CD9 clustering and formation of microvilli zippers between contacting cells regulates virus-induced cell fusion

Members of the tetraspanin family including CD9 contribute to the structural organization and plasticity of the plasma membrane. K41, a CD9-specific monoclonal antibody, inhibits the release of HIV-1 and canine distemper virus (CDV)- but not measles virus (MV)-induced cell–cell fusion. We now report that K41, which recognizes a conformational epitope on the large extracellular loop of CD9, induces rapid relocation and clustering of CD9 in net-like structures at cell–cell contact areas. High-resolution analyses revealed that CD9 clustering is accompanied by the formation of microvilli that protrude from either side of adjacent cell surfaces, thus forming structures like microvilli zippers. While the cellular CD9-associated proteins β₁-integrin and EWI-F were co-clustered with CD9 at cell–cell interfaces, viral proteins in infected cells were differentially affected. MV envelope proteins were detected within CD9 clusters, whereas CDV proteins were excluded from CD9 clusters. Thus, the tetraspanin CD9 can regulate cell–cell fusion by controlling the access of the fusion machinery to cell contact areas.     

Invasive species alter ontogenetic shifts in the trophic ecology of Lake Sturgeon (<Emphasis Type="Italic">Acipenser fulvescens</Emphasis>) in the Niagara River and Lake Ontario

Species invasions can alter food web structure and change ecosystem-level functioning, but it is often unclear how these invasions may affect the life history of native species. The Lake Sturgeon (Acipenser fulvescens), a large long-lived native fish species in the Great Lakes, has increased in abundance in the lower Niagara River and nearby Lake Ontario during a period of invasive species-induced ecosystem change precipitated most recently by Dreissenid mussels (Driessena polymorpha and Driessena bugensis) and Round Goby (Neogobius melanostomus). Material taken from cross-sections of archived pectoral spines from Niagara River Lake Sturgeon captured in 1998–2000 and 2010–2012 were analyzed for stable isotopes across discrete growth zones to provide an ontogenetic assessment of diet, and diet analysis of Lake Sturgeon captured in 2014 was conducted to assess the contribution of invasive prey. Round Goby was the most important Lake Sturgeon prey item (86% by weight) in 2014, which corroborated results of δ¹⁵N and δ¹³C. Lake Sturgeon captured after the invasion of Round Goby exhibited ontogenetic changes in δ¹⁵N that differed from pre-Round Goby patterns, though this effect was weaker for δ¹³C. Values of δ¹⁵N from spine growth zones indicated non-linear increases in trophic position with age and increased rate of δ¹⁵N enrichment after the Round Goby invasion. We conclude that Round Goby establishment in western Lake Ontario changed the feeding ecology of Lake Sturgeon, which may have a positive effect on population growth for this native species.     

Habitat change in braided flood plains (Tagliamento, NE-Italy)

1. Relative changes and age distribution of habitats were investigated in the active channel of a bar‐braided and an island‐braided reach of the Tagliamento River (NE‐Italy). Between September 1999 and January 2002, six habitat types were delineated with a differential Global Positioning System on five dates following floods of different magnitude. Overlay maps were employed to calculate age and relative change of habitats. We established exponential decay rates (k‐values) for islands and major aquatic habitats. 2. Relative changes of all aquatic habitats combined were up to 82% between survey dates in the bar‐braided flood plain, with a cumulative rate of 85% over the 2.5‐year period. Relative habitat changes in the island‐braided flood plain were lower with a cumulative change of almost 60% during the study period. In the bar‐braided flood plain significant exponential decay relationships were established for channels, alluvial channels, backwaters, and ponds. 3. Half‐lives were particularly short for backwaters and ponds. In the island‐braided reach, significant relationships existed for channels and alluvial channels. The half‐lives of channels and alluvial channels increased with the presence of vegetated islands. Relative habitat composition within the active corridor remained almost constant, supporting the applicability of the shifting mosaic steady state model to braided floodplain ecosystems. 4. Our results indicate that under natural conditions aquatic floodplain habitats can be highly dynamic over short time‐scales. Even small water level fluctuations (‘flow pulses’) can lead to major habitat changes with important consequences for the fauna and flora.     

Microbial Thiocyanate Utilization under Highly Alkaline Conditions

Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS⁻) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division Proteobacteria (the Halomonas group for the heterotrophs and the genus Thioalkalivibrio for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus Thioalkalivibrio. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genus Thioalkalivibrio according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase activity which converted cyanate (CNO⁻) to ammonia and CO₂. On the other hand, cyanase activity either was absent or was present at very low levels in the autotrophic strains grown on thiocyanate as the sole energy and N source. As a result, large amounts of cyanate were found to accumulate in the media during utilization of thiocyanate at pH 10 in batch and thiocyanate-limited continuous cultures. This is a first direct proof of a “cyanate pathway” in pure cultures of thiocyanate-degrading bacteria. Since it is relatively stable under alkaline conditions, cyanate is likely to play a role as an N buffer that keeps the alkaliphilic bacteria safe from inhibition by free ammonia, which otherwise would reach toxic levels during dissimilatory degradation of thiocyanate.     

A microsatellite linkage map of the European sea bass Dicentrarchus labrax L

A genetic linkage map of the European sea bass (Dicentrarchus labrax) was constructed from 174 microsatellite markers, including 145 new markers reported in this study. The mapping panel was derived from farmed sea bass from the North Adriatic Sea and consisted of a single family including both parents and 50 full-sib progeny (biparental diploids). A total of 162 microsatellites were mapped in 25 linkage groups. Eleven loci represent type I (coding) markers; 2 loci are located within the peptide Y (linkage group 1) and cytochrome P450 aromatase (linkage group 6) genes. The sex-averaged map spans 814.5 cM of the sea bass genome. The female map covers 905.9 cM, whereas the male map covers only 567.4 cM. The constructed map represents the first linkage map of European sea bass, one of the most important aquaculture species in Europe.     

Action Possibility Judgments of People with Varying Motor Abilities Due to Spinal Cord Injury

Predictions about one''s own action capabilities as well as the action capabilities of others are thought to be based on a simulation process involving linked perceptual and motor networks. Given the central role of motor experience in the formation of these networks, one''s present motor capabilities are thought to be the basis of their perceptual judgments about actions. However, it remains unknown whether the ability to form these action possibility judgments is affected by performance related changes in the motor system. To determine if judgments of action capabilities are affected by long-term changes in one''s own motor capabilities, participants with different degrees of upper-limb function due to their level (cervical vs. below cervical) of spinal cord injury (SCI) were tested on a perceptual-motor judgment task. Participants observed apparent motion videos of reciprocal aiming movements with varying levels of difficulty. For each movement, participants determined the shortest movement time (MT) at which they themselves and a young adult could perform the task while maintaining accuracy. Participants also performed the task. Analyses of MTs revealed that perceptual judgments for participant''s own movement capabilities were consistent with their actual performance- people with cervical SCI had longer judged and actual MTs than people with below cervical SCI. However, there were no between-group differences in judged MTs for the young adult. Although it is unclear how the judgments were adjusted (altered simulation vs. threshold modification), the data reveal that people with different motor capabilities due to SCI are not completely biased by their present capabilities and can effectively adjust their judgments to estimate the actions of others.     

The serine protease marapsin is expressed in stratified squamous epithelia and is up-regulated in the hyperproliferative epidermis of psoriasis and regenerating wounds

The trypsin-like serine protease marapsin is a member of the large protease gene cluster at human chromosome 16p13.3, which also contains the structurally related proteases testisin, tryptase epsilon, tryptase gamma, and EOS. To gain insight into the biological functions of marapsin, we undertook a detailed gene expression analysis. It showed that marapsin expression was restricted to tissues containing stratified squamous epithelia and was absent or only weakly expressed in all other tissues, including the pancreas. Marapsin was constitutively expressed in nonkeratinizing stratified squamous epithelia of human esophagus, tonsil, cervix, larynx, and cornea. In the keratinizing stratified squamous epidermis of skin, however, its expression was induced only during epidermal hyperproliferation, such as in psoriasis and in murine wound healing. In fact, marapsin was the second most strongly up-regulated protease in psoriatic lesions, where expression was localized to the upper region of the hyperplastic epidermis. Similarly, in the hyperproliferative epithelium of regenerating murine skin wounds, marapsin localized to the suprabasal layers, where keratinocytes undergo squamous differentiation. The transient up-regulation of marapsin, which closely correlated with re-epithelialization, was virtually absent in a genetic mouse model of delayed wound closure. These results suggested a function during the process of re-epithelialization. Furthermore, in reconstituted human epidermis, a model system of epidermal differentiation, members of the IL-20 subfamily of cytokines, such as IL-22, induced marapsin expression. Consistent with a physiologic role in marapsin regulation, IL-22 was also strongly expressed in re-epithelializing skin wounds. Marapsin's restricted expression, localization, and cytokine-inducible expression suggest a role in the terminal differentiation of keratinocytes in hyperproliferating squamous epithelia.     

Formation of Syncytia Is Repressed by Tetraspanins in Human Immunodeficiency Virus Type 1-Producing Cells

In vitro propagation studies have established that human immunodeficiency virus type 1 (HIV-1) is most efficiently transmitted at the virological synapse that forms between producer and target cells. Despite the presence of the viral envelope glycoprotein (Env) and CD4 and chemokine receptors at the respective surfaces, producer and target cells usually do not fuse with each other but disengage after the viral particles have been delivered, consistent with the idea that syncytia, at least in vitro, are not required for HIV-1 spread. Here, we tested whether tetraspanins, which are well known regulators of cellular membrane fusion processes that are enriched at HIV-1 exit sites, regulate syncytium formation. We found that overexpression of tetraspanins in producer cells leads to reduced syncytium formation, while downregulation has the opposite effect. Further, we document that repression of Env-induced cell-cell fusion by tetraspanins depends on the presence of viral Gag, and we demonstrate that fusion repression requires the recruitment of Env by Gag to tetraspanin-enriched microdomains (TEMs). However, sensitivity to fusion repression by tetraspanins varied for different viral strains, despite comparable recruitment of their Envs to TEMs. Overall, these data establish tetraspanins as negative regulators of HIV-1-induced cell-cell fusion, and they start delineating the requirements for this regulation.The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) is incorporated into released virus particles and enables the virus to attach to and fuse with target cells in order to initiate the infectious cycle. Before Env mediates the fusion of viral and cellular membranes, i.e., while it is still incorporated in the plasma membrane of the infected cell, it drives the adhesion between virus producer cell and target cells, which gives rise to the formation of the so-called virological synapse (VS) (21, 24, 35, 36). The VS shares certain characteristics with the immunological synapse, including an accumulation of specific cellular membrane proteins and lipids (see, e.g., reference 5), and it provides efficient and secure transfer of virus particles from infected to uninfected cells (8). Importantly, the two adhering cells, like the pre- and postsynaptic cells that form an immunological synapse, typically do not fuse during such cell-to-cell transfer events. At first glance this seems surprising, as HIV-1 Env, unlike many other viral envelope proteins, can induce membrane fusion at physiological pH. Also, adhesion of producer and target cell, which can be initiated when the uropod of the infected cell contacts the uninfected cell (8), followed by reorganization of the cytoskeleton (25) and formation of full-fledged synapses, can extend over minutes (see, e.g., reference 20). This process should allow enough time to trigger cell-cell fusion. However, it is now well established that newly synthesized Env is efficiently internalized upon its arrival at the host cell plasma membrane, unless it is recruited into budding structures by viral Gag (see, e.g., reference 11; also discussed in references 3 and 6). Further, and likely also contributing to the prevention of producer-target cell fusion, immature Gag at the host cell plasma membrane represses Env-driven fusion, and this repression is lost only once Gag is processed in released virions (9, 22, 23, 31, 50). Finally, because syncytia are clearly not required for the transmission of virus from cell to cell in vitro and are possibly detrimental to virus spread in vivo, we hypothesize that HIV-1 cooperates with cellular membrane proteins to prevent cell-cell fusion.Members of a group of cellular proteins known as tetraspanins play an important role as regulators of cellular fusion processes, including myotube formation and fertilization (28, 30, 44; reviewed in, e.g., reference 17). As membrane organizers, these proteins homo- and heteromultimerize and associate with other cellular proteins to form variably sized but discrete microdomains, the so-called tetraspanin-enriched microdomains (TEMs) (29) (also called TERMs [1] or TEAs [12]). Knowledge of the molecular mechanisms through which tetraspanins regulate the fusion of cellular membranes is still lacking, though the available evidence strongly suggests (i) that these proteins are not themselves fusogens but rather that they coordinate the fusion activity of other cellular proteins and (ii) that they can act both as positive and negative regulators of cellular fusion processes. For instance, several in vivo studies unequivocally showed that CD9 expression in oocytes is essential for sperm-egg fusion (27, 28, 30), but CD9 and CD81 ablation in monocytes enhances the formation of multinucleated phagocytes that are involved in immune defense against certain microbes (45). Interestingly, the same two tetraspanins are also known to regulate virus-induced fusion processes. CD9 is involved in regulating cell-cell fusion driven by canine distemper virus, as the anti-CD9 antibody K41 inhibits syncytium formation by this virus (42), and CD81 is a necessary cofactor for infection of cells by hepatitis C virus (see, e.g., references 2 and 52). Finally, tetraspanins on uninfected (target) cells inhibit HIV-1-induced cell-cell fusion (14). This fusion regulation is likely due to interactions of CD9 and CD81 with CD4 and coreceptors at the surface of target cells, though the tetraspanin CD63 has also been implicated in the trafficking of CXCR4 to the plasma membrane (51).Because tetraspanins in HIV-1-producing cells are enriched at budding sites (4, 10, 13, 15, 33, 46, 49) and at the VS (26), we hypothesized that they regulate Env-driven fusion at the VS. Here, we document that tetraspanins in HIV-1-producing cells can indeed restrict syncytium formation. We also define some of the requirements for this fusion inhibition, thus laying the necessary groundwork for future mechanistic analyses. In addition, the characterization of cell-cell fusion regulation parameters in this study will allow the fusion-inhibitory activities to be distinguished from other regulatory functions exerted by tetraspanins, such as the modulation of virion infectivity and the regulation of cell-to-cell transmission of HIV-1.