Geometric and topological properties of protein structures, including surface pockets, interior cavities and cross channels, are of fundamental importance for proteins to carry out their functions. Computed Atlas of Surface Topography of proteins (CASTp) is a web server that provides online services for locating, delineating and measuring these geometric and topological properties of protein structures. It has been widely used since its inception in 2003. In this article, we present the latest version of the web server, CASTp 3.0. CASTp 3.0 continues to provide reliable and comprehensive identifications and quantifications of protein topography. In addition, it now provides: (i) imprints of the negative volumes of pockets, cavities and channels, (ii) topographic features of biological assemblies in the Protein Data Bank, (iii) improved visualization of protein structures and pockets, and (iv) more intuitive structural and annotated information, including information of secondary structure, functional sites, variant sites and other annotations of protein residues. The CASTp 3.0 web server is freely accessible at http://sts.bioe.uic.edu/castp/.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch–seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

Here we report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties and cellular resolution input–output mapping, integrated through cross-modal computational analysis. Our results advance the collective knowledge and understanding of brain cell-type organization1–5. First, our study reveals a unified molecular genetic landscape of cortical cell types that integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a consensus taxonomy of transcriptomic types and their hierarchical organization that is conserved from mouse to marmoset and human. Third, in situ single-cell transcriptomics provides a spatially resolved cell-type atlas of the motor cortex. Fourth, cross-modal analysis provides compelling evidence for the transcriptomic, epigenomic and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types. We further present an extensive genetic toolset for targeting glutamatergic neuron types towards linking their molecular and developmental identity to their circuit function. Together, our results establish a unifying and mechanistic framework of neuronal cell-type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties.

Mammalian brain cells show remarkable diversity in gene expression, anatomy and function, yet the regulatory DNA landscape underlying this extensive heterogeneity is poorly understood. Here we carry out a comprehensive assessment of the epigenomes of mouse brain cell types by applying single-nucleus DNA methylation sequencing1,2 to profile 103,982 nuclei (including 95,815 neurons and 8,167 non-neuronal cells) from 45 regions of the mouse cortex, hippocampus, striatum, pallidum and olfactory areas. We identified 161 cell clusters with distinct spatial locations and projection targets. We constructed taxonomies of these epigenetic types, annotated with signature genes, regulatory elements and transcription factors. These features indicate the potential regulatory landscape supporting the assignment of putative cell types and reveal repetitive usage of regulators in excitatory and inhibitory cells for determining subtypes. The DNA methylation landscape of excitatory neurons in the cortex and hippocampus varied continuously along spatial gradients. Using this deep dataset, we constructed an artificial neural network model that precisely predicts single neuron cell-type identity and brain area spatial location. Integration of high-resolution DNA methylomes with single-nucleus chromatin accessibility data3 enabled prediction of high-confidence enhancer–gene interactions for all identified cell types, which were subsequently validated by cell-type-specific chromatin conformation capture experiments4. By combining multi-omic datasets (DNA methylation, chromatin contacts, and open chromatin) from single nuclei and annotating the regulatory genome of hundreds of cell types in the mouse brain, our DNA methylation atlas establishes the epigenetic basis for neuronal diversity and spatial organization throughout the mouse cerebrum.

Superoxide produced by the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is essential for host defense. Enzyme activation requires translocation of p67phox, p47phox, and Rac-GTP to flavocytochrome b 558 in phagocyte membranes. To examine the regulation of phagocytosis-induced superoxide production, flavocytochrome b558, p47phox, p67phox, and the FcγIIA receptor were expressed from stable transgenes in COS7 cells. The resulting COSphoxFcγR cells produce high levels of superoxide when stimulated with phorbol ester and efficiently ingest immunoglobulin (Ig)G-coated erythrocytes, but phagocytosis did not activate the NADPH oxidase. COS7 cells lack p40phox, whose role in the NADPH oxidase is poorly understood. p40phox contains SH3 and phagocyte oxidase and Bem1p (PB1) domains that can mediate binding to p47phox and p67phox, respectively, along with a PX domain that binds to phosphatidylinositol-3-phosphate (PI(3)P), which is generated in phagosomal membranes. Expression of p40phox was sufficient to activate superoxide production in COSphoxFcγR phagosomes. FcγIIA-stimulated NADPH oxidase activity was abrogated by point mutations in p40phox that disrupt PI(3)P binding, or by simultaneous mutations in the SH3 and PB1 domains. Consistent with an essential role for PI(3)P in regulating the oxidase complex, phagosome NADPH oxidase activation in primary macrophages ingesting IgG-coated beads was inhibited by phosphatidylinositol 3 kinase inhibitors to a much greater extent than phagocytosis itself. Hence, this study identifies a role for p40phox and PI(3)P in coupling FcγR-mediated phagocytosis to activation of the NADPH oxidase.

The phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b 558 and cytosolic p67 phox , p47 phox , and p40 phox subunits that undergo membrane translocation upon cellular activation. The function of p40 phox , which binds p67 phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40 phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40 phox in Fc␥R-induced oxidase activation, we used immunofluorescence and real-time imaging of Fc␥R-induced phagocytosis. YFP-tagged p67 phox and p40 phox translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67 phox and p47 phox accumulation on nascent and internalized phagosomes did not require p40 phox or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40 phox PI3P-binding domain or wortmannin. Translocation of p40 phox to nascent phagosomes required binding to p67 phox but not PI3P, although the loss of PI3P binding reduced p40 phox retention after phagosome internalization. We conclude that p40 phox functions primarily to regulate Fc␥R-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization. (Blood. 2008;112:3867-3877) IntroductionPhagocytic leukocytes are the front-line cellular defense against microbial attack, and are mobilized rapidly to the sites of infection where they ingest and kill opsonized microorganisms. The NADPH oxidase complex plays a central role in this process, as its assembly and activation on phagosomal membranes generate superoxide, the precursor of potent microbicidal oxidants. The importance of this enzyme is demonstrated by genetic defects in the NADPH oxidase complex that cause chronic granulomatous disease (CGD), characterized by recurrent severe and potentially lethal bacterial and fungal infections. 1 The NADPH oxidase includes the membrane-integrated flavocytochrome b, composed of gp91 phox and p22 phox , and the cytosolic components p47 phox , p67 phox , p40 phox , and Rac, a Rho-family GTPase, which translocate to flavocytochrome b upon cellular stimulation to activate superoxide production. [2][3][4] Segregation of regulatory components to the cytosol in resting cells facilitates the temporal and spatial regulation of NADPH oxidase activity. The p67 phox subunit is a Rac-GTP effector 2-4 containing a domain that activates electron transport through the flavocytochrome. 5 In resting cells, p67 phox is associated with p40 phox via complementary PB1 (phagocyte oxidase and Bem1p) motifs present in each protein. 2,[6][7][8] p67 phox is also linked to p47 phox via a high-affinity interaction involving an SH3 domain and a proline-rich region, respectively, in the C-termini of...

23The primary motor cortex (M1) is essential for voluntary fine motor control and is functionally conserved 24 across mammals. Using high-throughput transcriptomic and epigenomic profiling of over 450,000 single 25 nuclei in human, marmoset monkey, and mouse, we demonstrate a broadly conserved cellular makeup 26 of this region, whose similarity mirrors evolutionary distance and is consistent between the 27 transcriptome and epigenome. The core conserved molecular identity of neuronal and non-neuronal 28 types allowed the generation of a cross-species consensus cell type classification and inference of 29 conserved cell type properties across species. Despite overall conservation, many species 30 specializations were apparent, including differences in cell type proportions, gene expression, DNA 31 methylation, and chromatin state. Few cell type marker genes were conserved across species, 32 providing a short list of candidate genes and regulatory mechanisms responsible for conserved features 33 of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic 34 classification allowed the Patch-seq identification of layer 5 (L5) corticospinal Betz cells in non-human 35 primate and human and characterization of their highly specialized physiology and anatomy. These 36 findings highlight the robust molecular underpinnings of cell type diversity in M1 across mammals and 37 point to the genes and regulatory pathways responsible for the functional identity of cell types and their 38 species-specific adaptations. 39 40 distinguished on the basis of regions of open chromatin or DNA methylation 5,9,10 . Furthermore, several 48 recent studies have shown that transcriptomically-defined cell types can be aligned across species 2,11-49 13 , indicating that these methods provide a path to quantitatively study evolutionary conservation and 50 divergence at the level of cell types. However, application of these methods has been highly 51 fragmented to date. Human and mouse comparisons have been performed in different cortical regions, 52 using single-cell (with biases in cell proportions) versus single-nucleus (with biases in transcript 53 makeup) analysis, and most single-cell transcriptomic and epigenomic studies have been performed 54 independently. 55 56The primary motor cortex (MOp in mouse, M1 in human and non-human primates, all referred to as M1 57 herein) provides an ideal cortical region to address questions about cellular evolution in rodents and 58 primates by integrating these approaches. Unlike the primary visual cortex (V1), which is highly 59 specialized in primates, or frontal and temporal association areas, whose homologues in rodents 60 remain poorly defined, M1 is essential for fine motor control and is functionally conserved across 61 placental mammals. M1 is an agranular cortex, lacking a defined L4, although neurons with L4-like 62properties have been described 14 . L5 of carnivore and primate M1 contains exceptionally large 63 "giganto-cellular" corticospinal neurons (Betz c...

The mating of budding yeast depends on chemotropism, a fundamental cellular process. The two yeast mating types secrete peptide pheromones that bind to GPCRs on cells of the opposite type. Cells find and contact a partner by determining the direction of the pheromone source and polarizing their growth toward it. Actin-directed secretion to the chemotropic growth site (CS) generates a mating projection. When pheromone-stimulated cells are unable to sense a gradient, they form mating projections where they would have budded in the next cell cycle, at a position called the default polarity site (DS). Numerous models have been proposed to explain yeast gradient sensing, but none address how cells reliably switch from the intrinsically determined DS to the gradient-aligned CS, despite a weak spatial signal. Here we demonstrate that, in mating cells, the initially uniform receptor and G protein first polarize to the DS, then redistribute along the plasma membrane until they reach the CS. Our data indicate that signaling, polarity, and trafficking proteins localize to the DS during assembly of what we call the gradient tracking machine (GTM). Differential activation of the receptor triggers feedback mechanisms that bias exocytosis upgradient and endocytosis downgradient, thus enabling redistribution of the GTM toward the pheromone source. The GTM stabilizes when the receptor peak centers at the CS and the endocytic machinery surrounds it. A computational model simulates GTM tracking and stabilization and correctly predicts that its assembly at a single site contributes to mating fidelity.