Despite major advances in understanding the molecular and genetic basis of cancer, metastasis remains the cause of >90% of cancer-related mortality. Understanding metastasis initiation and progression is critical to developing new therapeutic strategies to treat and prevent metastatic disease. Prevailing theories hypothesize that metastases are seeded by rare tumour cells with unique properties, which may function like stem cells in their ability to initiate and propagate metastatic tumours. However, the identity of metastasis-initiating cells in human breast cancer remains elusive, and whether metastases are hierarchically organized is unknown. Here we show at the single-cell level that early stage metastatic cells possess a distinct stem-like gene expression signature. To identify and isolate metastatic cells from patient-derived xenograft models of human breast cancer, we developed a highly sensitive fluorescence-activated cell sorting (FACS)-based assay, which allowed us to enumerate metastatic cells in mouse peripheral tissues. We compared gene signatures in metastatic cells from tissues with low versus high metastatic burden. Metastatic cells from low-burden tissues were distinct owing to their increased expression of stem cell, epithelial-to-mesenchymal transition, pro-survival, and dormancy-associated genes. By contrast, metastatic cells from high-burden tissues were similar to primary tumour cells, which were more heterogeneous and expressed higher levels of luminal differentiation genes. Transplantation of stem-like metastatic cells from low-burden tissues showed that they have considerable tumour-initiating capacity, and can differentiate to produce luminal-like cancer cells. Progression to high metastatic burden was associated with increased proliferation and MYC expression, which could be attenuated by treatment with cyclin-dependent kinase (CDK) inhibitors. These findings support a hierarchical model for metastasis, in which metastases are initiated by stem-like cells that proliferate and differentiate to produce advanced metastatic disease.

Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block--although compositionally identical--engage in different local sets of interactions and conformations.

Earlier spring leaf unfolding is a frequently observed response of plants to climate warming. Many deciduous tree species require chilling for dormancy release, and warming-related reductions in chilling may counteract the advance of leaf unfolding in response to warming. Empirical evidence for this, however, is limited to saplings or twigs in climate-controlled chambers. Using long-term in situ observations of leaf unfolding for seven dominant European tree species at 1,245 sites, here we show that the apparent response of leaf unfolding to climate warming (ST, expressed in days advance of leaf unfolding per °C warming) has significantly decreased from 1980 to 2013 in all monitored tree species. Averaged across all species and sites, ST decreased by 40% from 4.0 ± 1.8 days °C(-1) during 1980-1994 to 2.3 ± 1.6 days °C(-1) during 1999-2013. The declining ST was also simulated by chilling-based phenology models, albeit with a weaker decline (24-30%) than observed in situ. The reduction in ST is likely to be partly attributable to reduced chilling. Nonetheless, other mechanisms may also have a role, such as 'photoperiod limitation' mechanisms that may become ultimately limiting when leaf unfolding dates occur too early in the season. Our results provide empirical evidence for a declining ST, but also suggest that the predicted strong winter warming in the future may further reduce ST and therefore result in a slowdown in the advance of tree spring phenology.

Haematopoietic stem cells (HSCs) reside in a perivascular niche but the specific location of this niche remains controversial. HSCs are rare and few can be found in thin tissue sections or upon live imaging, making it difficult to comprehensively localize dividing and non-dividing HSCs. Here, using a green fluorescent protein (GFP) knock-in for the gene Ctnnal1 in mice (hereafter denoted as α-catulin(GFP)), we discover that α-catulin(GFP) is expressed by only 0.02% of bone marrow haematopoietic cells, including almost all HSCs. We find that approximately 30% of α-catulin-GFP(+)c-kit(+) cells give long-term multilineage reconstitution of irradiated mice, indicating that α-catulin-GFP(+)c-kit(+) cells are comparable in HSC purity to cells obtained using the best markers currently available. We optically cleared the bone marrow to perform deep confocal imaging, allowing us to image thousands of α-catulin-GFP(+)c-kit(+) cells and to digitally reconstruct large segments of bone marrow. The distribution of α-catulin-GFP(+)c-kit(+) cells indicated that HSCs were more common in central marrow than near bone surfaces, and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted leptin receptor positive (Lepr(+)) and Cxcl12(high) niche cells, and approximately 85% of HSCs were within 10 μm of a sinusoidal blood vessel. Most HSCs, both dividing (Ki-67(+)) and non-dividing (Ki-67(-)), were distant from arterioles, transition zone vessels, and bone surfaces. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Lepr(+)Cxcl12(high) cells throughout the bone marrow.

The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages. We identified the first examples of proteins produced by phages that inhibit a CRISPR-Cas system. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR-Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. In vivo, this anti-CRISPR can convert the CRISPR-Cas system into a transcriptional repressor, providing the first example-to our knowledge-of modulation of CRISPR-Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR-Cas function.

The altered activity of the fructose transporter GLUT5, an isoform of the facilitated-diffusion glucose transporter family, has been linked to disorders such as type 2 diabetes and obesity. GLUT5 is also overexpressed in certain tumour cells, and inhibitors are potential drugs for these conditions. Here we describe the crystal structures of GLUT5 from Rattus norvegicus and Bos taurus in open outward- and open inward-facing conformations, respectively. GLUT5 has a major facilitator superfamily fold like other homologous monosaccharide transporters. On the basis of a comparison of the inward-facing structures of GLUT5 and human GLUT1, a ubiquitous glucose transporter, we show that a single point mutation is enough to switch the substrate-binding preference of GLUT5 from fructose to glucose. A comparison of the substrate-free structures of GLUT5 with occluded substrate-bound structures of Escherichia coli XylE suggests that, in addition to global rocker-switch-like re-orientation of the bundles, local asymmetric rearrangements of carboxy-terminal transmembrane bundle helices TM7 and TM10 underlie a 'gated-pore' transport mechanism in such monosaccharide transporters.

HIV-1 Nef, a protein important for the development of AIDS, has well-characterized effects on host membrane trafficking and receptor downregulation. By an unidentified mechanism, Nef increases the intrinsic infectivity of HIV-1 virions in a host-cell-dependent manner. Here we identify the host transmembrane protein SERINC5, and to a lesser extent SERINC3, as a potent inhibitor of HIV-1 particle infectivity that is counteracted by Nef. SERINC5 localizes to the plasma membrane, where it is efficiently incorporated into budding HIV-1 virions and impairs subsequent virion penetration of susceptible target cells. Nef redirects SERINC5 to a Rab7-positive endosomal compartment and thereby excludes it from HIV-1 particles. The ability to counteract SERINC5 was conserved in Nef encoded by diverse primate immunodeficiency viruses, as well as in the structurally unrelated glycosylated Gag from murine leukaemia virus. These examples of functional conservation and convergent evolution emphasize the fundamental importance of SERINC5 as a potent anti-retroviral factor.

HIV-1 Nef and the unrelated mouse leukaemia virus glycosylated Gag (glycoGag) strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of both SERINC3 and SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivity. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4(+) T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS.

Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the monoclonal antibodies cetuximab and panitumumab, which prevent epidermal growth factor receptor (EGFR) activation. Recent studies have identified alterations in KRAS and other genes as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in colorectal cancer and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in colorectal cancer on response to anti-EGFR antibody therapy, here we perform complete exome sequence and copy number analyses of 129 patient-derived tumour grafts and targeted genomic analyses of 55 patient tumours, all of which were KRAS wild-type. We analysed the response of tumours to anti-EGFR antibody blockade in tumour graft models and in clinical settings and functionally linked therapeutic responses to mutational data. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumours with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumour graft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluating response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and delineate new avenues for intervention in managing colorectal cancer.

Although all sensory circuits ascend to higher brain areas where stimuli are represented in sparse, stimulus-specific activity patterns, relatively little is known about sensory coding on the descending side of neural circuits, as a network converges. In insects, mushroom bodies have been an important model system for studying sparse coding in the olfactory system, where this format is important for accurate memory formation. In Drosophila, it has recently been shown that the 2,000 Kenyon cells of the mushroom body converge onto a population of only 34 mushroom body output neurons (MBONs), which fall into 21 anatomically distinct cell types. Here we provide the first, to our knowledge, comprehensive view of olfactory representations at the fourth layer of the circuit, where we find a clear transition in the principles of sensory coding. We show that MBON tuning curves are highly correlated with one another. This is in sharp contrast to the process of progressive decorrelation of tuning in the earlier layers of the circuit. Instead, at the population level, odour representations are reformatted so that positive and negative correlations arise between representations of different odours. At the single-cell level, we show that uniquely identifiable MBONs display profoundly different tuning across different animals, but that tuning of the same neuron across the two hemispheres of an individual fly was nearly identical. Thus, individualized coordination of tuning arises at this level of the olfactory circuit. Furthermore, we find that this individualization is an active process that requires a learning-related gene, rutabaga. Ultimately, neural circuits have to flexibly map highly stimulus-specific information in sparse layers onto a limited number of different motor outputs. The reformatting of sensory representations we observe here may mark the beginning of this sensory-motor transition in the olfactory system.

The factors shaping cometary nuclei are still largely unknown, but could be the result of concurrent effects of evolutionary and primordial processes. The peculiar bilobed shape of comet 67P/Churyumov-Gerasimenko may be the result of the fusion of two objects that were once separate or the result of a localized excavation by outgassing at the interface between the two lobes. Here we report that the comet's major lobe is enveloped by a nearly continuous set of strata, up to 650 metres thick, which are independent of an analogous stratified envelope on the minor lobe. Gravity vectors computed for the two lobes separately are closer to perpendicular to the strata than those calculated for the entire nucleus and adjacent to the neck separating the two lobes. Therefore comet 67P/Churyumov-Gerasimenko is an accreted body of two distinct objects with 'onion-like' stratification, which formed before they merged. We conclude that gentle, low-velocity collisions occurred between two fully formed kilometre-sized cometesimals in the early stages of the Solar System. The notable structural similarities between the two lobes of comet 67P/Churyumov-Gerasimenko indicate that the early-forming cometesimals experienced similar primordial stratified accretion, even though they formed independently.

Neurotransmitter-gated ion channels of the Cys-loop receptor family are essential mediators of fast neurotransmission throughout the nervous system and are implicated in many neurological disorders. Available X-ray structures of prokaryotic and eukaryotic Cys-loop receptors provide tremendous insights into the binding of agonists, the subsequent opening of the ion channel, and the mechanism of channel activation. Yet the mechanism of inactivation by antagonists remains unknown. Here we present a 3.0 Å X-ray structure of the human glycine receptor-α3 homopentamer in complex with a high affinity, high-specificity antagonist, strychnine. Our structure allows us to explore in detail the molecular recognition of antagonists. Comparisons with previous structures reveal a mechanism for antagonist-induced inactivation of Cys-loop receptors, involving an expansion of the orthosteric binding site in the extracellular domain that is coupled to closure of the ion pore in the transmembrane domain.