The mammalian vomeronasal organ encodes pheromone information about gender, reproductive status, genetic background and individual differences. It remains unknown how pheromone information interacts to trigger innate behaviors. In this study, we identify vomeronasal receptors responsible for detecting female pheromones. A sub-group of V1re clade members recognizes gender-identifying cues in female urine. Multiple members of the V1rj clade are cognate receptors for urinary estrus signals, as well as for sulfated estrogen (SE) compounds. In both cases, the same cue activates multiple homologous receptors, suggesting redundancy in encoding female pheromone cues. Neither gender-specific cues nor SEs alone are sufficient to promote courtship behavior in male mice, whereas robust courtship behavior can be induced when the two cues are applied together. Thus, integrated action of different female cues is required in pheromone-triggered mating behavior. These results suggest a gating mechanism in the vomeronasal circuit in promoting specific innate behavior.DOI: http://dx.doi.org/10.7554/eLife.03025.001.

The evolutionary rate of proteins involved in obligate protein-protein interactions is slower and the degree of coevolution higher than that for nonobligate protein-protein interactions. The coevolution of the proteins involved in certain nonobligate interactions is, however, essential to cell survival. To gain insight into the coevolution of one such nonobligate protein pair, the cytosolic ribonuclease inhibitor (RI) proteins and secretory pancreatic-type ribonucleases from cow (Bos taurus) and human (Homo sapiens) were produced in Escherichia coli and purified, and their physicochemical properties were analyzed. The two intraspecies complexes were found to be extremely tight (bovine Kd = 0.69 fM; human Kd = 0.34 fM). Human RI binds to its cognate ribonuclease (RNase 1) with 100-fold greater affinity than to the bovine homologue (RNase A). In contrast, bovine RI binds to RNase 1 and RNase A with nearly equal affinity. This broader specificity is consistent with there being more pancreatic-type ribonucleases in cows (20) than humans (13). Human RI (32 cysteine residues) also has 4-fold less resistance to oxidation by hydrogen peroxide than does bovine RI (29 cysteine residues). This decreased oxidative stability of human RI, which is caused largely by Cys74, implies a larger role for human RI as an antioxidant. The conformational and oxidative stabilities of both RIs increase upon complex formation with ribonucleases. Thus, RI has evolved to maintain its inhibition of invading ribonucleases, even when confronted with extreme environmental stress. That role appears to take precedence over its role in mediating oxidative damage.

Targeting small-molecule fluorescent indicators using genetically encoded protein tags yields new hybrid sensors for biological imaging. Optimization of such systems requires redesign of the synthetic indicator to allow cell-specific targeting without compromising the photophysical properties or cellular performance of the small-molecule probe. We developed a bright and sensitive Ca indicator by systematically exploring the relative configuration of dye and chelator, which can be targeted using the HaloTag self-labeling tag system. Our "isomeric tuning" approach is generalizable, yielding a far-red targetable indicator to visualize Ca fluxes in the primary cilium.

How pioneer factors interface with chromatin to promote accessibility for transcription control is poorly understood in vivo. Here, we directly visualize chromatin association by the prototypical GAGA pioneer factor (GAF) in live Drosophila hemocytes. Single-particle tracking reveals that most GAF is chromatin bound, with a stable-binding fraction showing nucleosome-like confinement residing on chromatin for more than 2 min, far longer than the dynamic range of most transcription factors. These kinetic properties require the full complement of GAF's DNA-binding, multimerization and intrinsically disordered domains, and are autonomous from recruited chromatin remodelers NURF and PBAP, whose activities primarily benefit GAF's neighbors such as Heat Shock Factor. Evaluation of GAF kinetics together with its endogenous abundance indicates that, despite on-off dynamics, GAF constitutively and fully occupies major chromatin targets, thereby providing a temporal mechanism that sustains open chromatin for transcriptional responses to homeostatic, environmental and developmental signals.

The measurement of ion concentrations and fluxes inside living cells is key to understanding cellular physiology. Fluorescent indicators that can infiltrate and provide intel on the cellular environment are critical tools for biological research. Developing these molecular informants began with the seminal work of Racker and colleagues ( (1979) 18, 2210), who demonstrated the passive loading of fluorescein in living cells to measure changes in intracellular pH. This work continues, employing a mix of old and new tradecraft to create innovative agents for monitoring ions inside living systems.

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

The Polycomb PRC1 plays essential roles in development and disease pathogenesis. Targeting of PRC1 to chromatin is thought to be mediated by the Cbx family proteins (Cbx2/4/6/7/8) binding to histone H3 with a K27me3 modification (H3K27me3). Despite this prevailing view, the molecular mechanisms of targeting remain poorly understood. Here, by combining live-cell single-molecule tracking (SMT) and genetic engineering, we reveal that H3K27me3 contributes significantly to the targeting of Cbx7 and Cbx8 to chromatin, but less to Cbx2, Cbx4, and Cbx6. Genetic disruption of the complex formation of PRC1 facilitates the targeting of Cbx7 to chromatin. Biochemical analyses uncover that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit. Live-cell SMT of Cbx7 mutants demonstrates that Cbx7 is targeted to chromatin by co-recognizing of H3K27me3 and DNA. Our data suggest a novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes.

One-third of the mammalian proteome is comprised of transmembrane and secretory proteins that are synthesized on endoplasmic reticulum (ER). Here, we investigate the spatial distribution and regulation of mRNAs encoding these membrane and secretory proteins (termed "secretome" mRNAs) through live cell, single molecule tracking to directly monitor the position and translation states of secretome mRNAs on ER and their relationship to other organelles. Notably, translation of secretome mRNAs occurred preferentially near lysosomes on ER marked by the ER junction-associated protein, Lunapark. Knockdown of Lunapark reduced the extent of secretome mRNA translation without affecting translation of other mRNAs. Less secretome mRNA translation also occurred when lysosome function was perturbed by raising lysosomal pH or inhibiting lysosomal proteases. Secretome mRNA translation near lysosomes was enhanced during amino acid deprivation. Addition of the integrated stress response inhibitor, ISRIB, reversed the translation inhibition seen in Lunapark knockdown cells, implying an eIF2 dependency. Altogether, these findings uncover a novel coordination between ER and lysosomes, in which local release of amino acids and other factors from ER-associated lysosomes patterns and regulates translation of mRNAs encoding secretory and membrane proteins.