Mammalian spermatozoa gain competence to fertilize an oocyte as they travel through the female reproductive tract. This process is accompanied by an elevation of sperm intracellular calcium and a membrane hyperpolarization. The latter is evoked by K(+) efflux; however, the molecular identity of the potassium channel of human spermatozoa (hKSper) is unknown. Here, we characterize hKSper, reporting that it is regulated by intracellular calcium but is insensitive to intracellular alkalinization. We also show that human KSper is inhibited by charybdotoxin, iberiotoxin, and paxilline, while mouse KSper is insensitive to these compounds. Such unique properties suggest that the Slo1 ion channel is the molecular determinant for hKSper. We show that Slo1 is localized to the sperm flagellum and is inhibited by progesterone. Inhibition of hKSper by progesterone may depolarize the spermatozoon to open the calcium channel CatSper, thus raising [Ca(2+)] to produce hyperactivation and allowing sperm to fertilize an oocyte. DOI:http://dx.doi.org/10.7554/eLife.01009.001.

Songbirds use auditory feedback to learn and maintain their songs, but how feedback interacts with vocal motor circuitry remains unclear. A potential site for this interaction is the song premotor nucleus HVC, which receives auditory input and contains neurons (HVCX cells) that innervate an anterior forebrain pathway (AFP) important to feedback-dependent vocal plasticity. Although the singing-related output of HVCX cells is unaltered by distorted auditory feedback (DAF), deafening gradually weakens synapses on HVCX cells, raising the possibility that they integrate feedback only at subthreshold levels during singing. Using intracellular recordings in singing zebra finches, we found that DAF failed to perturb singing-related synaptic activity of HVCX cells, although many of these cells responded to auditory stimuli in non-singing states. Moreover, in vivo multiphoton imaging revealed that deafening-induced changes to HVCX synapses require intact AFP output. These findings support a model in which the AFP accesses feedback independent of HVC. DOI: http://dx.doi.org/10.7554/eLife.01833.001.

In embryonic stem cells (ESCs), the Tip60 histone acetyltransferase activates genes required for proliferation and silences genes that promote differentiation. Here we show that the class II histone deacetylase Hdac6 co-purifies with Tip60-p400 complex from ESCs. Hdac6 is necessary for regulation of most Tip60-p400 target genes, particularly those repressed by the complex. Unlike differentiated cells, where Hdac6 is mainly cytoplasmic, Hdac6 is largely nuclear in ESCs, neural stem cells (NSCs), and some cancer cell lines, and interacts with Tip60-p400 in each. Hdac6 localizes to promoters bound by Tip60-p400 in ESCs, binding downstream of transcription start sites. Surprisingly, Hdac6 does not appear to deacetylate histones, but rather is required for Tip60-p400 binding to many of its target genes. Finally, we find that, like canonical subunits of Tip60-p400, Hdac6 is necessary for robust ESC differentiation. These data suggest that Hdac6 plays a major role in the modulation of Tip60-p400 function in stem cells. DOI: http://dx.doi.org/10.7554/eLife.01557.001.

Natural selection of variants within the Arabidopsis thaliana circadian clock can be attributed to adaptation to varying environments. To define a basis for such variation, we examined clock speed in a reporter-modified Bay-0 x Shakdara recombinant inbred line and localized heritable variation. Extensive variation led us to identify EARLY FLOWERING3 (ELF3) as a major quantitative trait locus (QTL). The causal nucleotide polymorphism caused a short-period phenotype under light and severely dampened rhythm generation in darkness, and entrainment alterations resulted. We found that ELF3-Sha protein failed to properly localize to the nucleus, and its ability to accumulate in darkness was compromised. Evidence was provided that the ELF3-Sha allele originated in Central Asia. Collectively, we showed that ELF3 protein plays a vital role in defining its light-repressor action in the circadian clock and that its functional abilities are largely dependent on its cellular localization.

Decision making often involves a tradeoff between speed and accuracy. Previous studies indicate that neural activity in the lateral intraparietal area (LIP) represents the gradual accumulation of evidence toward a threshold level, or evidence bound, which terminates the decision process. The level of this bound is hypothesized to mediate the speed-accuracy tradeoff. To test this, we recorded from LIP while monkeys performed a motion discrimination task in two speed-accuracy regimes. Surprisingly, the terminating threshold levels of neural activity were similar in both regimes. However, neurons recorded in the faster regime exhibited stronger evidence-independent activation from the beginning of decision formation, effectively reducing the evidence-dependent neural modulation needed for choice commitment. Our results suggest that control of speed vs accuracy may be exerted through changes in decision-related neural activity itself rather than through changes in the threshold applied to such neural activity to terminate a decision.

As the major mechanism of plant growth and morphogenesis, cell elongation is controlled by many hormonal and environmental signals. How these signals are coordinated at the molecular level to ensure coherent cellular responses remains unclear. In this study, we illustrate a molecular circuit that integrates all major growth-regulating signals, including auxin, brassinosteroid, gibberellin, light, and temperature. Analyses of genome-wide targets, genetic and biochemical interactions demonstrate that the auxin-response factor ARF6, the light/temperature-regulated transcription factor PIF4, and the brassinosteroid-signaling transcription factor BZR1, interact with each other and cooperatively regulate large numbers of common target genes, but their DNA-binding activities are blocked by the gibberellin-inactivated repressor RGA. In addition, a tripartite HLH/bHLH module feedback regulates PIFs and additional bHLH factors that interact with ARF6, and thereby modulates auxin sensitivity according to developmental and environmental cues. Our results demonstrate a central growth-regulation circuit that integrates hormonal, environmental, and developmental controls of cell elongation in Arabidopsis hypocotyl.

Mineral malnutrition stemming from undiversified plant-based diets is a top global challenge. In C3 plants (e.g., rice, wheat), elevated concentrations of atmospheric carbon dioxide (eCO2) reduce protein and nitrogen concentrations, and can increase the total non-structural carbohydrates (TNC; mainly starch, sugars). However, contradictory findings have obscured the effect of eCO2 on the ionome-the mineral and trace-element composition-of plants. Consequently, CO2-induced shifts in plant quality have been ignored in the estimation of the impact of global change on humans. This study shows that eCO2 reduces the overall mineral concentrations (-8%, 95% confidence interval: -9.1 to -6.9, p<0.00001) and increases TNC:minerals > carbon:minerals in C3 plants. The meta-analysis of 7761 observations, including 2264 observations at state of the art FACE centers, covers 130 species/cultivars. The attained statistical power reveals that the shift is systemic and global. Its potential to exacerbate the prevalence of 'hidden hunger' and obesity is discussed.DOI: http://dx.doi.org/10.7554/eLife.02245.001.

Brown adipose tissue (BAT) plays an essential role in metabolic homeostasis by dissipating energy via thermogenesis through uncoupling protein 1 (UCP1). Previously, we reported that the TATA-binding protein associated factor 7L (TAF7L) is an important regulator of white adipose tissue (WAT) differentiation. In this study, we show that TAF7L also serves as a molecular switch between brown fat and muscle lineages in vivo and in vitro. In adipose tissue, TAF7L-containing TFIID complexes associate with PPARγ to mediate DNA looping between distal enhancers and core promoter elements. Our findings suggest that the presence of the tissue-specific TAF7L subunit in TFIID functions to promote long-range chromatin interactions during BAT lineage specification.

The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, to release E2F transcription factors. However, this model remains unproven biochemically and the biologically active form(s) of Rb remains unknown. In this study, we find that Rb is exclusively mono-phosphorylated in early G1 phase by cyclin D:Cdk4/6. Mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but show preferential E2F binding patterns. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by quantum hyper-phosphorylation. Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription, whereas cells undergoing differentiation utilize un-phosphorylated Rb. These observations fundamentally change our understanding of G1 cell cycle progression and show that mono-phosphorylated Rb, generated by cyclin D:Cdk4/6, is the only Rb isoform in early G1 phase.

The ESCRT machinery along with the AAA+ ATPase Vps4 drive membrane scission for trafficking into multivesicular bodies in the endocytic pathway and for the topologically related processes of viral budding and cytokinesis, but how they accomplish this remains unclear. Using deep-etch electron microscopy, we find that endogenous ESCRT-III filaments stabilized by depleting cells of Vps4 create uniform membrane-deforming conical spirals which are assemblies of specific ESCRT-III heteropolymers. To explore functional roles for ESCRT-III filaments, we examine HIV-1 Gag-mediated budding of virus-like particles and find that depleting Vps4 traps ESCRT-III filaments around nascent Gag assemblies. Interpolating between the observed structures suggests a new role for Vps4 in separating ESCRT-III from Gag or other cargo to allow centripetal growth of a neck constricting ESCRT-III spiral.

Biological homeostasis invokes modulatory responses aimed at stabilizing internal conditions. Using tunable photo- and mechano-stimulation, we identified two distinct categories of homeostatic responses during the sleep-like state of Caenorhabditis elegans (lethargus). In the presence of weak or no stimuli, extended motion caused a subsequent extension of quiescence. The neuropeptide Y receptor homolog, NPR-1, and an inhibitory neuropeptide known to activate it, FLP-18, were required for this process. In the presence of strong stimuli, the correlations between motion and quiescence were disrupted for several minutes but homeostasis manifested as an overall elevation of the time spent in quiescence. This response to strong stimuli required the function of the DAF-16/FOXO transcription factor in neurons, but not that of NPR-1. Conversely, response to weak stimuli did not require the function of DAF-16/FOXO. These findings suggest that routine homeostatic stabilization of sleep may be distinct from homeostatic compensation following a strong disturbance.

The HIV-1 protein Rev controls a critical step in viral replication by mediating the nuclear export of unspliced and singly-spliced viral mRNAs. Multiple Rev subunits assemble on the Rev Response Element (RRE), a structured region present in these RNAs, and direct their export through the Crm1 pathway. Rev-RRE assembly occurs via several Rev oligomerization and RNA-binding steps, but how these steps are coordinated to form an export-competent complex is unclear. Here, we report the first crystal structure of a Rev dimer-RRE complex, revealing a dramatic rearrangement of the Rev-dimer upon RRE binding through re-packing of its hydrophobic protein-protein interface. Rev-RNA recognition relies on sequence-specific contacts at the well-characterized IIB site and local RNA architecture at the second site. The structure supports a model in which the RRE utilizes the inherent plasticity of Rev subunit interfaces to guide the formation of a functional complex.

Many insect species are host-obligate specialists. The evolutionary mechanism driving the adaptation of a species to a toxic host is, however, intriguing. We analyzed the tight association of Drosophila sechellia to its sole host, the fruit of Morinda citrifolia, which is toxic to other members of the melanogaster species group. Molecular polymorphisms in the dopamine regulatory protein Catsup cause infertility in D. sechellia due to maternal arrest of oogenesis. In its natural host, the fruit compensates for the impaired maternal dopamine metabolism with the precursor l-DOPA, resuming oogenesis and stimulating egg production. l-DOPA present in morinda additionally increases the size of D. sechellia eggs, what in turn enhances early fitness. We argue that the need of l-DOPA for successful reproduction has driven D. sechellia to become an M. citrifolia obligate specialist. This study illustrates how an insect's dopaminergic system can sustain ecological adaptations by modulating ontogenesis and development.

Zebrafish larvae show characteristic prey capture behavior in response to small moving objects. The neural mechanism used to recognize objects as prey remains largely unknown. We devised a machine learning behavior classification system to quantify hunting kinematics in semi-restrained animals exposed to a range of virtual stimuli. Two-photon calcium imaging revealed a small visual area, AF7, that was activated specifically by the optimal prey stimulus. This pretectal region is innervated by two types of retinal ganglion cells, which also send collaterals to the optic tectum. Laser ablation of AF7 markedly reduced prey capture behavior. We identified neurons with arbors in AF7 and found that they projected to multiple sensory and premotor areas: the optic tectum, the nucleus of the medial longitudinal fasciculus (nMLF) and the hindbrain. These findings indicate that computations in the retina give rise to a visual stream which transforms sensory information into a directed prey capture response.

Embryonic polarity of invertebrates, amphibians and fish is specified largely by maternal determinants, which fixes cell fates early in development. In contrast, amniote embryos remain plastic and can form multiple individuals until gastrulation. How is their polarity determined? In the chick embryo, the earliest known factor is cVg1 (homologous to mammalian growth differentiation factor 1, GDF1), a transforming growth factor beta (TGFβ) signal expressed posteriorly before gastrulation. A molecular screen to find upstream regulators of cVg1 in normal embryos and in embryos manipulated to form twins now uncovers the transcription factor Pitx2 as a candidate. We show that Pitx2 is essential for axis formation, and that it acts as a direct regulator of cVg1 expression by binding to enhancers within neighbouring genes. Pitx2, Vg1/GDF1 and Nodal are also key actors in left-right asymmetry, suggesting that the same ancient polarity determination mechanism has been co-opted to different functions during evolution.

The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50° kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface.

How different helicase families with a conserved catalytic 'helicase core' evolved to function on varied RNA and DNA substrates by diverse mechanisms remains unclear. In this study, we used Mss116, a yeast DEAD-box protein that utilizes ATP to locally unwind dsRNA, to investigate helicase specificity and mechanism. Our results define the molecular basis for the substrate specificity of a DEAD-box protein. Additionally, they show that Mss116 has ambiguous substrate-binding properties and interacts with all four NTPs and both RNA and DNA. The efficiency of unwinding correlates with the stability of the 'closed-state' helicase core, a complex with nucleotide and nucleic acid that forms as duplexes are unwound. Crystal structures reveal that core stability is modulated by family-specific interactions that favor certain substrates. This suggests how present-day helicases diversified from an ancestral core with broad specificity by retaining core closure as a common catalytic mechanism while optimizing substrate-binding interactions for different cellular functions.

Histone modifiers play essential roles in controlling transcription and organizing eukaryotic genomes into functional domains. Here, we show that Set1, the catalytic subunit of the highly conserved Set1C/COMPASS complex responsible for histone H3K4 methylation (H3K4me), behaves as a repressor of the transcriptome largely independent of Set1C and H3K4me in the fission yeast Schizosaccharomyces pombe. Intriguingly, while Set1 is enriched at highly expressed and repressed loci, Set1 binding levels do not generally correlate with the levels of transcription. We show that Set1 is recruited by the ATF/CREB homolog Atf1 to heterochromatic loci and promoters of stress-response genes. Moreover, we demonstrate that Set1 coordinates with the class II histone deacetylase Clr3 in heterochromatin assembly at prominent chromosomal landmarks and repression of the transcriptome that includes Tf2 retrotransposons, noncoding RNAs, and regulators of development and stress-responses. Our study delineates a molecular framework for elucidating the functional links between transcriptome control and chromatin organization.

Loss of Foxc1 is associated with Dandy-Walker malformation, the most common human cerebellar malformation characterized by cerebellar hypoplasia and an enlarged posterior fossa and fourth ventricle. Although expressed in the mouse posterior fossa mesenchyme, loss of Foxc1 non-autonomously induces a rapid and devastating decrease in embryonic cerebellar ventricular zone radial glial proliferation and concurrent increase in cerebellar neuronal differentiation. Subsequent migration of cerebellar neurons is disrupted, associated with disordered radial glial morphology. In vitro, SDF1α, a direct Foxc1 target also expressed in the head mesenchyme, acts as a cerebellar radial glial mitogen and a chemoattractant for nascent Purkinje cells. Its receptor, Cxcr4, is expressed in cerebellar radial glial cells and conditional Cxcr4 ablation with Nes-Cre mimics the Foxc1-/- cerebellar phenotype. SDF1α also rescues the Foxc1-/- phenotype. Our data emphasizes that the head mesenchyme exerts a considerable influence on early embryonic brain development and its disruption contributes to neurodevelopmental disorders in humans.

The steps from HIV-1 cytoplasmic entry until integration of the reverse transcribed genome are currently enigmatic. They occur in ill-defined reverse-transcription- and pre-integration-complexes (RTC, PIC) with various host and viral proteins implicated. In this study, we report quantitative detection of functional RTC/PIC by labeling nascent DNA combined with detection of viral integrase. We show that the viral CA (capsid) protein remains associated with cytoplasmic RTC/PIC but is lost on nuclear PIC in a HeLa-derived cell line. In contrast, nuclear PIC were almost always CA-positive in primary human macrophages, indicating nuclear import of capsids or capsid-like structures. We further show that the CA-targeted inhibitor PF74 exhibits a bimodal mechanism, blocking RTC/PIC association with the host factor CPSF6 and nuclear entry at low, and abrogating reverse transcription at high concentrations. The newly developed system is ideally suited for studying retroviral post-entry events and the roles of host factors including DNA sensors and signaling molecules.