Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched.

Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.

Cryo-EM Structures of the Magnesium Channel CorA Reveal Symmetry Break upon Gating.

CorA, the major Mg(2+) uptake system in prokaryotes, is gated by intracellular Mg(2+) (KD ∼ 1-2 mM). X-ray crystallographic studies of CorA show similar conformations under Mg(2+)-bound and Mg(2+)-free conditions, but EPR spectroscopic studies reveal large Mg(2+)-driven quaternary conformational changes. Here, we determined cryo-EM structures of CorA in the Mg(2+)-bound closed conformation and in two open Mg(2+)-free states at resolutions of 3.8, 7.1, and 7.1 Å, respectively. In the absence of bound Mg(2+), four of the five subunits are displaced to variable extents (∼ 10-25 Å) by hinge-like motions as large as ∼ 35° at the stalk helix. The transition between a single 5-fold symmetric closed state and an ensemble of low Mg(2+), open, asymmetric conformational states is, thus, the key structural signature of CorA gating. This mechanism is likely to apply to other structurally similar divalent ion channels.

Balancing act of a leading strand DNA polymerase-specific domain and its exonuclease domain promotes genome-wide sister replication fork symmetry.

Pol2 is the leading-strand DNA polymerase in budding yeast. Here we describe an antagonism between its conserved POPS (Pol2 family-specific catalytic core peripheral subdomain) and exonuclease domain and the importance of this antagonism in genome replication. We show that multiple defects caused by POPS mutations, including impaired growth and DNA synthesis, genome instability, and reliance on other genome maintenance factors, were rescued by exonuclease inactivation. Single-molecule data revealed that the rescue stemmed from allowing sister replication forks to progress at equal rates. Our data suggest that balanced activity of Pol2's POPS and exonuclease domains is vital for genome replication and stability.

The evolving tale of Pol2 function.

DNA replication is complex and highly regulated, and DNA replication errors can lead to human diseases such as cancer. DNA polymerase ε (polε) is a key player in DNA replication and contains a large subunit called POLE, which possesses both a DNA polymerase domain and a 3'-5' exonuclease domain (EXO). Mutations at the EXO domain and other missense mutations on POLE with unknown significance have been detected in a variety of human cancers. Based on cancer genome databases, Meng and colleagues (pp. 74-79) previously identified several missense mutations in POPS (pol2 family-specific catalytic core peripheral subdomain), and mutations at the conserved residues of yeast Pol2 (pol2-REL) showed reduced DNA synthesis and growth. In this issue of Genes & Development, Meng and colleagues (pp. 74-79) found unexpectedly that mutations at the EXO domain rescue the growth defects of pol2-REL. They further discovered that EXO-mediated polymerase backtracking impedes forward movement of the enzyme when POPS is defective, revealing a novel interplay between the EXO domain and POPS of Pol2 for efficient DNA synthesis. Additional molecular insight into this interplay will likely inform the impact of cancer-associated mutations found in both the EXO domain and POPS on tumorigenesis and uncover future novel therapeutic strategies.

Brain Lateralization: A Comparative Perspective.

Comparative studies on brain asymmetry date back to the 19th century but then largely disappeared due to the assumption that lateralization is uniquely human. Since the reemergence of this field in the 1970s, we learned that left-right differences of brain and behavior exist throughout the animal kingdom and pay off in terms of sensory, cognitive, and motor efficiency. Ontogenetically, lateralization starts in many species with asymmetrical expression patterns of genes within the Nodal cascade that set up the scene for later complex interactions of genetic, environmental, and epigenetic factors. These take effect during different time points of ontogeny and create asymmetries of neural networks in diverse species. As a result, depending on task demands, left- or right-hemispheric loops of feedforward or feedback projections are then activated and can temporarily dominate a neural process. In addition, asymmetries of commissural transfer can shape lateralized processes in each hemisphere. It is still unclear if interhemispheric interactions depend on an inhibition/excitation dichotomy or instead adjust the contralateral temporal neural structure to delay the other hemisphere or synchronize with it during joint action. As outlined in our review, novel animal models and approaches could be established in the last decades, and they already produced a substantial increase of knowledge. Since there is practically no realm of human perception, cognition, emotion, or action that is not affected by our lateralized neural organization, insights from these comparative studies are crucial to understand the functions and pathologies of our asymmetric brain.

Rare variants and handedness: spotlight on TUBB4B.

Twin studies suggest that additive genetic effects account for about a quarter of the variance in handedness. Recently, Schijven et al. used exome-wide sequencing to provide evidence for a role of rare protein-coding variants in handedness. These included the gene encoding beta-tubulin, TUBB4B, suggesting that microtubules are relevant for handedness ontogenesis.

Natural reversal of cavefish heart asymmetry is controlled by Sonic Hedgehog effects on the left-right organizer.

The direction of left-right visceral asymmetry is conserved in vertebrates. Deviations of the standard asymmetric pattern are rare, and the underlying mechanisms are not understood. Here, we use the teleost Astyanax mexicanus, consisting of surface fish with normal left-oriented heart asymmetry and cavefish with high levels of reversed right-oriented heart asymmetry, to explore natural changes in asymmetry determination. We show that Sonic Hedgehog (Shh) signaling is increased at the posterior midline, Kupffer's vesicle (the teleost left-right organizer) is enlarged and contains longer cilia, and the number of dorsal forerunner cells is increased in cavefish. Furthermore, Shh increase in surface fish embryos induces asymmetric changes resembling the cavefish phenotype. Asymmetric expression of the Nodal antagonist Dand5 is equalized or reversed in cavefish, and Shh increase in surface fish mimics changes in cavefish dand5 asymmetry. Shh decrease reduces the level of right-oriented heart asymmetry in cavefish. Thus, naturally occurring modifications in cavefish heart asymmetry are controlled by the effects of Shh signaling on left-right organizer function.

On the origins of developmental robustness: modeling buffering mechanisms against cell-level noise.

During development, cells are subject to stochastic fluctuations in their positions (i.e. cell-level noise) that can potentially lead to morphological noise (i.e. stochastic differences between morphologies that are expected to be equal, e.g. the right and left sides of bilateral organisms). In this study, we explore new and existing hypotheses on buffering mechanisms against cell-level noise. Many of these hypotheses focus on how the boundaries between territories of gene expression remain regular and well defined, despite cell-level noise and division. We study these hypotheses and how irregular territory boundaries lead to morphological noise. To determine the consistency of the different hypotheses, we use a general computational model of development: EmbryoMaker. EmbryoMaker can implement arbitrary gene networks regulating basic cell behaviors (contraction, adhesion, etc.), signaling and tissue biomechanics. We found that buffering mechanisms based on the orientation of cell divisions cannot lead to regular boundaries but that other buffering mechanisms can (homotypic adhesion, planar contraction, non-dividing boundaries, constant signaling and majority rule hypotheses). We also explore the effects of the shape and size of the territories on morphological noise.

The Drosophila AWP1 ortholog Doctor No regulates JAK/STAT signaling for left-right asymmetry in the gut by promoting receptor endocytosis.

Many organs of Drosophila show stereotypical left-right (LR) asymmetry; however, the underlying mechanisms remain elusive. Here, we have identified an evolutionarily conserved ubiquitin-binding protein, AWP1/Doctor No (Drn), as a factor required for LR asymmetry in the embryonic anterior gut. We found that drn is essential in the circular visceral muscle cells of the midgut for JAK/STAT signaling, which contributes to the first known cue for anterior gut lateralization via LR asymmetric nuclear rearrangement. Embryos homozygous for drn and lacking its maternal contribution showed phenotypes similar to those with depleted JAK/STAT signaling, suggesting that Drn is a general component of JAK/STAT signaling. Absence of Drn resulted in specific accumulation of Domeless (Dome), the receptor for ligands in the JAK/STAT signaling pathway, in intracellular compartments, including ubiquitylated cargos. Dome colocalized with Drn in wild-type Drosophila. These results suggest that Drn is required for the endocytic trafficking of Dome, which is a crucial step for activation of JAK/STAT signaling and the subsequent degradation of Dome. The roles of AWP1/Drn in activating JAK/STAT signaling and in LR asymmetric development may be conserved in various organisms.

Translational control of furina by an RNA regulon is important for left-right patterning, heart morphogenesis and cardiac valve function.

Heart development is a complex process that requires asymmetric positioning of the heart, cardiac growth and valve morphogenesis. The mechanisms controlling heart morphogenesis and valve formation are not fully understood. The pro-convertase FurinA functions in heart development across vertebrates. How FurinA activity is regulated during heart development is unknown. Through computational analysis of the zebrafish transcriptome, we identified an RNA motif in a variant FurinA transcript harbouring a long 3' untranslated region (3'UTR). The alternative 3'UTR furina isoform is expressed prior to organ positioning. Somatic deletions in the furina 3'UTR lead to embryonic left-right patterning defects. Reporter localisation and RNA-binding assays show that the furina 3'UTR forms complexes with the conserved RNA-binding translational repressor, Ybx1. Conditional ybx1 mutant embryos show premature and increased Furin reporter expression, abnormal cardiac morphogenesis and looping defects. Mutant ybx1 hearts have an expanded atrioventricular canal, abnormal sino-atrial valves and retrograde blood flow from the ventricle to the atrium. This is similar to observations in humans with heart valve regurgitation. Thus, the furina 3'UTR element/Ybx1 regulon is important for translational repression of FurinA and regulation of heart development.

Functional and structural asymmetry suggest a unifying principle for catalysis in membrane-bound pyrophosphatases.

Membrane-bound pyrophosphatases (M-PPases) are homodimeric primary ion pumps that couple the transport of Na+- and/or H+ across membranes to the hydrolysis of pyrophosphate. Their role in the virulence of protist pathogens like Plasmodium falciparum makes them an intriguing target for structural and functional studies. Here, we show the first structure of a K+-independent M-PPase, asymmetric and time-dependent substrate binding in time-resolved structures of a K+-dependent M-PPase and demonstrate pumping-before-hydrolysis by electrometric studies. We suggest how key residues in helix 12, 13, and the exit channel loops affect ion selectivity and K+-activation due to a complex interplay of residues that are involved in subunit-subunit communication. Our findings not only explain ion selectivity in M-PPases but also why they display half-of-the-sites reactivity. Based on this, we propose, for the first time, a unified model for ion-pumping, hydrolysis, and energy coupling in all M-PPases, including those that pump both Na+ and H+.

Large-scale analysis of structural brain asymmetries in schizophrenia via the ENIGMA consortium.

Left-right asymmetry is an important organizing feature of the healthy brain that may be altered in schizophrenia, but most studies have used relatively small samples and heterogeneous approaches, resulting in equivocal findings. We carried out the largest case-control study of structural brain asymmetries in schizophrenia, with MRI data from 5,080 affected individuals and 6,015 controls across 46 datasets, using a single image analysis protocol. Asymmetry indexes were calculated for global and regional cortical thickness, surface area, and subcortical volume measures. Differences of asymmetry were calculated between affected individuals and controls per dataset, and effect sizes were meta-analyzed across datasets. Small average case-control differences were observed for thickness asymmetries of the rostral anterior cingulate and the middle temporal gyrus, both driven by thinner left-hemispheric cortices in schizophrenia. Analyses of these asymmetries with respect to the use of antipsychotic medication and other clinical variables did not show any significant associations. Assessment of age- and sex-specific effects revealed a stronger average leftward asymmetry of pallidum volume between older cases and controls. Case-control differences in a multivariate context were assessed in a subset of the data (N = 2,029), which revealed that 7% of the variance across all structural asymmetries was explained by case-control status. Subtle case-control differences of brain macrostructural asymmetry may reflect differences at the molecular, cytoarchitectonic, or circuit levels that have functional relevance for the disorder. Reduced left middle temporal cortical thickness is consistent with altered left-hemisphere language network organization in schizophrenia.

Hemispheric asymmetry in cortical thinning reflects intrinsic organization of the neurotransmitter systems and homotopic functional connectivity.

Hemispheric lateralization and its origins have been of great interest in neuroscience for over a century. The left-right asymmetry in cortical thickness may stem from differential maturation of the cerebral cortex in the two hemispheres. Here, we investigated the spatial pattern of hemispheric differences in cortical thinning during adolescence, and its relationship with the density of neurotransmitter receptors and homotopic functional connectivity. Using longitudinal data from IMAGEN study (N = 532), we found that many cortical regions in the frontal and temporal lobes thinned more in the right hemisphere than in the left. Conversely, several regions in the occipital and parietal lobes thinned less in the right (vs. left) hemisphere. We then revealed that regions thinning more in the right (vs. left) hemispheres had higher density of neurotransmitter receptors and transporters in the right (vs. left) side. Moreover, the hemispheric differences in cortical thinning were predicted by homotopic functional connectivity. Specifically, regions with stronger homotopic functional connectivity showed a more symmetrical rate of cortical thinning between the left and right hemispheres, compared with regions with weaker homotopic functional connectivity. Based on these findings, we suggest that the typical patterns of hemispheric differences in cortical thinning may reflect the intrinsic organization of the neurotransmitter systems and related patterns of homotopic functional connectivity.

Developmental bias predicts 60 million years of wing shape evolution.

The degree to which developmental biases affect trait evolution is subject to much debate. Here, we first quantify fluctuating asymmetry as a measure of developmental variability, i.e., the propensity of developmental systems to create some phenotypic variants more often than others, and show that it predicts phenotypic and standing genetic variation as well as deep macroevolutionary divergence in wing shape in sepsid flies. Comparing our data to the findings of a previous study demonstrates that developmental variability in the sepsid fly Sepsis punctum strongly aligns with mutational, standing genetic, and macroevolutionary variation in the Drosophilidae--a group that diverged from the sepsid lineage ca. 64 My ago. We also find that developmental bias in S. punctum wing shape aligns with the effects of allometry, but less so with putatively adaptive thermal plasticity and population differentiation along latitude. Our findings demonstrate that developmental bias in fly wings predicts evolvability and macroevolutionary trajectories on a much greater scale than previously appreciated but also suggest that causal explanations for such alignments may go beyond simple constraint hypotheses.

A spatiotemporal barrier formed by Follistatin is required for left-right patterning.

How left-right (LR) asymmetry emerges in a patterning field along the anterior-posterior axis remains an unresolved problem in developmental biology. Left-biased Nodal emanating from the LR organizer propagates from posterior to anterior (PA) and establishes the LR pattern of the whole embryo. However, little is known about the regulatory mechanism of the PA spread of Nodal and its asymmetric activation in the forebrain. Here, we identify bilaterally expressed Follistatin (Fst) as a regulator blocking the propagation of the zebrafish Nodal ortholog Southpaw (Spaw) in the right lateral plate mesoderm (LPM), and restricting Spaw transmission in the left LPM to facilitate the establishment of a robust LR asymmetric Nodal patterning. In addition, Fst inhibits the Activin-Nodal signaling pathway in the forebrain thus preventing Nodal activation prior to the arrival, at a later time, of Spaw emanating from the left LPM. This contributes to the orderly propagation of asymmetric Nodal activation along the PA axis. The LR regulation function of Fst is further confirmed in chick and frog embryos. Overall, our results suggest that a robust LR patterning emerges by counteracting a Fst barrier formed along the PA axis.

Development of the Fetal Brain Corticocortical Structural Network during the Second-to-Third Trimester Based on Diffusion MRI.

During the second-to-third trimester, the neuronal pathways of the fetal brain experience rapid development, resulting in the complex architecture of the interwired network at birth. While diffusion MRI-based tractography has been employed to study the prenatal development of structural connectivity network (SCN) in preterm neonatal and postmortem fetal brains, the in utero development of SCN in the normal fetal brain remains largely unknown. In this study, we utilized in utero dMRI data from human fetuses of both sexes between 26 and 38 gestational weeks to investigate the developmental trajectories of the fetal brain SCN, focusing on intrahemispheric connections. Our analysis revealed significant increases in global efficiency, mean local efficiency, and clustering coefficient, along with significant decrease in shortest path length, while small-worldness persisted during the studied period, revealing balanced network integration and segregation. Widespread short-ranged connectivity strengthened significantly. The nodal strength developed in a posterior-to-anterior and medial-to-lateral order, reflecting a spatiotemporal gradient in cortical network connectivity development. Moreover, we observed distinct lateralization patterns in the fetal brain SCN. Globally, there was a leftward lateralization in network efficiency, clustering coefficient, and small-worldness. The regional lateralization patterns in most language, motor, and visual-related areas were consistent with prior knowledge, except for Wernicke's area, indicating lateralized brain wiring is an innate property of the human brain starting from the fetal period. Our findings provided a comprehensive view of the development of the fetal brain SCN and its lateralization, as a normative template that may be used to characterize atypical development.

CPLANE protein INTU regulates growth and patterning of the mouse lungs through cilia-dependent Hh signaling.

Congenital lung malformations are fatal at birth in their severe forms. Prevention and early intervention of these birth defects require a comprehensive understanding of the molecular mechanisms of lung development. We find that the loss of inturned (Intu), a cilia and planar polarity effector gene, severely disrupts growth and branching morphogenesis of the mouse embryonic lungs. Consistent with our previous results indicating an important role for Intu in ciliogenesis and hedgehog (Hh) signaling, we find greatly reduced number of primary cilia in both the epithelial and mesenchymal tissues of the lungs. We also find significantly reduced expression of Gli1 and Ptch1, direct targets of Hh signaling, suggesting disruption of cilia-dependent Hh signaling in Intu mutant lungs. An agonist of the Hh pathway activator, smoothened, increases Hh target gene expression and tubulogenesis in explanted wild type, but not Intu mutant, lungs, suggesting impaired Hh signaling response underlying lung morphogenetic defects in Intu mutants. Furthermore, removing both Gli2 and Intu completely abolishes branching morphogenesis of the lung, strongly supporting a mechanism by which Intu regulates lung growth and patterning through cilia-dependent Hh signaling. Moreover, a transcriptomics analysis identifies around 200 differentially expressed genes (DEGs) in Intu mutant lungs, including known Hh target genes Gli1, Ptch1/2 and Hhip. Genes involved in muscle differentiation and function are highly enriched among the DEGs, consistent with an important role of Hh signaling in airway smooth muscle differentiation. In addition, we find that the difference in gene expression between the left and right lungs diminishes in Intu mutants, suggesting an important role of Intu in asymmetrical growth and patterning of the mouse lungs.

Mechanism of GTPase activation of a prokaryotic small Ras-like GTPase MglA by an asymmetrically interacting MglB dimer.

Cell polarity oscillations in Myxococcus xanthus motility are driven by a prokaryotic small Ras-like GTPase, mutual gliding protein A (MglA), which switches from one cell pole to the other in response to extracellular signals. MglA dynamics is regulated by MglB, which functions both as a GTPase activating protein (GAP) and a guanine nucleotide exchange factor (GEF) for MglA. With an aim to dissect the asymmetric role of the two MglB protomers in the dual GAP and GEF activities, we generated a functional MglAB complex by coexpressing MglB with a linked construct of MglA and MglB. This strategy enabled us to generate mutations of individual MglB protomers (MglB1 or MglB2 linked to MglA) and delineate their role in GEF and GAP activities. We establish that the C-terminal helix of MglB1, but not MglB2, stimulates nucleotide exchange through a site away from the nucleotide-binding pocket, confirming an allosteric mechanism. Interaction between the N-terminal ß-strand of MglB1 and ß0 of MglA is essential for the optimal GEF activity of MglB. Specific residues of MglB2, which interact with Switch-I of MglA, partially contribute to its GAP activity. Thus, the role of the MglB2 protomer in the GAP activity of MglB is limited to restricting the conformation of MglA active site loops. The direct demonstration of the allosteric mechanism of GEF action provides us new insights into the regulation of small Ras-like GTPases, a feature potentially present in many uncharacterized GEFs.

Consensus, controversies, and conundrums of P4-ATPases: The emerging face of eukaryotic lipid flippases.

The cryo-EM resolution revolution has heralded a new era in our understanding of eukaryotic lipid flippases with a rapidly growing number of high-resolution structures. Flippases belong to the P4 family of ATPases (type IV P-type ATPases) that largely follow the reaction cycle proposed for the more extensively studied cation-transporting P-type ATPases. However, unlike the canonical P-type ATPases, no flippase cargos are transported in the phosphorylation half-reaction. Instead of being released into the intracellular or extracellular milieu, lipid cargos are transported to their destination at the inner leaflet of the membrane. Recent flippase structures have revealed multiple conformational states during the lipid transport cycle. Nonetheless, critical conformational states capturing the lipid cargo "in transit" are still missing. In this review, we highlight the amazing structural advances of these lipid transporters, discuss various perspectives on catalytic and regulatory mechanisms in the literature, and shed light on future directions in further deciphering the detailed molecular mechanisms of lipid flipping.

Planar cell polarity-dependent asymmetric organization of microtubules for polarized positioning of the basal body in node cells.

For left-right symmetry breaking in the mouse embryo, the basal body must become positioned at the posterior side of node cells, but the precise mechanism for this has remained unknown. Here, we examined the role of microtubules (MTs) and actomyosin in this basal body positioning. Exposure of mouse embryos to agents that stabilize or destabilize MTs or F-actin impaired such positioning. Active myosin II was detected at the anterior side of node cells before the posterior shift of the basal body, and this asymmetric activation was lost in Prickle and dachsous mutant embryos. The organization of basal-body associated MTs (baMTs) was asymmetric between the anterior and posterior sides of node cells, with anterior baMTs extending horizontally and posterior baMTs extending vertically. This asymmetry became evident after polarization of the PCP core protein Vangl1 and before the posterior positioning of the basal body, and it also required the PCP core proteins Prickle and dachsous. Our results suggest that the asymmetry in baMT organization may play a role in correct positioning of the basal body for left-right symmetry breaking.