controversial. For example, pterobranchs have sometimes been compared to ancestral echinoderms. Molecular data identify enteropneusts as paraphyletic, and harrimaniids as the sister group of pterobranchs. Recent molecular phylogenies suggest that enteropneusts are probably basal within hemichordates, contrary to previous views, but otherwise provide little guidance as to the nature of the primitive hemichordate. In addition, the hemichordate fossil record is almost entirely restricted to peridermal skeletons of pterobranchs, notably graptolites. Owing to their low preservational potentials, fossil enteropneusts are exceedingly rare, and throw no light on either hemichordate phylogeny or the proposed harrimaniid–pterobranch transition. Here we describe an enteropneust, Spartobranchus tenuis (Walcott, 1911), from the Middle Cambrian-period (Series 3, Stage 5) Burgess Shale. It is remarkably similar to the extant harrimaniids, but differs from all known enteropneusts in that it is associated with a fibrous tube that is sometimes branched. We suggest that this is the precursor of the pterobranch periderm, and supports the hypothesis that pterobranchs are miniaturized and derived from an enteropneust-like worm. It also shows that the periderm was acquired before size reduction and acquisition of feeding tentacles, and that coloniality emerged through aggregation of individuals, perhaps similar to the Cambrian rhabdopleurid Fasciculitubus. The presence of both enteropneusts and pterobranchs in Middle Cambrian strata, suggests that hemichordates originated at the onset of the Cambrian explosion.

doi: 10.1038/nature12017

進化:保存された卵胞の化石から明らかになった鳥類繁殖行動の初期進化

Preservation of ovarian follicles reveals early evolution of avian reproductive behaviour p.507

The two groups of archosaurs, crocodilians and birds, form an extant phylogenetic bracket for understanding the reproductive behaviour of dinosaurs. This behaviour is inferred from preserved nests and eggs, and even gravid individuals. Data indicate that many ‘avian’ traits were already present in Paraves—the clade that includes birds and their close relatives—and that the early evolution of the modern avian form of reproduction was already well on its way. Like living neornithine birds, non-avian maniraptorans had daily oviposition and asymmetrical eggs with complex shell microstructure, and were known to protect their clutches. However, like crocodilians, non-avian maniraptorans had two active oviducts (one present in living birds), relatively smaller eggs, and may not have turned their eggs in the way that living birds do. Here we report on the first discovery of fossilized mature or nearly mature ovarian follicles, revealing a previously undocumented stage in dinosaur reproduction: reproductively active females near ovulation. Preserved in a specimen of the long bony-tailed Jeholornis and two enantiornithine birds from the Early Cretaceous period lacustrine Jehol Biota in northeastern China, these discoveries indicate that basal birds only had one functional ovary, but retained primitive morphologies as a result of their lower metabolic rate relative to living birds. They also indicate that basal birds reached sexual maturity before skeletal maturity, as in crocodiles and paravian dinosaurs. Differences in follicular morphology between Jeholornis and the enantiornithines are interpreted as forming an evolutionary gradient from the reproductive condition in paravian dinosaurs towards neornithine birds. Furthermore, differences between the two enantiornithines indicate that this lineage might also have evolved advanced reproductive traits in parallel to the neornithine lineage.

doi: 10.1038/nature11985

遺伝学:複製と転写の衝突により加速される遺伝子進化

Accelerated gene evolution through replication–transcription conflicts p.512

Several mechanisms that increase the rate of mutagenesis across the entire genome have been identified; however, how the rate of evolution might be promoted in individual genes is unclear. Most genes in bacteria are encoded on the leading strand of replication. This presumably avoids the potentially detrimental head-on collisions that occur between the replication and transcription machineries when genes are encoded on the lagging strand. Here we identify the ubiquitous (core) genes in Bacillus subtilis and determine that 17% of them are on the lagging strand. We find a higher rate of point mutations in the core genes on the lagging strand compared with those on the leading strand, with this difference being primarily in the amino-acid-changing (nonsynonymous) mutations. We determine that, overall, the genes under strong negative selection against amino-acid-changing mutations tend to be on the leading strand, co-oriented with replication. In contrast, on the basis of the rate of convergent mutations, genes under positive selection for amino-acid-changing mutations are more commonly found on the lagging strand, indicating faster adaptive evolution in many genes in the head-on orientation. Increased gene length and gene expression amounts are positively correlated with the rate of accumulation of nonsynonymous mutations in the head-on genes, suggesting that the conflict between replication and transcription could be a driving force behind these mutations. Indeed, using reversion assays, we show that the difference in the rate of mutagenesis of genes in the two orientations is transcription dependent. Altogether, our findings indicate that head-on replication–transcription conflicts are more mutagenic than co-directional conflicts and that these encounters can significantly increase adaptive structural variation in the coded proteins. We propose that bacteria, and potentially other organisms, promote faster evolution of specific genes through orientation-dependent encounters between DNA replication and transcription.

doi: 10.1038/nature11989

細胞生物学:多能性転写回路におけるTFIIDの中心的役割

A central role for TFIID in the pluripotent transcription circuitry p.516

Embryonic stem (ES) cells are pluripotent and characterized by open chromatin and high transcription levels, achieved through auto-regulatory and feed-forward transcription factor loops. ES-cell identity is maintained by a core of factors including Oct4 (also known as Pou5f1), Sox2, Klf4, c-Myc (OSKM) and Nanog, and forced expression of the OSKM factors can reprogram somatic cells into induced pluripotent stem cells (iPSCs) resembling ES cells. These gene-specific factors for RNA-polymerase-II-mediated transcription recruit transcriptional cofactors and chromatin regulators that control access to and activity of the basal transcription machinery on gene promoters. How the basal transcription machinery is involved in setting and maintaining the pluripotent state is unclear. Here we show that knockdown of the transcription factor IID (TFIID) complex affects the pluripotent circuitry in mouse ES cells and inhibits reprogramming of fibroblasts. TFIID subunits and the OSKM factors form a feed-forward loop to induce and maintain a stable transcription state. Notably, transient expression of TFIID subunits greatly enhanced reprogramming. These results show that TFIID is critical for transcription-factor-mediated reprogramming. We anticipate that, by creating plasticity in gene expression programs, transcription complexes such as TFIID assist reprogramming into different cellular states.

doi: 10.1038/nature11970

構造生物学:Photorhabdus luminescens毒素の注射器様の注入機構

A syringe-like injection mechanism in Photorhabdus luminescens toxins p.520

Photorhabdus luminescens is an insect pathogenic bacterium that is symbiotic with entomopathogenic nematodes. On invasion of insect larvae, P. luminescens is released from the nematodes and kills the insect through the action of a variety of virulence factors including large tripartite ABC-type toxin complexes (Tcs). Tcs are typically composed of TcA, TcB and TcC proteins and are biologically active only when complete. Functioning as ADP-ribosyltransferases, TcC proteins were identified as the actual functional components that induce actin-clustering, defects in phagocytosis and cell death. However, little is known about the translocation of TcC into the cell by the TcA and TcB components. Here we show that TcA in P. luminescens (TcdA1) forms a transmembrane pore and report its structure in the prepore and pore state determined by cryoelectron microscopy. We find that the TcdA1 prepore assembles as a pentamer forming an α-helical, vuvuzela-shaped channel less than 1.5 nanometres in diameter surrounded by a large outer shell. Membrane insertion is triggered not only at low pH as expected, but also at high pH, explaining Tc action directly through the midgut of insects. Comparisons with structures of the TcdA1 pore inserted into a membrane and in complex with TcdB2 and TccC3 reveal large conformational changes during membrane insertion, suggesting a novel syringe-like mechanism of protein translocation. Our results demonstrate how ABC-type toxin complexes bridge a membrane to insert their lethal components into the cytoplasm of the host cell. We believe that the proposed mechanism is characteristic of the whole ABC-type toxin family. This explanation of toxin translocation is a step towards understanding the host–pathogen interaction and the complex life cycle of P. luminescens and other pathogens, including human pathogenic bacteria, and serves as a strong foundation for the development of biopesticides.

doi: 10.1038/nature11987

免疫:組織常在型M2様マクロファージの分化におけるTrib1の重要な役割

Critical role of Trib1 in differentiation of tissue-resident M2-like macrophages p.524

Macrophages consist of at least two subgroups, M1 and M2 (refs 1, 2, 3). Whereas M1 macrophages are proinflammatory and have a central role in host defence against bacterial and viral infections, M2 macrophages are associated with responses to anti-inflammatory reactions, helminth infection, tissue remodelling, fibrosis and tumour progression. Trib1 is an adaptor protein involved in protein degradation by interacting with COP1 ubiquitin ligase. Genome-wide association studies in humans have implicated TRIB1 in lipid metabolism. Here we show that Trib1 is critical for the differentiation of F4/80+MR+ tissue-resident macrophages—that share characteristics with M2 macrophages (which we term M2-like macrophages)—and eosinophils but not for the differentiation of M1 myeloid cells. Trib1 deficiency results in a severe reduction of M2-like macrophages in various organs, including bone marrow, spleen, lung and adipose tissues. Aberrant expression of C/EBPα in Trib1-deficient bone marrow cells is responsible for the defects in macrophage differentiation. Unexpectedly, mice lacking Trib1 in haematopoietic cells show diminished adipose tissue mass accompanied by evidence of increased lipolysis, even when fed a normal diet. Supplementation of M2-like macrophages rescues the pathophysiology, indicating that a lack of these macrophages is the cause of lipolysis. In response to a high-fat diet, mice lacking Trib1 in haematopoietic cells develop hypertriglyceridaemia and insulin resistance, together with increased proinflammatory cytokine gene induction. Collectively, these results demonstrate that Trib1 is critical for adipose tissue maintenance and suppression of metabolic disorders by controlling the differentiation of tissue-resident M2-like macrophages.

doi: 10.1038/nature11930

植物科学:CLASPを介して起こる表層微小管の配置が、PINの極性化軸を誘導する

CLASP-mediated cortical microtubule organization guides PIN polarization axis p.529

Recent evidence indicates a correlation between orientation of the plant cortical microtubule cytoskeleton and localization of polar cargoes. However, the molecules and mechanisms that create this correlation have remained unknown. Here we show that, in Arabidopsis thaliana, the microtubule orientation regulators CLASP and MAP65 (refs 3, 4) control the abundance of polarity regulator PINOID kinase at the plasma membrane. By localized upregulation of clathrin-dependent endocytosis at cortical microtubule- and clathrin-rich domains orthogonal to the axis of polarity, PINOID accelerates the removal of auxin transporter PIN proteins from those sites. This mechanism links directional microtubule organization to the polar localization of auxin transporter PIN proteins, and clarifies how microtubule-enriched cell sides are kept distinct from polar delivery domains. Our results identify the molecular machinery that connects microtubule organization to the regulation of the axis of PIN polarization.

doi: 10.1038/nature11980

細胞生物学:コンホメーション変化に関わるバイオセンサーによって明らかになったエンドソームからのGPCRシグナル伝達の存在

Conformational biosensors reveal GPCR signalling from endosomes p.534

A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response. Evidence supporting these various latter hypotheses is indirect or subject to alternative interpretation, and it remains unknown if endosome-localized GPCRs are even present in an active form. Here we describe the application of conformation-specific single-domain antibodies (nanobodies) to directly probe activation of the β2-adrenoceptor, a prototypical GPCR, and its cognate G protein, Gs (ref. 12), in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational change in vivo.

doi: 10.1038/nature12000

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