In their active GTP-bound form, Rab proteins interact with proteins termed effector molecules. In this study, we have thoroughly characterized a Rab effector domain that is present in proteins of the Mical and EHBP families, both known to act in endosomal trafficking. Within our study, we show that these effectors display a preference for Rab8 family proteins (Rab8, 10, 13 and 15) and that some of the effector domains can bind two Rab proteins via separate binding sites. Structural analysis allowed us to explain the specificity towards Rab8 family members and the presence of two similar Rab binding sites that must have evolved via gene duplication. This study is the first to thoroughly characterize a Rab effector protein that contains two separate Rab binding sites within a single domain, allowing Micals and EHBPs to bind two Rabs simultaneously, thus suggesting previously unknown functions of these effector molecules in endosomal trafficking.

Planarians regenerate all body parts after injury, including the central nervous system (CNS). We capitalized on this distinctive trait and completed a gene expression-guided functional screen to identify factors that regulate diverse aspects of neural regeneration in Schmidtea mediterranea. Our screen revealed molecules that influence neural cell fates, support the formation of a major connective hub, and promote reestablishment of chemosensory behavior. We also identified genes that encode signaling molecules with roles in head regeneration, including some that are produced in a previously uncharacterized parenchymal population of cells. Finally, we explored genes downregulated during planarian regeneration and characterized, for the first time, glial cells in the planarian CNS that respond to injury by repressing several transcripts. Collectively, our studies revealed diverse molecules and cell types that underlie an animal's ability to regenerate its brain.

The pre-placodal ectoderm, marked by the expression of the transcription factor Six1 and its co-activator Eya1, develops into placodes and ultimately into many cranial sensory organs and ganglia. Using RNA-Seq in Xenopus laevis we screened for presumptive direct placodal target genes of Six1 and Eya1 by overexpressing hormone-inducible constructs of Six1 and Eya1 in pre-placodal explants, and blocking protein synthesis before hormone-inducing nuclear translocation of Six1 or Eya1. Comparing the transcriptome of explants with non-induced controls, we identified hundreds of novel Six1/Eya1 target genes with potentially important roles for placode development. Loss-of-function studies confirmed that target genes encoding known transcriptional regulators of progenitor fates (e.g. Sox2, Hes8) and neuronal/sensory differentiation (e.g. Ngn1, Atoh1, Pou4f1, Gfi1) require Six1 and Eya1 for their placodal expression. Our findings provide insights into the gene regulatory network regulating placodal neurogenesis downstream of Six1 and Eya1 suggesting new avenues of research into placode development and disease.

How movements are selected is a fundamental question in systems neuroscience. While many studies have elucidated the sensorimotor transformations underlying stimulus-guided movements, less is known about how internal goals - critical drivers of goal-directed behavior - guide movements. The basal ganglia are known to bias movement selection according to value, one form of internal goal. Here, we examine whether other internal goals, in addition to value, also influence movements via the basal ganglia. We designed a novel task for mice that dissociated equally rewarded internally-specified and stimulus-guided movements, allowing us to test how each engaged the basal ganglia. We found that activity in the substantia nigra pars reticulata, a basal ganglia output, predictably differed preceding internally-specified and stimulus-guided movements. Incorporating these results into a simple model suggests that internally-specified movements may be facilitated relative to stimulus-guided movements by basal ganglia processing.

Herbivore natural enemies protect plants by regulating herbivore populations. Whether they can alter the behavior of their prey to increase predation success is unknown. We investigate if and how infection by the entomopathogenic nematode Heterorhabditis bacteriophora changes the behavior of healthy larvae of the western corn rootworm (Diabrotica virgifera), a major pest of maize. We found that nematode-infected rootworm cadavers are attractive to rootworm larvae, and that this behavior increases nematode reproductive success. Nematode-infected rootworms release distinct volatile bouquets, including the unusual volatile butylated hydroxytoluene (BHT). BHT alone attracts rootworms, and increases nematode reproductive success. A screen of different nematode and herbivore species shows that attraction of healthy hosts to nematode-infected cadavers is widespread and likely involves species-specific volatile cues. This study reveals a new facet of the biology of herbivore natural enemies that boosts their predation success by increasing the probability of host encounters.

A longstanding question is how influenza virus evolves to escape human immunity, which is polyclonal and can target many distinct epitopes. Here, we map how all amino-acid mutations to influenza's major surface protein affect viral neutralization by polyclonal human sera. The serum of some individuals is so focused that it selects single mutations that reduce viral neutralization by over an order of magnitude. However, different viral mutations escape the sera of different individuals. This individual-to-individual variation in viral escape mutations is not present among ferrets that have been infected just once with a defined viral strain. Our results show how different single mutations help influenza virus escape the immunity of different members of the human population, a phenomenon that could shape viral evolution and disease susceptibility.

The human brain recurrently prioritizes task-relevant over task-irrelevant visual information. A central question is whether multiple objects can be prioritized simultaneously. To answer this, we let observers search for two colored targets among distractors. Crucially, we independently varied the number of target colors that observers anticipated, and the number of target colors actually used to distinguish the targets in the display. This enabled us to dissociate the preparation of selection mechanisms from the actual engagement of such mechanisms. Multivariate classification of electroencephalographic activity allowed us to track selection of each target separately across time. The results revealed only small neural and behavioral costs associated with preparing for selecting two objects, but substantial costs when engaging in selection. Further analyses suggest this cost is the consequence of neural competition resulting in limited parallel processing, rather than a serial bottleneck. The findings bridge diverging theoretical perspectives on capacity limitations of feature-based attention.

The principles underlying the animacy organization of the ventral temporal cortex (VTC) remain hotly debated, with recent evidence pointing to an animacy continuum rather than a dichotomy. What drives this continuum? According to the visual categorization hypothesis, the continuum reflects the degree to which animals contain animal-diagnostic features. By contrast, the agency hypothesis posits that the continuum reflects the degree to which animals are perceived as (social) agents. Here, we tested both hypotheses with a stimulus set in which visual categorizability and agency were dissociated based on representations in convolutional neural networks and behavioral experiments. Using fMRI, we found that visual categorizability and agency explained independent components of the animacy continuum in VTC. Modeled together, they fully explained the animacy continuum. Finally, clusters explained by visual categorizability were localized posterior to clusters explained by agency. These results show that multiple organizing principles, including agency, underlie the animacy continuum in VTC.

Genes can encode multiple isoforms, broadening their functions and providing a molecular substrate to evolve phenotypic diversity. Evolution of isoform function is a potential route to adapt to new environments. Here we show that de novo, beneficial alleles in the nurf-1 gene became fixed in two laboratory lineages of C. elegans after isolation from the wild in 1951, before methods of cryopreservation were developed. nurf-1 encodes an ortholog of BPTF, a large (>300 kD) multidomain subunit of the NURF chromatin remodeling complex. Using CRISPR-Cas9 genome editing and transgenic rescue, we demonstrate that in C. elegans, nurf-1 has split into two, largely non-overlapping isoforms (NURF-1.D and NURF-1.B, which we call Yin and Yang, respectively) that share only two of 26 exons. Both isoforms are essential for normal gametogenesis but have opposite effects on male/female gamete differentiation. Reproduction in hermaphrodites, which involves production of both sperm and oocytes, requires a balance of these opposing Yin and Yang isoforms. Transgenic rescue and genetic position of the fixed mutations suggest that different isoforms are modified in each laboratory strain. In a related clade of Caenorhabditis nematodes, the shared exons have duplicated, resulting in the split of the Yin and Yang isoforms into separate genes, each containing approximately 200 amino acids of duplicated sequence that has undergone accelerated protein evolution following the duplication. Associated with this duplication event is the loss of two additional nurf-1 transcripts, including the long-form transcript and a newly identified, highly expressed transcript encoded by the duplicated exons. We propose these lost transcripts are non-functional side products necessary to transcribe the Yin and Yang transcripts in the same cells. Our work demonstrates how gene sharing, through the production of multiple isoforms, can precede the creation of new, independent genes.

Kinesin-1 and cytoplasmic dynein are microtubule (MT) motors that transport intracellular cargoes. It remains unclear how these motors move along MTs densely coated with obstacles of various sizes in the cytoplasm. Here, we tested the ability of single and multiple motors to bypass synthetic obstacles on MTs in vitro. Contrary to previous reports, we found that single mammalian dynein is highly capable of bypassing obstacles. Single human kinesin-1 motors fail to avoid obstacles, consistent with their inability to take sideways steps on to neighboring MT protofilaments. Kinesins overcome this limitation when working in teams, bypassing obstacles as effectively as multiple dyneins. Cargos driven by multiple kinesins or dyneins are also capable of rotating around the MT to bypass large obstacles. These results suggest that multiplicity of motors is required not only for transporting cargos over long distances and generating higher forces, but also for maneuvering cargos on obstacle-coated MT surfaces.

The endoplasmic reticulum (ER) imports ATP and uses energy from ATP hydrolysis for protein folding and trafficking. However, little is known about how this vital ATP transport occurs across the ER membrane. Here, using three commonly used cell lines (CHO, INS1 and HeLa), we report that ATP enters the ER lumen through a cytosolic Ca2+-antagonized mechanism, or CaATiER (Ca2+-Antagonized Transport into ER). Significantly, we show that mitochondria supply ATP to the ER and a SERCA-dependent Ca2+ gradient across the ER membrane is necessary for ATP transport into the ER, through SLC35B1/AXER. We propose that under physiological conditions, increases in cytosolic Ca2+ inhibit ATP import into the ER lumen to limit ER ATP consumption. Furthermore, the ATP level in the ER is readily depleted by oxidative phosphorylation (OxPhos) inhibitors and that ER protein misfolding increases ATP uptake from mitochondria into the ER. These findings suggest that ATP usage in the ER may increase mitochondrial OxPhos while decreasing glycolysis, i.e. an 'anti-Warburg' effect.

Patients with liver diseases often suffer from chronic itch, yet the pruritogen(s) and receptor(s) remain largely elusive. Here, we identify bile acids as natural ligands for MRGPRX4. MRGPRX4 is expressed in human dorsal root ganglion (hDRG) neurons and co-expresses with itch receptor HRH1. Bile acids elicited Ca2+ responses in cultured hDRG neurons, and bile acids or a MRGPRX4 specific agonist induced itch in human subjects. However, a specific agonist for another bile acid receptor TGR5 failed to induce itch in human subjects and we find that human TGR5 is not expressed in hDRG neurons. Finally, we show positive correlation between cholestatic itch and plasma bile acids level in itchy patients and the elevated bile acids is sufficient to activate MRGPRX4. Taken together, our data strongly suggest that MRGPRX4 is a novel bile acid receptor that likely underlies cholestatic itch in human, providing a promising new drug target for anti-itch therapies.

We previously showed in Drosophila melanogaster embryos that low-affinity Ultrabithorax (Ubx)-responsive shavenbaby (svb) enhancers drive expression using localized transcriptional environments and that active svb enhancers on different chromosomes tended to colocalize (Tsai et al., 2017). Here, we test the hypothesis that these multi-enhancer 'hubs' improve phenotypic resilience to stress by buffering against decreases in transcription factor concentrations and transcriptional output. Deleting a redundant enhancer from the svb locus led to reduced trichome numbers in embryos raised at elevated temperatures. Using high-resolution fluorescence microscopy, we observed lower Ubx concentration and transcriptional output in this deletion allele. Transcription sites of the full svb cis-regulatory region inserted into a different chromosome colocalized with the svb locus, increasing Ubx concentration, the transcriptional output of svb, and partially rescuing the phenotype. Thus, multiple enhancers could reinforce a local transcriptional hub to buffer against environmental stresses and genetic perturbations, providing a mechanism for phenotypical robustness.

The non-destructive collection of ultrathin sections on silicon wafers for post-embedding staining and volumetric correlative light and electron microscopy traditionally requires exquisite manual skills and is tedious and unreliable. In MagC introduced here, sample blocks are augmented with a magnetic resin enabling the remote actuation and collection of hundreds of sections on wafer. MagC allowed the correlative visualization of neuroanatomical tracers within their ultrastructural volumetric electron microscopy context.

Flavodiiron proteins (FDPs) constitute a group of modular enzymes widespread in Bacteria, Archaea and Eukarya. Synechocystis sp. PCC 6803 has four FDPs (Flv1-4), which are essential for the photoprotection of photosynthesis. A direct comparison of light-induced O2 reduction (Mehler-like reaction) under high (3% CO2, HC) and low (air level CO2, LC) inorganic carbon conditions demonstrated that the Flv1/Flv3 heterodimer is solely responsible for an efficient steady-state O2 photoreduction under HC, with flv2 and flv4 expression strongly down-regulated. Conversely, under LC conditions, Flv1/Flv3 acts only as a transient electron sink, due to the competing withdrawal of electrons by the highly induced NDH-1 complex. Further, in vivo evidence is provided indicating that Flv2/Flv4 contributes to the Mehler-like reaction when naturally expressed under LC conditions, or, when artificially overexpressed under HC. The O2 photoreduction driven by Flv2/Flv4 occurs down-stream of PSI in a coordinated manner with Flv1/Flv3 and supports slow and steady-state O2 photoreduction.

Overexpression of anti-apoptotic proteins MCL1 and Bcl-xL are frequently observed in many cancers. Inhibitors targeting MCL1 are in clinical development, however numerous cancer models are intrinsically resistant to this approach. To discover mechanisms underlying resistance to MCL1 inhibition, we performed multiple flow-cytometry based genome-wide CRISPR screens interrogating two drugs that directly (MCL1i) or indirectly (CDK9i) target MCL1. Remarkably, both screens identified three components (CUL5, RNF7 and UBE2F) of a cullin-RING ubiquitin ligase complex (CRL5) that resensitized cells to MCL1 inhibition. We find that levels of the BH3-only pro-apoptotic proteins Bim and Noxa are proteasomally regulated by the CRL5 complex. Accumulation of Noxa caused by depletion of CRL5 components was responsible for re-sensitization to CDK9 inhibitor, but not MCL1 inhibitor. Discovery of a novel role of CRL5 in apoptosis and resistance to multiple types of anticancer agents suggests the potential to improve combination treatments.

The human visual system is tasked with recovering the different physical sources of optical structure that generate our retinal images. Separate research has focused on understanding how the visual system estimates (a) environmental sources of image structure and (b) blur induced by the eye's limited focal range, but little is known about how the visual system distinguishes environmental sources from optical defocus. Here, we present evidence that this is a fundamental perceptual problem and provide insights into how and when the visual system succeeds and fails in solving it. We show that fully focused surface shading can be misperceived as defocused and that optical blur can be misattributed to the material properties and shape of surfaces. We further reveal how these misperceptions depend on the relationship between shading gradients and sharp contours, and conclude that computations of blur are inherently linked to computations of surface shape, material, and illumination.

Action potentials trigger neurotransmitter release at active zones, specialized release sites in axons. Many neurons also secrete neurotransmitters or neuromodulators from their somata and dendrites. However, it is unclear whether somatodendritic release employs specialized sites for release, and the molecular machinery for somatodendritic release is not understood. Here, we identify an essential role for the active zone protein RIM in stimulated somatodendritic dopamine release in the midbrain. In mice in which RIMs are selectively removed from dopamine neurons, action potentials failed to evoke significant somatodendritic release detected via D2 receptor-mediated currents. Compellingly, spontaneous dopamine release was normal upon RIM knockout. Dopamine neuron morphology, excitability, and dopamine release evoked by amphetamine, which reverses dopamine transporters, were also unaffected. We conclude that somatodendritic release employs molecular scaffolds to establish secretory sites for rapid dopamine signaling during firing. In contrast, basal release that is independent of action potential firing does not require RIM.

Cultured mouse peritoneal macrophages release large numbers of ~30-nm cholesterol-rich particles. Here, we show that those particles represent fragments of the plasma membrane that are pulled away and left behind during the projection and retraction of filopodia and lamellipodia. Consistent with this finding, the particles are enriched in proteins found in focal adhesions, which attach macrophages to the substrate. The release of particles is abolished by blocking cell movement (either by depolymerizing actin with latrunculin A or by inhibiting myosin II with blebbistatin). Confocal microscopy and NanoSIMS imaging studies revealed that the plasma membrane-derived particles are enriched in 'accessible cholesterol' (a mobile pool of cholesterol detectable with the modified cytolysin ALO-D4) but not in sphingolipid-sequestered cholesterol [a pool detectable with ostreolysin A (OlyA)]. The discovery that macrophages release cholesterol-rich particles during cellular locomotion is likely relevant to cholesterol efflux and could contribute to extracellular cholesterol deposition in atherosclerotic plaques.

Regulators of G-protein Signaling are a conserved family of proteins required in various biological processes including cell differentiation. We previously demonstrated that Rgs12 is essential for osteoclast differentiation and its deletion in vivo protected mice against pathological bone loss. To characterize its mechanism in osteoclastogenesis, we selectively deleted Rgs12 in C57BL/6J mice targeting osteoclast precursors using LyzM-driven Cre mice or overexpressed Rgs12 in RAW264.7 cells. Rgs12 deletion in vivo led to an osteopetrotic phenotype evidenced by increased trabecular bone, decreased osteoclast number and activity but no change in osteoblast number and bone formation. Rgs12 overexpression increased osteoclast number and size, and bone resorption activity. Proteomics analysis of Rgs12-depleted osteoclasts identified an upregulation of antioxidant enzymes under the transcriptional regulation of Nrf2, the master regulator of oxidative stress. We confirmed an increase of Nrf2 activity and impaired reactive oxygen species production in Rgs12-deficient cells. Conversely, Rgs12 overexpression suppressed Nrf2 through a mechanism dependent on the 26S proteasome, and promoted RANKL-induced phosphorylation of ERK1/2 and NFκB, which was abrogated by antioxidant treatment. Our study therefore identified a novel role of Rgs12 in regulating Nrf2, thereby controlling cellular redox state and osteoclast differentiation.