AMPA receptor (AMPA-R) complexes consist of channel forming subunits, GluA1–4 and auxiliary proteins including TARPs, CNIHs, synDIG1, and CKAMP44, which can modulate AMPA-R function in specific ways. Combinatorial effects of four GluA subunits binding to various auxiliary subunits amplify the functional diversity of AMPA-Rs. The significance and magnitude of molecular diversity, however, remain elusive. To gain insight into the molecular complexity of AMPA and kainate receptors (KA-Rs), we compared the proteins that co-purify with each receptor type in rat brain. This interactome study identified the majority of known interacting proteins and more importantly, provides novel candidates for further studies. We validate the claudin homologue GSG1L as a novel binding protein and unique modulator of AMPA-R gating, as determined by detailed molecular, cellular, electrophysiological, and biochemical experiments. GSG1L extends the functional variety of AMPA-R complexes and further investigation of other candidates may reveal additional complexity of ionotropic glutamate receptor function.

Adlercreutzia equolifaciens gen. nov., sp. nov., an equol-producing bacterium isolated from human faeces, and emended description of the genus Eggerthella Nine strains capable of metabolizing isoflavones to equol were isolated from human faeces. Four of the strains were characterized by determining phenotypic and biochemical features and their phylogenetic position based on 16S rRNA gene sequence analysis. These strains were related to Eggerthella sinensis HKU14 T with about 93 % 16S rRNA gene sequence similarity; they were asaccharolytic, obligately anaerobic, non-spore-forming, non-motile and Gram-positive coccobacilli. In enzyme activity tests, arginine dihydrolase, arginine and leucine arylamidases were positive but nitrate reduction, urease and b-glucosidase were negative. The major menaquinone was DMMK-6 (dimethylmenaquinone-6), while that of members of the genus Eggerthella was MMK-6 (methylmenaquinone-6). Furthermore, the cell-wall peptidoglycan type of these strains was A1c, while that of members of the genus Eggerthella was A4c. On the basis of these data, a new genus, Adlercreutzia gen. nov., is proposed with one species, Adlercreutzia equolifaciens sp. , 2006). We isolated nine strains capable of metabolizing isoflavones to equol from human faeces. Seven of these strains could metabolize daidzein via dihydrodaidzein to equol, while the other two isolates could metabolize only dihydrodaidzein to equol. These strains were divided into four groups by 16S rRNA gene sequence analysis. Representative strains selected from each group were asaccharolytic, obligately anaerobic, non-spore-forming, non-motile and Gram-positive coccobacilli. Although these strains were genetically related to the genus Eggerthella, they did not belong to the genera Eggerthella, Slackia or Denitrobacterium from 16S rRNA gene sequence analysis.The presence of dimethylmenaquinone-6 (DMMK-6) as the predominant menaquinone of these strains is unique. Furthermore, the cell-wall peptidoglycan type of these strains was A1c, while that of members of the genus Eggerthella was A4c.Strains FJC-A10, FJC-A161, FJC-B9 T , FJC-B12, FJC-B15, FJC-B19, FJC-B20, FJC-D47 and FJC-D53 were cultivated for 3 days at 37 u C on BL agar (Nissui) with 5 % (v/v) horse blood. Coriobacterium glomerans JCM 10262 T was cultivated for 2 days at 30 u C on GAM agar (Nissui). Detailed menaquinone and fatty acid profiles of representative strains and related type strains and minimum-evolution and maximumparsimony 16S rRNA gene sequence-based trees are available as supplementary material with the online version of this paper.

Subunit assembly governs regulation of AMPA receptor (AMPA-R) synaptic delivery and determines biophysical parameters of the ion channel. However, little is known about the molecular pathways of this process. Here, we present single-particle EM three-dimensional structures of dimeric biosynthetic intermediates of the GluA2 subunit of AMPA-Rs. Consistent with the structures of intact tetramers, the N-terminal domains of the biosynthetic intermediates form dimers. Transmembrane domains also dimerize despite the two ligandbinding domains (LBDs) being separated. A significant difference was detected between the dimeric structures of the wild type and the L504Y mutant, a point mutation that blocks receptor trafficking and desensitization. In contrast to the wild type, whose LBD is separated, the LBD of the L504Y mutant was detected as a single density. Our results provide direct structural evidence that separation of the LBD within the intact dimeric subunits is critical for efficient tetramerization in the endoplasmic reticulum and further trafficking of AMPARs. The contribution of stargazin on the subunit assembly of AMPA-R was examined. Our data suggest that stargazin affects AMPA-R trafficking at a later stage of receptor maturation.

We used microarrays carrying most of the genes that are developmentally regulated in Dictyostelium to discover those that are preferentially expressed in prestalk cells. Prestalk cells are localized at the front of slugs and play crucial roles in morphogenesis and slug migration. Using whole-mount in situ hybridization, we were able to verify 104 prestalk genes. Three of these were found to be expressed only in cells at the very front of slugs, the PstA cell type. Another 10 genes were found to be expressed in the small number of cells that form a central core at the anterior, the PstAB cell type. The rest of the prestalk-specific genes are expressed in PstO cells, which are found immediately posterior to PstA cells but anterior to 80% of the slug that consists of prespore cells. Half of these are also expressed in PstA cells. At later stages of development, the patterns of expression of a considerable number of these prestalk genes changes significantly, allowing us to further subdivide them. Some are expressed at much higher levels during culmination, while others are repressed. These results demonstrate the extremely dynamic nature of cell-type-specific expression in Dictyostelium and further define the changing physiology of the cell types. One of the signals that affect gene expression in PstO cells is the hexaphenone DIF-1. We found that expression of about half of the PstO-specific genes were affected in a mutant that is unable to synthesize DIF-1, while the rest appeared to be DIF independent. These results indicate that differentiation of some aspects of PstO cells can occur in the absence of DIF-1.

Highlights d Most mouse Cre driver lines tested exhibited variable rates of germline recombination d Germline recombination exhibits parental sex bias and target locus selectivity d Similar principles apply to multiple organisms and recombinase systems d Guidelines are provided for detecting and minimizing unwanted germline recombination

The precise knowledge of the subunit assembly process of NMDA receptors (NMDA-Rs) is essential to understand the receptor architecture and underlying mechanism of channel function. Because NMDA-Rs are obligatory heterotetramers requiring the GluN1 subunit, it is critical to investigate how GluN1 and GluN2 type subunits coassemble into tetramers. By combining approaches in cell biology, biochemistry, single particle electron microscopy, and x-ray crystallography, we report the mechanisms and phenotypes of mutant GluN1 subunits that are defective in receptor maturation. The T110A mutation in the N-terminal domain (NTD) of the GluN1 promotes heterodimerization between the NTDs of GluN1 and GluN2, whereas the Y109C mutation in the adjacent residue stabilizes the homodimer of the NTD of GluN1. The crystal structure of the NTD of GluN1 revealed the mechanism underlying the biochemical properties of these mutants. Effects of these mutations on the maturation of heteromeric NMDA-Rs were investigated using a receptor trafficking assay. Our results suggest that the NTDs of the GluN1 subunit initially form homodimers and the subsequent dimer dissociation is critical for forming heterotetrameric NMDA-Rs containing GluN2 subunits, defining a molecular determinant for receptor assembly. The domain arrangement of the dimeric NTD of GluN1 is unique among the ionotropic glutamate receptors and predicts that the structure and mechanism around the NTDs of NMDA-Rs are different from those of the homologous AMPA and kainate receptors.

The mRNA encoding activity-regulated cytoskeleton-associated protein (Arc) is known to be targeted to dendritic regions that have received strong synaptic inputs. However, the cis-acting elements in Arc mRNA that mediate dendritic targeting have not been identified. To identify the dendritic targeting element (DTE) in rat Arc mRNA, we expressed reporter mRNAs containing various regions of Arc in primary hippocampal neurones and analysed their subcellular distribution by in situ hybridization. Here, we report that the 3'-untranslated region of rat Arc mRNA contains a 350-nucleotide DTE with strong dendritic targeting activity and another 370-nucleotide sequence with weaker dendritic targeting activity. The 350-nucleotide DTE does not share any obvious sequence similarity with other known DTEs previously reported.

Adherens junctions (AJs) play a role in mechanically connecting adjacent cells to maintain tissue structure, particularly in epithelial cells. The major cell–cell adhesion molecules at AJs are cadherins and nectins. Afadin binds to both nectins and α-catenin and recruits the cadherin-β-catenin complex to the nectin-based cell–cell adhesion site to form AJs. To explore the role of afadin in radial glial and ependymal cells in the brain, we generated mice carrying a nestin-Cre-mediated conditional knockout (cKO) of the afadin gene. Newborn afadin-cKO mice developed hydrocephalus and died neonatally. The afadin-cKO brain displayed enlarged lateral ventricles and cerebral aqueduct, resulting from stenosis of the caudal end of the cerebral aqueduct and obliteration of the ventral part of the third ventricle. Afadin deficiency further caused the loss of ependymal cells from the ventricular and aqueductal surfaces. During development, radial glial cells, which terminally differentiate into ependymal cells, scattered from the ventricular zone and were replaced by neurons that eventually covered the ventricular and aqueductal surfaces of the afadin-cKO midbrain. Moreover, the denuded ependymal cells were only occasionally observed in the third ventricle and the cerebral aqueduct of the afadin-cKO midbrain. Afadin was co-localized with nectin-1 and N-cadherin at AJs of radial glial and ependymal cells in the control midbrain, but these proteins were not concentrated at AJs in the afadin-cKO midbrain. Thus, the defects in the afadin-cKO midbrain most likely resulted from the destruction of AJs, because AJs in the midbrain were already established before afadin was genetically deleted. These results indicate that afadin is essential for the maintenance of AJs in radial glial and ependymal cells in the midbrain and is required for normal morphogenesis of the cerebral aqueduct and ventral third ventricle in the midbrain.