Specialized Sites Research Articles

Quantitative SUMO-1 Modification of a Vaccinia Virus Protein Is Required for Its Specific Localization and Prevents Its Self-Association

Vaccinia virus (VV), the prototype member of the Poxviridae, a family of large DNA viruses, carries out DNA replication in specialized cytoplasmic sites that are enclosed by the rough endoplasmic reticulum (ER). We show that the VV gene product of A40R is quantitatively modified by SUMO-1, which is required for its localization to the ER-enclosed replication sites. Expression of A40R lacking SUMO-1 induced the formation of rod-shaped cytoplasmic aggregates. The latter likely consisted of polymers of nonsumoylated protein, because unmodified A40R interacted with itself, but not with the SUMO-1-conjugated protein. Using a bacterial sumoylation system, we furthermore show that unmodified A40R is mostly insoluble, whereas the modified form is completely soluble. By electron microscopy, the A40R rods seen in cells were associated with the cytosolic side of the ER and induced the apposition of several ER cisternae. A40R is the first example of a poxvirus protein to acquire SUMO-1. Its quantitative SUMO-1 modification is required for its proper localization to the viral "mini-nuclei" and prevents its self-association. The ability of the nonsumoylated A40R to bring ER membranes close together could suggest a role in the fusion of ER cisternae when these coalesce to enclose the VV replication sites.

Meiotic Recombination in Drosophila Females Depends on Chromosome Continuity Between Genetically Defined Boundaries

In the pairing-site model, specialized regions on each chromosome function to establish meiotic homolog pairing. Analysis of these sites could provide insights into the mechanism used by Drosophila females to form a synaptonemal complex (SC) in the absence of meiotic recombination. These specialized sites were first established on the X chromosome by noting that there were barriers to crossover suppression caused by translocation heterozygotes. These sites were genetically mapped and proposed to be pairing sites. By comparing the cytological breakpoints of third chromosome translocations to their patterns of crossover suppression, we have mapped two sites on chromosome 3R. We have performed experiments to determine if these sites have a role in meiotic homolog pairing and the initiation of recombination. Translocation heterozygotes exhibit reduced gene conversion within the crossover-suppressed region, consistent with an effect on the initiation of meiotic recombination. To determine if homolog pairing is disrupted in translocation heterozygotes, we used fluorescent in situ hybridization to measure the extent of homolog pairing. In wild-type oocytes, homologs are paired along their entire lengths prior to accumulation of the SC protein C(3)G. Surprisingly, translocation heterozygotes exhibited homolog pairing similar to wild type within the crossover-suppressed regions. This result contrasted with our observations of c(3)G mutant females, which were found to be defective in pairing. We propose that each Drosophila chromosome is divided into several domains by specialized sites. These sites are not required for homolog pairing. Instead, the initiation of meiotic recombination requires continuity of the meiotic chromosome structure within each of these domains.

The Complementary and Alternative Medicine Information Source Book

The Complementary and Alternative Medicine Information Source Book

Retrovirus budding.

Human immunodeficiency virus (HIV) and other retroviruses acquire their envelopes and spread infection by budding through the limiting membranes of producer cells. To facilitate budding, retroviruses usurp a cellular pathway that is normally used to create vesicles that bud into late endosomal compartments called multivesicular bodies (MVB). Research on yeast and human MVB biogenesis has led to the identification of 25 human proteins that are required for vesicle formation and for HIV-1 budding, and has produced a working model for sequential recruitment of these proteins during MVB vesicle formation. Retroviruses can redirect this machinery to the plasma membrane and leave the cell in a single step or, alternatively, can bud directly into MVB compartments and then exit cells via the exosome pathway. Remarkably, virus release from both the plasma membrane and MVB compartments can occur directionally into specialized sites of cell-to-cell contact called virological synapses. Thus retroviruses have evolved elaborate mechanisms for escaping the cell and maximizing their chances of infecting a new host.

The architecture of the active zone in the presynaptic nerve terminal.

Active zones are highly specialized sites for release of neurotransmitter from presynaptic nerve terminals. The architecture of the active zone is exquisitely designed to facilitate the regulated tethering, docking, and fusing of the synaptic vesicles with the plasma membrane. Here we present our view of the structural and molecular organization of active zones across species and propose that all active zones are organized according to a common principle in which the structural differences correlate with the kinetics of transmitter release.

Xin repeats define a novel actin-binding motif.

Xin is a protein that is expressed during early developmental stages of cardiac and skeletal muscles. Immunolocalization studies indicated a peripheral localization in embryonic mouse heart, where Xin localizes with beta-catenin and N-cadherin. In adult tissues, Xin is found primarily in the intercalated discs of cardiomyocytes and the myotendinous junctions of skeletal muscle cells, both specialized attachment sites of the myofibrillar ends to the sarcolemma. A large part of the Xin protein consists of unique 16 amino acid repeats with unknown function. We have investigated the characteristics of the Xin repeats by transfection experiments and actin-binding assays and ascertained that, upon expression in cultured cells, these repeats bind to and stabilize the actin-based cytoskeleton. In vitro co-sedimentation assays with skeletal muscle actin indicated that they not only directly bind actin filaments, but also have the capability of arranging microfilaments into networks that sediment upon low-speed centrifugation. Very similar repeats were also found in 'Xin-repeat protein 2' (XIRP2), a novel protein that seems to be expressed mainly in striated muscles. Human XIRP2 contains 28 Xin repeats with properties identical to those of Xin. We conclude that the Xin repeats define a novel, repetitive actin-binding motif present in at least two different muscle proteins. These Xin-repeat proteins therefore constitute the first two members of a novel family of actin-binding proteins.

Keratin 2e: A Marker for Murine Nipple Epidermis

Mesenchyme-derived signals influence the unique keratinization and appendage formation programs in specialized skin regions. Interactions between primary mammary mesenchyme and epidermal cells result in the formation of the nipple; however, it is unclear whether this represents a site of regionally specialized epidermis. We profiled the ultrastructure and keratin expression of the murine nipple, and the ventral skin of the K14-parathyroid hormone-related protein (PTHrP) transgenic mouse, which models nipple formation. We found the murine nipple and ventral K14-PTHrP epidermis display expanded suprabasal and granular layers, as well as a thickened cornified layer compared to ventral skin of wild-type littermates. We also observed increased levels of filaggrin in extracts from the ventral epidermis of the K14-PTHrP mouse when compared to that of wild-type littermates. Keratin 2e, previously reported to be expressed in various specialized epidermal sites in the mouse, is expressed in the nipple and the ventral skin of the K14-PTHrP mouse. Keratinocytes grown from the ventral epidermis of the K14-PTHrP mouse or wild-type littermates exhibited identical expression of epidermal markers in vitro, suggesting that the modulated differentiation profile observed in the nipple or the ventral K14-PTHrP skin was dependent on interactions with fibroblasts. The lack of appendages, altered stratification pattern and expression of a specialized keratin suggests that the murine nipple is an example of regionally specialized epidermis.

Ultrastructural analysis of transitional endoplasmic reticulum and pre-Golgi intermediates: a highway for cars and trucks.

Cargo selection and export from the endoplasmic reticulum occurs at specialized sites in cells. Export complexes consist of transitional elements of the endoplasmic reticulum and pre-Golgi intermediates. It is generally assumed that 60 to 80 nm initially COPII-coated vesicles derived from the transitional endoplasmic reticulum are the main carriers for transport of cargo to the Golgi apparatus. We have analyzed on serial ultrathin sections the transitional endoplasmic reticulum and pre-Golgi intermediates of beta cells of islets of Langerhans in mouse pancreas. In addition to Golgi-associated complexes, others were observed in the periphery of the cells or close to the nuclear envelope. Upon three-dimensional reconstruction, non-coated ribosome-free tubules with an average diameter of 115 nm (range 60-195 nm) and a length of up to 500 nm were detected in the pre-Golgi intermediates in addition to small vesiculo-tubular elements. Furthermore, evidence was found that the large tubular elements may directly arise from transitional elements of the endoplasmic reticulum. In a given cell, pre-Golgi intermediates were found to be composed solely of small vesiculo-tubular elements or additionally of tubules or solely of tubules. Immunogold labeling for proinsulin indicated that the large tubular elements contained cargo and thus appear to take part in ER-to-Golgi transport.

Mismatch Repair Regulates Homologous Recombination, but Has Little Influence on Antigenic Variation, in Trypanosoma brucei

Antigenic variation is critical in the life of the African trypanosome, as it allows the parasite to survive in the face of host immunity and enhance its transmission to other hosts. Much of trypanosome antigenic variation uses homologous recombination of variant surface glycoprotein (VSG)-encoding genes into specialized transcription sites, but little is known about the processes that regulate it. Here we describe the effects on VSG switching when two central mismatch repair genes, MSH2 and MLH1, are mutated. We show that disruption of the parasite mismatch repair system causes an increased frequency of homologous recombination, both between perfectly matched DNA molecules and between DNA molecules with divergent sequences. Mismatch repair therefore provides an important regulatory role in homologous recombination in this ancient eukaryote. Despite this, the mismatch repair system has no detectable role in regulating antigenic variation, meaning that VSG switching is either immune to mismatch selection or that mismatch repair acts in a subtle manner, undetectable by current assays.

Roles of antioxidants on prolonged storage of avian spermatozoa in vivo and in vitro.

This review focuses on natural and assisted prevention against lipid peroxidation in avian spermatozoa. The presence of high levels of n-6 polyunsaturated fatty acids (PUFAs) in the plasma membrane creates favorable conditions for the formation of peroxidative products, a major cause of membrane damage which may ultimately impair male fertility. However, a complex antioxidant system involving vitamin C, vitamin E and GSH is naturally present in avian semen. Coupled with a battery of enzymatic defenses (e.g., SOD, GSH-Px either Se- or non-Se-dependent), this system acts to prevent or restrict the formation and propagation of peroxides. The presence of specialized sites dedicated to prolonged sperm storage in avian females raises the question of durable protection of sperm membranes against peroxidation. Preliminary observations have revealed the presence of a specific antioxidant system at these sites in which vitamin C could exert a major role. From a practical standpoint, the extensive use of artificial insemination in poultry, along with the emergence in some species of workable techniques to cryopreserve spermatozoa, demand better control of peroxidation occurring in the plasma membrane of spermatozoa before or during storage. Dietary supplementation with vitamin E is effective in limiting lipid peroxidation of sperm plasma membranes, both in chickens and turkeys. In addition, organic Se with or without vitamin E stimulates Se-GSH-Px activity in seminal plasma. Preliminary observations in female chickens have also revealed the effectiveness of dietary supplementation with vitamin E, organic selenium or both to sustain fertility in aging flocks.

The a-Subunit of the V-type H+-ATPase Interacts with Phosphofructokinase-1 in Humans

V-type or H+-ATPases are a family of ATP-dependent proton pumps that move protons across the plasma membrane at specialized sites such as kidney epithelial cells and osteoclasts as well as acidifying intracellular compartments. The 100-kDa polytopic a-subunit of this group of ATPases is suggested to play an important role in coupling the two functions of the pump, ATP hydrolysis and proton transport. In man, different a-subunit isoforms are encoded by four genes. ATP6V0A4 encodes a4, which is expressed apically in alpha-intercalated cells in both human and mouse kidney. We sought binding partners for the C terminus of a4 in order to address its potential role in the H+-ATPase complex. Random peptide phage display analysis revealed a consensus motif (WLELRP) with almost complete homology to part of the enzyme phosphofructokinase 1 (PFK-1). Activity of this enzyme is the rate-limiting step in glycolysis. Specificity of a4 binding to this peptide was confirmed by enzyme-linked immunosorbent assay. Protein-protein interaction was further demonstrated by co-immunoprecipitation of a4 with PFK-1 from solubilized human kidney membrane proteins. An in vitro bead-bound PFK-1 pull-down assay showed that this interaction was also true for the ubiquitously expressed a1 subunit. Finally, PFK-1 co-immunolocalized with a4 in alpha-intercalated cells in the collecting ducts of human kidney. These findings indicate a direct link between V-type H+-ATPases and glycolysis via the C-terminal region of the a-subunit of the pump and suggest a novel regulatory mechanism between H+-ATPase function and energy supply. This interaction between the a-subunit and PFK-1 also provides new evidence that the C terminus of this subunit lies cytoplasmically in vivo.

Zinc is incorporated into cuticular “tools” after ecdysis: The time course of the zinc distribution in “tools” and whole bodies of an ant and a scorpion

An understanding of the developmental course of specialized accumulations in the cuticular “tools” of arthropods will give clues to the chemical form, function and biology of these accumulations as well as to their evolutionary history. Specimens from individuals representing a range of developmental stages were examined using MeV - Ion microscopy. We found that zinc, manganese, calcium and chlorine began to accumulate in the mandibular teeth of the ant Tapinoma sessile after pre-ecdysial tanning, and the zinc mostly after eclosion; peak measured zinc concentrations reached 16% of dry mass. Accumulations in the pedipalp teeth, tarsal claws, cheliceral teeth and sting (aculeus) of the scorpion Vaejovis spinigeris also began after pre-ecdysial tanning and more than 48 h after ecdysis of the second instars. Zinc may be deposited in the fully formed cuticle through a network of nanometer scale canals that we observed only in the metal bearing cuticle of both the ants and scorpions. In addition to the elemental analyses of cuticular “tools”, quantitative distribution maps for whole ants were obtained. The zinc content of the mandibular teeth was a small fraction of, and independent of, the total body content of zinc. We did not find specialized storage sites that were depleted when zinc was incorporated into the mandibular teeth. The similarities in the time course of zinc, manganese and calcium deposition in the cuticular “tools” of the ant (a hexapod arthropod) and those of the scorpion (a chelicerate arthropod) contribute to the evidence suggesting that heavy metal - halogen fortification evolved before these groups diverged.

Endothelial cell glycosylation: regulation and modulation of biological processes.

The concept of homing tissue specialization and tissue-specific lymphocyte recirculation that is fundamental to acquired immunity raised from the demonstration, by Gowans and Knight in 1964, that circulating leukocytes enter secondary lymphoid organs at specialized endothelial sites (Picker and Butcher, 1992), which was the starting point for many studies to understand specificity of cell homing. T memory lymphocytes were first shown to be able to recirculate to tissues where they came from; further on, B immunoblasts were shown to migrate preferentially into mucosal tissues (for a review, see Kunkel and Butcher, 2002). The regio-selectivity is indeed favorable to perform an immune response (Butcher and Picker, 1996): it helps meetings between antigens and lymphocytes which carry the antigen specific receptor, reducing cross reactivity and allowing site specialization of the immune responses. Indeed, integration and control of systemic immune responses depend on the regulated trafficking of lymphocytes. Homing process disperses the immuno-logical repertoire, directs lymphocyte subsets to the specialized microenvironments that control their differentiation, regulates their survival, and targets immune effector cells to sites of antigenic or microbial invasion.

Protein-protein interactions between large proteins: two-hybrid screening using a functionally classified library composed of long cDNAs.

Large proteins have multiple domains that are potentially capable of binding many kinds of partners. It is conceivable, therefore, that such proteins could function as an intricate framework of assembly protein complexes. To comprehensively study protein-protein interactions between large KIAA proteins, we have constructed a library composed of 1087 KIAA cDNA clones based on prior functional classifications done in silico. We were guided by two principles that raise the success rate for detecting interactions per tested combination: we avoided testing low-probability combinations, and reduced the number of potential false negatives that arise from the fact that large proteins cannot reliably be expressed in yeast. The latter was addressed by constructing a cDNA library comprised of random fragments encoding large proteins. Cytoplasmic domains of KIAA transmembrane proteins (>1000 amino acids) were used as bait for yeast two-hybrid screening. Our analyses reveal that several KIAA proteins bearing a transmembrane region have the capability of binding to other KIAA proteins containing domains (e.g., PDZ, SH3, rhoGEF, and spectrin) known to be localized to highly specialized submembranous sites, indicating that they participate in cellular junction formation, receptor or channel clustering, and intracellular signaling events. Our representative library should be a very useful resource for detecting previously unidentified interactions because it complements conventional expression libraries, which seldom contain large cDNAs.

Electrolyte and fluid secretion by cultured human inner medullary collecting duct cells.

Inner medullary collecting ducts (IMCD) are the final nephron segments through which urine flows. To investigate epithelial ion transport in human IMCD, we established primary cell cultures from initial (hIMCD(i)) and terminal (hIMCD(t)) inner medullary regions of human kidneys. AVP, PGE(2), and forskolin increased cAMP in both hIMCD(i) and hIMCD(t) cells. The effects of AVP and PGE2 were greatest in hIMCD(i); however, forskolin increased cAMP to the same extent in hIMCD(i) and hIMCD(t). Basal short-circuit current (I(SC)) of hIMCD(i) monolayers was 1.4 +/- 0.5 microA/cm2 and was inhibited by benzamil, a Na+ channel blocker. 8-Bromo-cAMP, AVP, PGE(2), and forskolin increased I(SC); the current was reduced by blocking PKA, apical Cl- channels, basolateral NKCC1 (a Na+ - K+ - 2Cl- cotransporter), and basolateral Cl-/HCO(3)(-) exchangers. In fluid transport studies, hIMCD(i) monolayers absorbed fluid in the basal state and forskolin reversed net fluid transport to secretion. In hIMCD(t) monolayers, basal current was not different from zero and cAMP had no effect on I(SC). We conclude that AVP and PGE2 stimulate cAMP-dependent Cl- secretion by hIMCD(i) cells, but not hIMCD(t) cells, in vitro. We suggest that salt secretion at specialized sites along human collecting ducts may be important in the formation of the final urine.

An Internet-based system for the commerce of medical devices.

The MEDIDEV service combines a central product catalog featuring basic information with Internet-connected websites and online catalogs hosting the detailed information. This approach is compatible with the manufacturers' requirement to maintain control over the detailed information of their products. At the same time, it allows the end user to efficiently choose from a potentially long list of products-those which mostly satisfy his requirements-and then to get more detailed information in the corresponding linked websites and on-line catalogs. The manufacturers have two alternative ways for updating the information stored in the Portal. They can use a human-operated and HTML-based interface or an automated and CORBA-based interface. Thus, the timeliness and consistency of the Portal information is ensured. The adoption of Internet standard technologies (Java, CORBA, XML) has maintained a high level of platform independence and interoperability. The current version has been developed for Windows NT, while the final version is currently ported to a HP UNIX machine. Special care has been taken to support both major Internet browsers: Internet Explorer and Netscape Navigator. The implementation of XML for the data presentation has led to a reduced and optimized transmission of data from the Portal to the user's browser. Only the new data are transmitted as XML documents each time, while formatting information is once loaded at the browser. The separation of data from the formatting information leads to another advantage. It is a common practice today for horizontal portals, which are targeting a wide variety of interests, to subcontract content from other, more specialized portals or sites (vertical portals). The MEDIDEV Portal can provide its data to other sites as XML documents that will be parsed and displayed with their own look and feel. This opens up new marketing opportunities.

FIGQY phosphorylation defines discrete populations of L1 cell adhesion molecules at sites of cell-cell contact and in migrating neurons.

Phosphorylation of neurofascin, a member of the L1 family of cell adhesion molecules (L1 CAMs), at the conserved FIGQY-tyrosine abolishes the ankyrin-neurofascin interaction. This study provides the first evidence, in Drosophila melanogaster and vertebrates, for the physiological occurrence of FIGQY phosphorylation in L1 family members. FIGQY tyrosine phosphorylation is localized at specialized cell junctions, including paranodes of sciatic nerve, neuromuscular junctions of adult rats and Drosophila embryos, epidermal muscle attachment sites of Drosophila, and adherens junctions of developing epithelial cells of rat and Drosophila. In addition, FIGQY-phosphorylated L1 CAMs are abundantly expressed in regions of neuronal migration and axon extension, including the embryonic cortex, the neonatal cerebellum and the rostral migratory stream, a region of continued neurogenesis and migration throughout adulthood in the rat. Based on our results, physiological FIGQY-tyrosine phosphorylation of the L1 family likely regulates adhesion molecule-ankyrin interactions establishing ankyrin-free and ankyrin-containing microdomains and participates in an ankyrin-independent intracellular signaling pathway at specialized sites of intercellular contact in epithelial and nervous tissue.

Enteric co-innervation of striated muscle fibers in the esophagus: just a "hangover"?

Striated muscle of the esophagus was until recently considered to consist of "classical" skeletal muscle fibers innervated by cholinergic vagal motoneurons. The recently described co-innervation originating from enteric neurons expressing nNOS, VIP, NPY, and galanin added a new dimension of complexity. The aim of this study was to summarize current knowledge about, and to get further hints as to the possible function of enteric co-innervation of striated esophageal muscle fibers. Aldehyde fixed rat esophagi were processed for immunocytochemistry for CGRP or VAChT (to demonstrate vagal motor terminals), nNOS/NADPH-d, VIP, NPY, and galanin (to demonstrate enteric terminals), met-enkephalin, mu opiate receptor, muscarinic receptors m1-3, soluble guanylyl cyclase, and cGMP dependent kinase type I and II. Motor endplates were visualized using fluorochrome tagged alpha-bungarotoxin to label nicotinic receptors, or with AChE histochemistry. Besides light and confocal laser scanning microscopy, immuno electron microscopy was also employed. Up to 80% of motor endplates were co-innervated. In addition to nNOS, VIP, NPY, and galanin, many enteric terminals in esophageal motor endplates expressed met-enkephalin. Some appeared to stain for the muscarinic m(2) receptor. There was prominent immunostaining for the micro opioid receptor in the sarcolemma at both junctional and extrajunctional sites. Immunostaining for soluble guanylyl cyclase was prominent immediately beneath the clusters of nicotinic receptors. Enteric varicosities and vagal terminals intermingled in motor endplates often without intervening teloglial processes. During ontogeny, initially high co-innervation rates were reduced to adult levels in a cranio-caudally progressing manner. We conclude that, in addition to a possible nitrergic, VIP-, NPY-, and galaninergic modulation of neuromuscular transmission by enteric neurons, opioidergic mechanisms could play a role. On the other hand, cholinergic influence on enteric neurons may be exerted also by the nucleus ambiguus via motor endplates, in addition to the input from the dorsal motor nucleus. The observations that enteric nerve fibers contact striated muscle fibers at specialized sites, i.e., motor endplates, and that these contacts appear in an ordered cranio-caudal sequence after cholinergic motor endplates have been established point to a specific function in neuronal control of esophageal muscle rather than to be an unspecific "hangover" from the smooth muscle past of this organ.

Tensin Can Induce JNK and p38 Activation

Cells organize diverse types of specialized adhesion sites upon attachment to extracellular matrix (ECM) components. One of the physiological roles of such cell–ECM interactions is to initiate and regulate adhesion-mediated signal transduction responses. The association of cells with fibronectin fibrils has been shown to regulate the JNK and p38 signaling pathways. We tested whether tensin, a cytoskeletal component localized to both focal contacts and fibronectin-associated fibrillar adhesions, can induce these signaling pathways. We found that tensin overexpression resulted in activation of both the c-Jun amino-terminal kinase (JNK) and p38 pathways. Tensin-mediated JNK activation was independent of the activities of the small GTP binding proteins Rac and Cdc42, but did depend on SEK, a kinase involved in the JNK pathway. We suggest that tensin may directly activate the JNK and p38 pathways, acting downstream or independent of the activities of the small GTP binding proteins Rac and Cdc42.

The junction-associated protein AF-6 interacts and clusters with specific Eph receptor tyrosine kinases at specialized sites of cell-cell contact in the brain.

The AF-6/afadin protein, which contains a single PDZ domain, forms a peripheral component of cell membranes at specialized sites of cell-cell junctions. To identify potential receptor-binding targets of AF-6 we screened the PDZ domain of AF-6 against a range of COOH-terminal peptides selected from receptors having potential PDZ domain-binding termini. The PDZ domain of AF-6 interacts with a subset of members of the Eph subfamily of RTKs via its COOH terminus both in vitro and in vivo. Cotransfection of a green fluorescent protein-tagged AF-6 fusion protein with full-length Eph receptors into heterologous cells induces a clustering of the Eph receptors and AF-6 at sites of cell-cell contact. Immunohistochemical analysis in the adult rat brain reveals coclustering of AF-6 with Eph receptors at postsynaptic membrane sites of excitatory synapses in the hippocampus. Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor. The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates. AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.