Neuroligins are postsynaptic adhesion molecules that are essential for postsynaptic specialization and synaptic function. But the underlying molecular mechanisms of neuroligin functions remain unclear. We found that Drosophila Neuroligin 1 (DNlg1) regulates synaptic structure and function through WAVE regulatory complex (WRC)-mediated postsynaptic actin reorganization. The disruption of DNlg1, DNlg2, or their presynaptic partner neurexin (DNrx) led to a dramatic decrease in the amount of F-actin. Further study showed that DNlg1, but not DNlg2 or DNlg3, directly interacts with the WRC via its C-terminal interacting receptor sequence. That interaction is required to recruit WRC to the postsynaptic membrane to promote F-actin assembly. Furthermore, the interaction between DNlg1 and the WRC is essential for DNlg1 to rescue the morphological and electrophysiological defects in dnlg1 mutants. Our results reveal a novel mechanism by which the DNrx-DNlg1 trans-synaptic interaction coordinates structural and functional properties at the neuromuscular junction.

Grid cells in the entorhinal cortex allow for the precise decoding of position in space. Along with potentially playing an important role in navigation, grid cells have recently been hypothesized to make a general contribution to mental operations. A prerequisite for this hypothesis is that grid cell activity does not critically depend on physical movement. Here, we show that movement of covert attention, without any physical movement, also elicits spatial receptive fields with a triangular tiling of space. In monkeys trained to maintain central fixation while covertly attending to a stimulus moving in the periphery we identified a significant population (20/141, 14% neurons at a FDR <5%) of entorhinal cells with spatially structured receptive fields. This contrasts with recordings obtained in the hippocampus, where grid-like representations were not observed. Our results provide evidence that neurons in macaque entorhinal cortex do not rely on physical movement.

Although, in most animals, brain connectivity varies between individuals, behaviour is often similar across a species. What fundamental structural properties are shared across individual networks that define this behaviour? We describe a probabilistic model of connectivity in the hatchling Xenopus tadpole spinal cord which, when combined with a spiking model, reliably produces rhythmic activity corresponding to swimming. The probabilistic model allows calculation of structural characteristics that reflect common network properties, independent of individual network realisations. We use the structural characteristics to study examples of neuronal dynamics, in the complete network and various sub-networks, and this allows us to explain the basis for key experimental findings, and make predictions for experiments. We also study how structural and functional features differ between detailed anatomical connectomes and those generated by our new, simpler, model (meta-model).

Phosphorylation of the μ-opioid receptor (MOR) is known as a key step in desensitization and internalization but the role in the development of long-term tolerance at the cellular level is not known. Viral expression of wild type (exWT) and mutant MORs, where all phosphorylation sites on the C-terminus (Total Phosphorylation Deficient (TPD)) were mutated to alanine, were examined in locus coeruleus neurons in a MOR knockout rat. Both receptors activated potassium conductance similar to endogenous receptors in wild type animals. The exWT receptors, like endogenous receptors, acutely desensitized, internalized and, after chronic morphine treatment, displayed signs of tolerance. However, TPD receptors did not desensitize or internalize with agonist treatment. In addition the TPD receptors did not develop cellular tolerance following chronic morphine treatment. Thus C-terminal phosphorylation is necessary for the expression of acute desensitization, trafficking and one sign of long-term tolerance to morphine at the cellular level.

The immediate evolutionary space accessible to HIV is largely determined by how single amino acid mutations affect fitness. These mutational effects can shift as the virus evolves. However, the prevalence of such shifts in mutational effects remains unclear. Here, we quantify the effects on viral growth of all amino acid mutations to two HIV envelope (Env) proteins that differ at [Formula: see text]100 residues. Most mutations similarly affect both Envs, but the amino acid preferences of a minority of sites have clearly shifted. These shifted sites usually prefer a specific amino acid in one Env, but tolerate many amino acids in the other. Surprisingly, shifts are only slightly enriched at sites that have substituted between the Envs-and many occur at residues that do not even contact substitutions. Therefore, long-range epistasis can unpredictably shift Env's mutational tolerance during HIV evolution, although the amino acid preferences of most sites are conserved between moderately diverged viral strains.

Astrocytes play an important role in Rett syndrome (RTT) disease progression. Although the non-cell-autonomous effect of RTT astrocytes on neurons was documented, cell-autonomous phenotypes and mechanisms within RTT astrocytes are not well understood. We report that spontaneous calcium activity is abnormal in RTT astrocytes in vitro, in situ, and in vivo. Such abnormal calcium activity is mediated by calcium overload in the endoplasmic reticulum caused by abnormal store operated calcium entry, which is in part dependent on elevated expression of TRPC4. Furthermore, the abnormal calcium activity leads to excessive activation of extrasynaptic NMDA receptors (eNMDARs) on neighboring neurons and increased network excitability in Mecp2 knockout mice. Finally, both the abnormal astrocytic calcium activity and the excessive activation of eNMDARs are caused by Mecp2 deletion in astrocytes in vivo. Our findings provide evidence that abnormal calcium homeostasis is a key cell-autonomous phenotype in RTT astrocytes, and reveal its mechanism and consequence.

Type II topoisomerases manage DNA supercoiling and aid chromosome segregation using a complex, ATP-dependent duplex strand passage mechanism. Type IIB topoisomerases and their homologs support both archaeal/plant viability and meiotic recombination. Topo VI, a prototypical type IIB topoisomerase, comprises two Top6A and two Top6B protomers; how these subunits cooperate to engage two DNA segments and link ATP turnover to DNA transport is poorly understood. Using multiple biochemical approaches, we show that Top6B, which harbors the ATPase activity of topo VI, recognizes and exploits the DNA crossings present in supercoiled DNA to stimulate subunit dimerization by ATP. Top6B self-association in turn induces extensive DNA bending, which is needed to support duplex cleavage by Top6A. Our observations explain how topo VI tightly coordinates DNA crossover recognition and ATP binding with strand scission, providing useful insights into the operation of type IIB topoisomerases and related meiotic recombination and GHKL ATPase machineries.

The ultimate formation of a four-chambered heart allowing the separation of the pulmonary and systemic circuits was key for the evolutionary success of tetrapods. Complex processes of cell diversification and tissue morphogenesis allow the left and right cardiac compartments to become distinct but remain poorly understood. Here, we describe an unexpected laterality in the single zebrafish atrium analogous to that of the two atria in amniotes, including mammals. This laterality appears to derive from an embryonic antero-posterior asymmetry revealed by the expression of the transcription factor gene meis2b. In adult zebrafish hearts, meis2b expression is restricted to the left side of the atrium where it controls the expression of pitx2c, a regulator of left atrial identity in mammals. Altogether, our studies suggest that the multi-chambered atrium in amniotes arose from a molecular blueprint present before the evolutionary emergence of cardiac septation and provide insights into the establishment of atrial asymmetry.

CTCF and cohesin are key drivers of 3D-nuclear organization, anchoring the megabase-scale Topologically Associating Domains (TADs) that segment the genome. Here, we present and validate a computational method to predict cohesin-and-CTCF binding sites that form intra-TAD DNA loops. The intra-TAD loop anchors identified are structurally indistinguishable from TAD anchors regarding binding partners, sequence conservation, and resistance to cohesin knockdown; further, the intra-TAD loops retain key functional features of TADs, including chromatin contact insulation, blockage of repressive histone mark spread, and ubiquity across tissues. We propose that intra-TAD loops form by the same loop extrusion mechanism as the larger TAD loops, and that their shorter length enables finer regulatory control in restricting enhancer-promoter interactions, which enables selective, high-level expression of gene targets of super-enhancers and genes located within repressive nuclear compartments. These findings elucidate the role of intra-TAD cohesin-and-CTCF binding in nuclear organization associated with widespread insulation of distal enhancer activity.

Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.

Most eukaryotic parasites are obligately heteroxenous, requiring sequential infection of different host species in order to survive. Toxoplasma gondii is a rare exception to this rule, having a uniquely facultative heteroxenous life cycle. To understand the origins of this phenomenon, we compared development and stress responses in T. gondii to those of its its obligately heteroxenous relative, Hammondia hammondi and have identified multiple H. hammondi growth states that are distinct from those in T. gondii. Of these, the most dramatic difference was that H. hammondi was refractory to stressors that robustly induce cyst formation in T. gondii, and this was reflected most dramatically in its unchanging transcriptome after stress exposure. We also found that H. hammondi could be propagated in vitro for up to 8 days post-excystation, and we exploited this to generate the first ever transgenic H. hammondi line. Overall our data show that H. hammondi zoites grow as stringently regulated, unique life stages that are distinct from T. gondii tachyzoites, and implicate stress sensitivity as a potential developmental innovation that increased the flexibility of the T. gondii life cycle.

Perinatal brain injuries, including hippocampal lesions, cause lasting changes in dopamine function in rodents, but it is not known if this occurs in humans. We compared adults who were born very preterm with perinatal brain injury to those born very preterm without perinatal brain injury, and age-matched controls born at full term using [18F]-DOPA PET and structural MRI. Dopamine synthesis capacity was reduced in the perinatal brain injury group relative to those without brain injury (Cohen's d = 1.36, p=0.02) and the control group (Cohen's d = 1.07, p=0.01). Hippocampal volume was reduced in the perinatal brain injury group relative to controls (Cohen's d = 1.17, p=0.01) and was positively correlated with striatal dopamine synthesis capacity (r = 0.344, p=0.03). This is the first evidence in humans linking neonatal hippocampal injury to adult dopamine dysfunction, and provides a potential mechanism linking early life risk factors to adult mental illness.

CHARGE syndrome is caused by heterozygous mutations in the chromatin remodeler, CHD7, and is characterized by a set of malformations that, on clinical grounds, were historically postulated to arise from defects in neural crest formation during embryogenesis. To better delineate neural crest defects in CHARGE syndrome, we generated induced pluripotent stem cells (iPSCs) from two patients with typical syndrome manifestations, and characterized neural crest cells differentiated in vitro from these iPSCs (iPSC-NCCs). We found that expression of genes associated with cell migration was altered in CHARGE iPSC-NCCs compared to control iPSC-NCCs. Consistently, CHARGE iPSC-NCCs showed defective delamination, migration and motility in vitro, and their transplantation in ovo revealed overall defective migratory activity in the chick embryo. These results support the historical inference that CHARGE syndrome patients exhibit defects in neural crest migration, and provide the first successful application of patient-derived iPSCs in modeling craniofacial disorders.

Gene expression evolution through gene regulatory network (GRN) changes has gained appreciation as a driver of morphological evolution. However, understanding how GRNs evolve is hampered by finding relevant cis-regulatory element (CRE) mutations, and interpreting the protein-DNA interactions they alter. We investigated evolutionary changes in the duplicated Bric-à-brac (Bab) transcription factors and a key Bab target gene in a GRN underlying the novel dimorphic pigmentation of D. melanogaster and its relatives. It has remained uncertain how Bab was integrated within the pigmentation GRN. Here, we show that the ancestral transcription factor activity of Bab gained a role in sculpting sex-specific pigmentation through the evolution of binding sites in a CRE of the pigment-promoting yellow gene. This work demonstrates how a new trait can evolve by incorporating existing transcription factors into a GRN through CRE evolution, an evolutionary path likely to predominate newly evolved functions of transcription factors.

Cells respond to stress by remodeling their transcriptome through transcription and degradation. Xrn1p-dependent degradation in P-bodies is the most prevalent decay pathway, yet, P-bodies may facilitate not only decay, but also act as a storage compartment. However, which and how mRNAs are selected into different degradation pathways and what determines the fate of any given mRNA in P-bodies remain largely unknown. We devised a new method to identify both common and stress-specific mRNA subsets associated with P-bodies. mRNAs targeted for degradation to P-bodies, decayed with different kinetics. Moreover, the localization of a specific set of mRNAs to P-bodies under glucose deprivation was obligatory to prevent decay. Depending on its client mRNA, the RNA-binding protein Puf5p either promoted or inhibited decay. Furthermore, the Puf5p-dependent storage of a subset of mRNAs in P-bodies under glucose starvation may be beneficial with respect to chronological lifespan.

Single-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce 'Spot-On', an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants.

Ring-forming AAA+ chaperones exert ATP-fueled substrate unfolding by threading through a central pore. This activity is potentially harmful requiring mechanisms for tight repression and substrate-specific activation. The AAA+ chaperone ClpC with the peptidase ClpP forms a bacterial protease essential to virulence and stress resistance. The adaptor MecA activates ClpC by targeting substrates and stimulating ClpC ATPase activity. We show how ClpC is repressed in its ground state by determining ClpC cryo-EM structures with and without MecA. ClpC forms large two-helical assemblies that associate via head-to-head contacts between coiled-coil middle domains (MDs). MecA converts this resting state to an active planar ring structure by binding to MD interaction sites. Loss of ClpC repression in MD mutants causes constitutive activation and severe cellular toxicity. These findings unravel an unexpected regulatory concept executed by coiled-coil MDs to tightly control AAA+ chaperone activity.

Intracellular parasitism often results in gene loss, genome reduction, and dependence upon the host for cellular functioning. Rozellomycota is a clade comprising many such parasites and is related to the diverse, highly reduced, animal parasites, Microsporidia. We sequenced the nuclear and mitochondrial genomes of Paramicrosporidium saccamoebae [Rozellomycota], an intranuclear parasite of amoebae. A canonical fungal mitochondrial genome was recovered from P. saccamoebae that encodes genes necessary for the complete oxidative phosphorylation pathway including Complex I, differentiating it from most endoparasites including its sequenced relatives in Rozellomycota and Microsporidia. Comparative analysis revealed that P. saccamoebae shares more gene content with distantly related Fungi than with its closest relatives, suggesting that genome evolution in Rozellomycota and Microsporidia has been affected by repeated and independent gene losses, possibly as a result of variation in parasitic strategies (e.g. host and subcellular localization) or due to multiple transitions to parasitism.

How tissue-resident macrophages (TRM) impact adaptive immune responses remains poorly understood. We report novel mechanisms by which TRMs regulate T cell activities at tissue sites. These mechanisms are mediated by the complement receptor of immunoglobulin family (CRIg). Using animal models for autoimmune type 1 diabetes (T1D), we found that CRIg+ TRMs formed a protective barrier surrounding pancreatic islets. Genetic ablation of CRIg exacerbated islet inflammation and local T cell activation. CRIg exhibited a dual function of attenuating early T cell activation and promoting the differentiation of Foxp3+ regulatory (Treg) cells. More importantly, CRIg stabilized the expression of Foxp3 in Treg cells, by enhancing their responsiveness to interleukin-2. The expression of CRIg in TRMs was postnatally regulated by gut microbial signals and metabolites. Thus, environmental cues instruct TRMs to express CRIg, which functions as an immune checkpoint molecule to regulate adaptive immunity and promote immune tolerance.

Odor-preferences are usually influenced by life experiences. However, the neural circuit mechanisms remain unclear. The medial olfactory tubercle (mOT) is involved in both reward and olfaction, whereas the ventral tegmental area (VTA) dopaminergic (DAergic) neurons are considered to be engaged in reward and motivation. Here, we found that the VTA (DAergic)-mOT pathway could be activated by different types of naturalistic rewards as well as odors in DAT-cre mice. Optogenetic activation of the VTA-mOT DAergic fibers was able to elicit preferences for space, location and neutral odor, while pharmacological blockade of the dopamine receptors in the mOT fully prevented the odor-preference formation. Furthermore, inactivation of the mOT-projecting VTA DAergic neurons eliminated the previously formed odor-preference and strongly affected the Go-no go learning efficiency. In summary, our results revealed that the VTA (DAergic)-mOT pathway mediates a variety of naturalistic reward processes and different types of preferences including odor-preference in mice.