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In many contexts, the problem arises of determining which of many candidate mutations is the most likely to be causative for some phenotype. It is desirable to have a way to evaluate this probability that relies as little as possible on previous knowledge, to avoid bias against discovering new genes or functions. We have isolated mutants with blocked cell cycle progression in Chlamydomonas and determined mutant genome sequences. Due to the intensity of UV mutagenesis required for efficient mutant collection, the mutants contain multiple mutations altering coding sequence. To provide a quantitative estimate of probability that each individual mutation in a given mutant is the causative one, we developed a Bayesian approach. The approach employs four independent indicators: sequence conservation of the mutated coding sequence with Arabidopsis; severity of the mutation relative to Chlamydomonas wild-type based on Blosum62 scores; meiotic mapping information for location of the causative mutation relative to known molecular markers; and, for a subset of mutants, the transcriptional profile of the candidate wild-type genes through the mitotic cell cycle. These indicators are statistically independent, and so can be combined quantitatively into a single probability calculation. We validate this calculation: recently isolated mutations that were not in the training set for developing the indicators, with high calculated probability of causality, are confirmed in every case by additional genetic data to indeed be causative. Analysis of best reciprocal BLAST (BRB) relationships among Chlamydomonas and other eukaryotes indicate that the temperature sensitive-lethal (Ts-lethal) mutants that our procedure recovers are highly enriched for fundamental cell-essential functions conserved broadly across plants and other eukaryotes, accounting for the high information content of sequence alignment to Arabidopsis.

Spectraplakins are large molecules that cross-link F-actin and microtubules (MTs). Mutations in spectraplakins yield defective cell polarization, aberrant focal adhesion dynamics, and dystonia. We present the 2.8 angstrom crystal structure of the hACF7 EF1-EF2-GAR MT-binding module and delineate the GAR residues critical for MT binding. The EF1-EF2 and GAR domains are autonomous domains connected by a flexible linker. The EF1-EF2 domain is an EF beta-scaffold with two bound Ca2+ ions that straddle an N-terminal a helix. The GAR domain has a unique alpha/beta sandwich fold that coordinates Zn2+. While the EF1-EF2 domain is not sufficient for MT binding, the GAR domain is and likely enhances EF1-EF2-MT engagement. Residues in a conserved basic patch, distal to the GAR domain's Zn2+-binding site, mediate MT binding.

Many Gram-negative bacterial pathogens use a syringe-like apparatus called a type III secretion system to inject virulence factors into host cells. Some of these effectors are enzymes that modify host proteins to subvert their normal functions. NleB is a glycosyltransferase that modifies host proteins with N-acetyl-Dglucosamine to inhibit antibacterial and inflammatory host responses. NleB is conserved among the attaching/effacing pathogens enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium. Moreover, Salmonella enterica strains encode up to three NleB orthologs named SseK1, SseK2, and SseK3. However, there are conflicting reports regarding the activities and host protein targets among the NleB/SseK orthologs. Therefore, here we performed in vitro glycosylation assays and cell culture experiments to compare the activities and substrate specificities of these effectors. SseK1, SseK3, EHEC NleB1, EPEC NleB1, and C. rodentium NleB blocked TNF-mediated NF-kappa B pathway activation, whereas SseK2 and NleB2 did not. C. rodentium NleB, EHEC NleB1, and SseK1 glycosylated host GAPDH. C. rodentium NleB, EHEC NleB1, EPEC NleB1, and SseK2 glycosylated the FADD (Fas-associated death domain protein). SseK3 and NleB2 were not active against either substrate. We also found that EHEC NleB1 glycosylated two GAPDH arginine residues, Arg197 and Arg200, and that these two residues were essential for GAPDH-mediated activation of TNF receptorassociated factor 2 ubiquitination. These results provide evidence that members of this highly conserved family of bacterial virulence effectors target different host protein substrates and exhibit distinct cellular modes of action to suppress host responses.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel evolved from an ATP-binding cassette transporter. CFTR channel gating is strictly coupled to phosphorylation and ATP hydrolysis. Previously, we reported essentially identical structures of zebrafish and human CFTR in the dephosphorylated, ATP-free form. Here, we present the structure of zebrafish CFTR in the phosphorylated, ATP-bound conformation, determined by cryoelectron microscopy to 3.4 angstrom resolution. Comparison of the two conformations shows major structural rearrangements leading to channel opening. The phosphorylated regulatory domain is disengaged from its inhibitory position; the nucleotide-binding domains (NBDs) form a "head-to-tail" dimer upon binding ATP; and the cytoplasmic pathway, found closed off in other ATP-binding cassette transporters, is cracked open, consistent with CFTR's unique channel function. Unexpectedly, the extracellular mouth of the ion pore remains closed, indicating that local movements of the transmembrane helices can control ion access to the pore even in the NBD-dimerized conformation.

Background: B cells undergo maturation and class-switching in response to antigen exposure and T-cell help. Early B-cell differentiation has not been defined in patients with early-onset atopic dermatitis (AD). Objective: We sought to define the frequency of B-cell subsets associated with progressive B-cell maturation and IgE class-switching. Methods: We studied 27 children and 34 adults with moderate-to-severe AD (mean SCORAD score, 55 and 65, respectively) and age-matched control subjects (15 children and 27 adults). IgD/CD27 and CD24/CD38 core gating systems and an 11-color flow cytometric panel were used to determine the frequencies of circulating B-cell subsets. Serum total and allergen-specific IgE (sIgE) levels were measured by using ImmunoCAP. Results: Compared with adults, children showed T-cell predominance in the skin. Circulating CD19(+) CD20(+) B-cell counts were lower in patients with pediatric AD than in control subjects (24% vs 33%, P = .04), whereas CD3(+) T-cell counts were higher (62% vs 52%, P = .05). A decreased B-cell/T-cell lymphocyte ratio with age was observed only in pediatric control subjects (r = -0.48, P = .07). In pediatric patients with AD, a positive correlation was observed between B-cell/T-cell ratio and nonswitched memory B-cell counts (r = 0.42, P = .03). Higher frequencies of positive sIgE levels were seen in pediatric patients with AD (P < .0001). Diverse sIgE levels correlated with SCORAD scores and age of pediatric patients with AD (P < .01). Positive correlations were observed between activated B-cell and memory T-cell counts (P < .02). In patients with AD, IgE sensitization to most allergens clustered with age, T(H)1, T(H)2, total IgE levels, and B-cell memory subsets. Conclusions: Peripheral B and T cells are altered in pediatric patients with early AD, but T cells predominate in skin lesions.

Protein O-mannosylation is found in yeast and metazoans, and a family of conserved orthologous protein O-mannosyltransferases is believed to initiate this important post-translational modification. We recently discovered that the cadherin superfamily carries O-linked mannose (O-Man) glycans at highly conserved residues in specific extracellular cadherin domains, and it was suggested that the function of E-cadherin was dependent on the O-Man glycans. Deficiencies in enzymes catalyzing O-Man biosynthesis, including the two human protein O-mannosyltransferases, POMT1 and POMT2, underlie a subgroup of congenital muscular dystrophies designated alpha-dystroglycanopathies, because deficient O-Man glycosylation of alpha-dystroglycan disrupts laminin interaction with alpha-dystroglycan and the extracellular matrix. To explore the functions of O-Man glycans on cadherins and protocadherins, we used a combinatorial gene-editing strategy in multiple cell lines to evaluate the role of the two POMTs initiating O-Man glycosylation and the major enzyme elongating O-Man glycans, the protein O-mannose alpha-1,2-N-acetylglucosaminyltransferase, POMGnT1. Surprisingly, O-mannosylation of cadherins and protocadherins does not require POMT1 and/or POMT2 in contrast to alpha-dystroglycan, and moreover, the O-Man glycans on cadherins are not elongated. Thus, the classical and evolutionarily conserved POMT O-mannosylation pathway is essentially dedicated to alpha-dystroglycan and a few other proteins, whereas a novel O-mannosylation process in mammalian cells is predicted to serve the large cadherin superfamily and other proteins.

Dysfunctions of hearing and balance are often irreversible in mammals owing to the inability of cells in the inner ear to proliferate and replace lost sensory receptors. To determine the molecular basis of this deficiency we have investigated the dynamics of growth and cellular proliferation in a murine vestibular organ, the utricle. Based on this analysis, we have created a theoretical model that captures the key features of the organ's morphogenesis. Our experimental data and model demonstrate that an elastic force opposes growth of the utricular sensory epithelium during development, confines cellular proliferation to the organ's periphery, and eventually arrests its growth. We find that an increase in cellular density and the subsequent degradation of the transcriptional cofactor Yap underlie this process. A reduction in mechanical constraints results in accumulation and nuclear translocation of Yap, which triggers proliferation and restores the utricle's growth; interfering with Yap's activity reverses this effect.

Hunger, driven by negative energy balance, elicits the search for and consumption of food. While this response is in part mediated by neurons in the hypothalamus, the role of specific cell types in other brain regions is less well defined. Here, we show that neurons in the dorsal raphe nucleus, expressing vesicular transporters for GABA or glutamate (hereafter, DRN Vgat and DRN VGLUT3 neurons), are reciprocally activated by changes in energy balance and that modulating their activity has opposite effects on feeding-DRN Vgat neurons increase, whereas DRN VGLUT3 neurons suppress, food intake. Furthermore, modulation of these neurons in obese (ob/ob) mice suppresses food intake and body weight and normalizes locomotor activity. Finally, using molecular profiling, we identify druggable targets in these neurons and show that local infusion of agonists for specific receptors on these neurons has potent effects on feeding. These data establish the DRN as an important node controlling energy balance.

Focal adhesion kinase (FAK) inhibitors have been developed as potential anticancer agents and are undergoing clinical trials. In vitro activation of the FAK kinase domain triggers autophosphorylation of Y397, Src activation, and subsequent phosphorylation of other FAK tyrosine residues. However, how FAK Y397 mutations affect FAK kinase-dead (KD) phenotypes in tumour angiogenesis in vivo is unknown. We developed three Pdgfb-iCre(ert)-driven endothelial cell (EC)-specific, tamoxifen-inducible homozygous mutant mouse lines: FAK wild-type (WT), FAK KD, and FAK double mutant (DM), i.e. KD with a putatively phosphomimetic Y397E mutation. These ECCre+;FAK(WT/WT), ECCre+;FAK(KD/KD) and ECCre+;FAK(DM/DM) mice were injected subcutaneously with syngeneic B16F0 melanoma cells. Tumour growth and tumour blood vessel functions were unchanged between ECCre+;FAK(WT/WT) and ECCre-;FAK(WT/WT) control mice. In contrast, tumour growth and vessel density were decreased in ECCre+;FAK(KD/KD) and ECCre+;FAK(DM/DM) mice, as compared with Cre-littermates. Despite no change in the percentage of perfused vessels or pericyte coverage in either genotype, tumour hypoxia was elevated in ECCre+;FAK(KD/KD) and ECCre+;FAK(DM/DM) mice. Furthermore, although ECCre+;FAK(KD/KD) mice showed reduced blood vessel leakage, ECCre+;FAK(DM/DM) and ECCre-;FAK(DM/DM) mice showed no difference in leakage. Mechanistically, fibronectin-stimulated Y397 autophosphorylation was reduced in Cre+;FAK(KD/KD) ECs as compared with Cre+;FAK(WT/WT) cells, with no change in phosphorylation of the known Src targets FAK-Y577, FAK-Y861, FAK-Y925, paxillin-Y118, p130Cas-Y410. Cre+;FAK(DM/DM) ECs showed decreased Src target phosphorylation levels, suggesting that the Y397E substitution actually disrupted Src activation. Reduced VE-cadherin-pY658 levels in Cre+;FAK(KD/KD) ECs were rescued in Cre+ FAK(DM/DM) ECs, corresponding with the rescue in vessel leakage in the ECCre+;FAK(DM/DM) mice. We show that EC-specific FAK kinase activity is required for tumour growth, angiogenesis, and vascular permeability. The ECCre+;FAK(DM/DM) mice restored the KD-dependent tumour vascular leakage observed in ECCre+;FAK(KD/KD) mice in vivo. This study opens new fields in in vivo FAK signalling. (C) 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Background: Germline heterozygous mutations in human signal transducer and activator of transcription 1 (STAT1) can cause loss of function (LOF), as in patients with Mendelian susceptibility to mycobacterial diseases, or gain of function (GOF), as in patients with chronic mucocutaneous candidiasis. LOF and GOF mutations are equally rare and can affect the same domains of STAT1, especially the coiled-coil domain (CCD) and DNA-binding domain (DBD). Moreover, 6% of patients with chronic mucocutaneous candidiasis with a GOF STAT1 mutation have mycobacterial disease, obscuring the functional significance of the identified STAT1 mutations. Current computational approaches, such as combined annotation-dependent depletion, do not distinguish LOF and GOF variants. Objective: We estimated variations in the CCD/DBD of STAT1. Methods: We mutagenized 342 individual wild-type amino acids in the CCD/DBD (45.6% of full-length STAT1) to alanine and tested the mutants for STAT1 transcriptional activity. Results: Of these 342 mutants, 201 were neutral, 30 were LOF, and 111 were GOF mutations in a luciferase assay. This assay system correctly estimated all previously reported LOF mutations (100%) and slightly fewer GOF mutations (78.1%) in the CCD/DBD of STAT1. We found that GOF alanine mutants occurred at the interface of the antiparallel STAT1 dimer, suggesting that they destabilize this dimer. This assay also precisely predicted the effect of 2 hypomorphic and dominant negative mutations, E157K and G250E, in the CCD of STAT1 that we found in 2 unrelated patients with Mendelian susceptibility to mycobacterial diseases. Conclusion: The systematic alanine-scanning assay is a useful tool to estimate the GOF or LOF status and the effect of heterozygous missense mutations in STAT1 identified in patients with severe infectious diseases, including mycobacterial and fungal diseases.