The sec6/8 complex is located at neurite outgrowth and axonal synapse-assembly domains

The molecules that specify domains on the neuronal plasma membrane for the delivery and accumulation of vesicles during neurite outgrowth and synapse formation are unknown. We investigated the role of the sec6/8 complex, a set of proteins that specifies vesicle targeting sites in yeast and epithelial cells, in neuronal membrane trafficking. This complex was found in layers of developing rat brain undergoing synaptogenesis. In cultured hippocampal neurons, the sec6/8 complex was present in regions of ongoing membrane addition: the tips of growing neurites, filopodia, and growth cones. In young axons, the sec6/8 complex was also confined to periodic domains of the plasma membrane. The distribution of synaptotagmin, synapsin1, sec6, and FM1-43 labeling in cultured neurons suggested that the plasma membrane localization of the sec6/8 complex preceded the arrival of synaptic markers and was downregulated in mature synapses. We propose that the sec6/8 complex specifies sites for targeting vesicles at domains of neurite outgrowth and potential active zones during synaptogenesis.     

Role of the yeast Gin4p protein kinase in septin assembly and the relationship between septin assembly and septin function

To identify septin-interacting proteins in Saccharomyces cerevisiae, we screened for mutations that are synthetically lethal with a cdc12 septin mutation. One of the genes identified was GIN4, which encodes a protein kinase related to Hsl1p/Nik1p and Ycl024Wp in S. cerevisiae and to Nim1p/Cdr1p and Cdr2p in Schizosaccharomyces pombe. The Gin4p kinase domain displayed a two-hybrid interaction with the COOH-terminal portion of the Cdc3p septin, and Gin4p colocalized with the septins at the mother-bud neck. This localization depended on the septins and on the COOH-terminal (nonkinase) region of Gin4p, and overproduction of this COOH-terminal region led to a loss of septin organization and associated morphogenetic defects. We detected no effect of deleting YCL024W, either alone or in combination with deletion of GIN4. Deletion of GIN4 was not lethal but led to a striking reorganization of the septins accompanied by morphogenetic abnormalities and a defect in cell separation; however, remarkably, cytokinesis appeared to occur efficiently. Two other proteins that localize to the neck in a septin-dependent manner showed similar reorganizations and also appeared to remain largely functional. The septin organization observed in gin4Delta vegetative cells resembles that seen normally in cells responding to mating pheromone, and no Gin4p was detected in association with the septins in such cells. The organization of the septins observed in gin4Delta cells and in cells responding to pheromone appears to support some aspects of the model for septin organization suggested previously by Field et al. (Field, C.M., O. Al-Awar, J. Rosenblatt, M.L. Wong, B. Alberts, and T.J. Mitchison. 1996. J. Cell Biol. 133:605-616).     

Cadherin-8 protein expression in gray matter structures and nerve fibers of the neonatal and adult mouse brain

The topological distribution of mouse cadherin-8 protein in the neonatal and adult mouse brain was studied immunohistochemically using a rabbit antiserum. Cadherin-8 expression was restricted to several areas in neonatal brains constituting particular neural circuits, i.e. the limbic system, the basal ganglia-thalamocortical circuit, and the cerebellum and related nuclei. In addition, the nerve fibers linking some of the cadherin-8-positive areas, i.e. the habenulo-interpeduncular tract, decussation of the dorsal tegmentum, the medial longitudinal fasciculus, transverse pontine fibers, the brachium conjunctivum and the inferior cerebellar peduncle were cadherin-8 positive, as were the spinal tract of the trigeminal nerve, oculomotor nerve, facial nerve and trigeminal nerve. Cadherin-8 expression also showed a patch-like distribution in the intermediate gray layer of the superior colliculus, resembling acetylcholinesterase-rich patches in allocation. Segmentally organized cadherin-8-positive areas were found in the neonatal cerebellar Purkinje cell layer. Some nuclei and fibers in the brainstem and cerebellum, expressing cadherin-8 at neonatal stages, were also stained in the adult mouse brain. These findings suggest that cadherin-8 is involved in the formation of particular neural circuits by connecting areas expressing this molecule with positive nerve fibers, and indicate its possible implication in subdivisional organization in the superior colliculus and cerebellum.     

Characterization of interactions between the mammalian polycomb-group proteins Enx1/EZH2 and EED suggests the existence of different mammalian polycomb-group protein complexes

In Drosophila melanogaster, the Polycomb-group (PcG) and trithorax-group (trxG) genes have been identified as repressors and activators, respectively, of gene expression. Both groups of genes are required for the stable transmission of gene expression patterns to progeny cells throughout development. Several lines of evidence suggest a functional interaction between the PcG and trxG proteins. For example, genetic evidence indicates that the enhancer of zeste [E(z)] gene can be considered both a PcG and a trxG gene. To better understand the molecular interactions in which the E(z) protein is involved, we performed a two-hybrid screen with Enx1/EZH2, a mammalian homolog of E(z), as the target. We report the identification of the human EED protein, which interacts with Enx1/EZH2. EED is the human homolog of eed, a murine PcG gene which has extensive homology with the Drosophila PcG gene extra sex combs (esc). Enx1/EZH2 and EED coimmunoprecipitate, indicating that they also interact in vivo. However, Enx1/EZH2 and EED do not coimmunoprecipitate with other human PcG proteins, such as HPC2 and BMI1. Furthermore, unlike HPC2 and BMI1, which colocalize in nuclear domains of U-2 OS osteosarcoma cells, Enx1/EZH2 and EED do not colocalize with HPC2 or BMI1. Our findings indicate that Enx1/EZH2 and EED are members of a class of PcG proteins that is distinct from previously described human PcG proteins.     

Subunit composition of the peripherin/rds-rom-1 disk rim complex from rod photoreceptors: hydrodynamic evidence for a tetrameric quaternary structure

Peripherin/rds and rom-1 are homologous integral membrane protein subunits found as an oligomeric complex at the rim regions of rod and cone photoreceptor outer segment disks. These proteins are essential for the morphogenesis of normal outer segments and have been linked to a variety of human retinal degenerative diseases. Previous studies have suggested that disulfide-linked homodimers of peripherin/rds and rom-1 can associate noncovalently to form higher order structures. We have characterized the hydrodynamic properties of Triton X-100 solubilized peripherin/rds-rom-1 complexes from bovine ROS membranes by gel exclusion chromatography on Sepharose C1-6B and velocity sedimentation through H2O- and D2O-based sucrose gradients. A single hydrodynamic species is observed which has a Stokes radius of 6.2 nm, a sedimentation coefficient (S20,w) of 5.8 S, and a partial specific volume of 0.83 mL/g. From these data the molecular mass of the detergent-peripherin/rds-rom-1 complex is calculated to be 240 kDa. The protein component of this complex is estimated to be 135 kDa, providing direct evidence that the solubilized peripherin/rds-rom-1 complex is a tetramer. The abundance of this complex as measured by competitive ELISA and immunoaffinity purification is approximately 4% of total bovine ROS membrane protein and indicates that peripherin/rds-rom-1 tetramers are present at a relatively high average surface density (ca. 4100/ microns m2) at the rim surfaces of rod outer segment disks.     

Dual function of Cyk2, a cdc15/PSTPIP family protein, in regulating actomyosin ring dynamics and septin distribution

We previously showed that the budding yeast Saccharomyces cerevisiae assembles an actomyosin-based ring that undergoes a contraction-like size change during cytokinesis. To learn more about the biochemical composition and activity of this ring, we have characterized the in vivo distribution and function of Cyk2p, a budding yeast protein that exhibits significant sequence similarity to the cdc15/PSTPIP family of cleavage furrow proteins. Video microscopy of cells expressing green fluorescent protein (GFP)-tagged Cyk2p revealed that Cyk2p forms a double ring that coincides with the septins through most of the cell cycle. During cytokinesis, however, the Cyk2 double ring merges with the actomyosin ring and exhibits a contraction-like size change that is dependent on Myo1p. The septin double ring, in contrast, does not undergo the contraction-like size change but the separation between the two rings increases during cytokinesis. These observations suggest that the septin-containing ring is dynamically distinct from the actomyosin ring and that Cyk2p transits between the two types of structures. Gene disruption of CYK2 does not affect the assembly of the actomyosin ring but results in rapid disassembly of the ring during the contraction phase, leading to incomplete cytokinesis, suggesting that Cyk2p has an important function in modulating the stability of the actomyosin ring during contraction. Overexpression of Cyk2p also blocks cytokinesis, most likely due to a loss of the septins from the bud neck, indicating that Cyk2p may also play a role in regulating the localization of the septins.     

Subunit composition of brain voltage-gated potassium channels determined by hongotoxin-1, a novel peptide derived from Centruroides limbatus venom

Five novel peptidyl inhibitors of Shaker-type (Kv1) K+ channels have been purified to homogeneity from venom of the scorpion Centruroides limbatus. The complete primary amino acid sequence of the major component, hongotoxin-1 (HgTX1), has been determined and confirmed after expression of the peptide in Escherichia coli. HgTX1 inhibits 125I-margatoxin binding to rat brain membranes as well as depolarization-induced 86Rb+ flux through homotetrameric Kv1.1, Kv1. 2, and Kv1.3 channels stably transfected in HEK-293 cells, but it displays much lower affinity for Kv1.6 channels. A HgTX1 double mutant (HgTX1-A19Y/Y37F) was constructed to allow high specific activity iodination of the peptide. HgTX1-A19Y/Y37F and monoiodinated HgTX1-A19Y/Y37F are equally potent in inhibiting 125I-margatoxin binding to rat brain membranes as HgTX1 (IC50 values approximately 0.3 pM). 125I-HgTX1-A19Y/Y37F binds with subpicomolar affinities to membranes derived from HEK-293 cells expressing homotetrameric Kv1.1, Kv1.2, and Kv1.3 channels and to rat brain membranes (Kd values 0.1-0.25 pM, respectively) but with lower affinity to Kv1.6 channels (Kd 9.6 pM), and it does not interact with either Kv1.4 or Kv1.5 channels. Several subpopulations of native Kv1 subunit oligomers that contribute to the rat brain HgTX1 receptor have been deduced by immunoprecipitation experiments using antibodies specific for Kv1 subunits. HgTX1 represents a novel and useful tool with which to investigate subclasses of voltage-gated K+ channels and Kv1 subunit assembly in different tissues.     

Crystal structure of the human ubiquitin-like protein NEDD8 and interactions with ubiquitin pathway enzymes

The NEDD8/Rub1 class of ubiquitin-like proteins has been implicated in progression of the cell cycle from G1 into S phase. These molecules undergo a metabolism that parallels that of ubiquitin and involves specific interactions with many different proteins. We report here the crystal structure of recombinant human NEDD8 refined at 1.6-A resolution to an R factor of 21.9%. As expected from the high sequence similarity (57% identical), the NEDD8 structure closely resembles that reported previously for ubiquitin. We also show that recombinant human NEDD8 protein is activated, albeit inefficiently, by the ubiquitin-activating (E1) enzyme and that NEDD8 can be transferred from E1 to the ubiquitin conjugating enzyme E2-25K. E2-25K adds NEDD8 to a polyubiquitin chain with an efficiency similar to that of ubiquitin. A chimeric tetramer composed of three ubiquitins and one histidine-tagged NEDD8 binds to the 26 S proteasome with an affinity similar to that of tetraubiquitin. Seven residues that differ from the corresponding residues in ubiquitin, but are conserved between NEDD8 orthologs, are candidates for mediating interactions with NEDD8-specific partners. One such residue, Ala-72 (Arg in ubiquitin), is shown to perform a key role in selecting against reaction with the ubiquitin E1 enzyme, thereby acting to prevent the inappropriate diversion of NEDD8 into ubiquitin-specific pathways.     

Integrative levels, the brain, and the emergence of complex behavior.

The aim of this article is to bring clarity and unification to the question of how certain complex behaviors, such as feeding, learning, language, culture, and neural complexity, are related. Three critical ideas—the organizing principle of integrative levels, the tendency for increased complexity with evolutionary change, and the contextual nature of behavioral events—are central to the discussion. A theoretical framework is presented that synthesizes existing knowledge in a meaningful way. Data are drawn from the behavioral, neuroanatomical, cognitive, and linguistic sciences and integrated within an organized and unified theoretical perspective referred to as developmental dynamic systems theory. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Intermolecular and interdomain interactions of a dynamin-related GTP-binding protein, Dnm1p/Vps1p-like protein

Dnm1p/Vps1p-like protein (DVLP) is a mammalian member of the dynamin GTPase family, which is classified into subfamilies on the basis of the structural similarity. Mammalian dynamins constitute the dynamin subfamily. DVLP belongs to the Vps1 subfamily, which also includes yeast Vps1p and Dnm1p. Typical structural features that discriminate between members of the Vps1 and dynamin subfamilies are that the former lacks the pleckstrin homology and Pro-rich domains. Dynamin exists as tetramers under physiological salt conditions, whereas under low salt conditions, it can polymerize into spirals that resemble the collar structures seen at the necks of constricted coated pits. In this study, we found that DVLP is also oligomeric, probably tetrameric, under physiological salt conditions and forms sedimentable large aggregates under low salt conditions. The data indicate that neither the pleckstrin homology nor Pro-rich domain is required for the self-assembly. Analyses using the two-hybrid system and co-immunoprecipitation show that the N-terminal region containing the GTPase domain and a domain (DVH1) conserved across members of the dynamin and Vps1 subfamilies, can interact with the C-terminal region containing another conserved domain (DVH2). The data on the interdomain interaction of DVLP is compatible with the previous reports on the interdomain interaction of dynamin. Thus, the self-assembly mechanism of DVLP appears to resemble that of dynamin, suggesting that DVLP may also be involved in the formation of transport vesicles.     

The 77-kDa echinoderm microtubule-associated protein (EMAP) shares epitopes with the mammalian brain MAPs, MAP-2 and tau

Heat shock proteins not only can protect host cells against heat stress, they can also enable freeze tolerance as well. With respect to this unexpected feature, we are able to show that, at least in Escherichia coli, the heat shock proteins DnaK/DnaJ and GroEL play a very significant role. We found that the recovery rate of E. coli cultures that had been stored at -80 degreesC in the absence of any cryoprotectant was related to the abundance of these heat shock proteins accumulated before the freeze treatment. Before freezing, the DnaK in the bacterial cells was induced to accumulate to a level comparable to that produced in response to heat shock. After the freezing treatment, the recovery rate of the induced culture was very similar to that of the heat-shocked culture. Over production of GroEL was also protective but less effective. While freezing inevitably leads to protein denaturation, we propose that advance synthesis of DnaK/DnaJ and GroEL can accordingly prevent irreversible denaturation by chaperoning the unfolded polypeptides during freezing.     

Subcellular localization and ubiquitin-conjugating enzyme (E2) interactions of mammalian HECT family ubiquitin protein ligases

In most instances, the transfer of ubiquitin to target proteins is catalyzed by the action of ubiquitin protein ligases (E3s). Full-length cDNAs encoding murine E6-associated protein (mE6-AP) as well as Nedd-4, a protein that is homologous to E6-AP in its C terminus, were cloned. Nedd-4 and mouse E6-AP are both enzymatically active E3s and function with members of the UbcH5 family of E2s. Mouse E6-AP, like its human counterpart, ubiquitinates p53 in the presence of human papilloma virus E6 protein, while Nedd-4 does not. Consistent with its role in p53 ubiquitination, mE6-AP was found both in the nucleus and cytosol, while Nedd-4 was found only in the cytosol. Binding studies implicate a 150-amino acid region that is 40% identical between mE6-AP and Nedd-4 as a binding site for the C-terminal portion of an E2 enzyme (UbcH5B). Nedd-4 was determined to have a second nonoverlapping E2 binding site that recognizes the first 67 amino acids of UbcH5B but not the more C-terminal portion of this E2. These findings provide the first demonstration of physical interactions between mammalian E2s and E3s and establish that these interactions occur independently of ubiquitin and an intact E3 catalytic domain. Furthermore, the presence of two E2 binding sites within Nedd-4 suggests models for ubiquitination involving multiple E2 enzymes associated with E3s.     

Emopamil-binding protein, a mammalian protein that binds a series of structurally diverse neuroprotective agents, exhibits delta8-delta7 sterol isomerase activity in yeast

Delta8-delta7 sterol isomerase is an essential enzyme on the sterol biosynthesis pathway in eukaryotes. This endoplasmic reticulum-resident membrane protein catalyzes the conversion of delta8-sterols to their corresponding delta7-isomers. No sequence data for high eukaryote sterol isomerase being available so far, we have cloned a murine sterol isomerase-encoding cDNA by functional complementation of the corresponding deficiency in the yeast Saccharomyces cerevisiae. The amino acid sequence deduced from the cDNA open reading frame is highly similar to human emopamil-binding protein (EBP), a protein of unknown function that constitutes a molecular target for neuroprotective drugs. A yeast strain in which the sterol isomerase coding sequence has been replaced by that of human EBP or its murine homologue recovers the ability to convert delta8-sterol into delta7-sterol, both in vivo and in vitro. In these recombinant strains, both cell proliferation and the sterol isomerization reaction are inhibited by the high affinity EBP ligand trifluoperazine, as is the case in mammalian cells but not in wild type yeast cell. In contrast, the recombinant strains are much less susceptible to the sterol inhibition effect of haloperidol and fenpropimorph, as compared with wild type yeast strains. Our results strongly suggest that EBP and delta8-delta7 sterol isomerase are identical proteins in mammals.     

Fusion of the MLL gene with two different genes, AF-6 and AF-5alpha, by a complex translocation involving chromosomes 5, 6, 8 and 11 in infant leukemia

We analysed a complex translocation involving chromosomes 5, 6, 8 and 11 in a case of infant leukemia. Molecular analysis of the MLL gene revealed that MLL was fused with two different genes, AF-6 on chromosome 6q27 and AF-5alpha. AF-5alpha, the 11th partner gene fused with MLL, is a novel gene mapped to chromosome 5q12, which encodes a 31 kDa protein of 269 amino acids and contains a possible nuclear targeting sequence, a potential leucine zipper dimerization motif and an alpha-helical coiled-coil domain. In situ hybridization and molecular cloning analyses demonstrated that two different types of chromosomal recombination had occurred in the cells. One was a three-way translocation among chromosomes 6, 8 and 11, and the other was an insertion of a chromosome 5-derived segment into the breakpoint of chromosomes 8 and 11. Accordingly, the karyotype was defined as del(5)(q11.2q12), der(6)t(6;8) (q27;q11.2), der(8)(8pter-->8q11.2::5q11.2-->5q12::11q23-->++ +11qter), der(11)t(6;11) (q27;q23). Thus, the MLL gene created two different fusion mRNAs, since the chromosome 11 split into two different chromosomes 5 and 6. This is the first report demonstrating fusion of the MLL gene with two different genes by a complex translocation.