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Hypersaline environments with salt concentrations up to NaCl saturation are inhabited by a great diversity of microorganisms belonging to the three domains of life. They all must cope with the low water activity of their environment, but different strategies exist to provide osmotic balance of the cells' cytoplasm with the salinity of the medium. One option used by many halophilic Archaea and a few representatives of the Bacteria is to accumulate salts, mainly KCl and to adapt the entire intracellular machinery to function in the presence of molar concentrations of salts. A more widespread option is the synthesis or accumulation of organic osmotic, so-called compatible solutes. Here, we review the mechanisms of osmotic adaptation in a number of model organisms, including the KCl accumulating Halobacterium salinarum (Archaea) and Salinibacter ruber (Bacteria), Halomonas elongata as a representative of the Bacteria that synthesize organic osmotic solutes, eukaryotic microorganisms including the unicellular green alga Dunaliella salina and the black yeasts Hortaea werneckii and the basidiomycetous Wallemia ichthyophaga, which use glycerol and other compatible solutes. The strategies used by these model organisms and by additional halophilic microorganisms presented are then compared to obtain an integrative picture of the adaptations to life at high salt concentrations in the microbial world.

PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures, using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering detector analysis. We show for the first time that numerous PKH26 nanoparticles (nine out of ten PKH26-positive particles) are formed during ultracentrifugation-based exosome staining, which are almost indistinguishable from PKH26-labeled exosomes in terms of size, surface area, and fluorescence intensity. When PKH26-labeled exosomes were purified through sucrose, PKH26 nanoparticles were differentiated from PKH26-labeled exosomes based on their reduced size. However, PKH26 nanoparticles were only physically removed from PKH26-labeled exosomes when separated on a sucrose gradient, and at the expense of low PKH26-labeled exosome recovery. Overall, low PKH26-positive particle recovery is characteristic of filtration-based exosome staining. Importantly, PKH26 nanoparticles are internalized by primary astrocytes into similar subcellular compartments as PKH26-labeled exosomes. Altogether, PKH26 nanoparticles can result in false-positive signals for stained EVs that can compromise the interpretation of EV internalization. Thus, for use in EV uptake and functional studies, sucrose-gradient-based isolation should be the method of choice to obtain PKH26-labeled exosomes devoid of PKH26 nanoparticles.

HIV buds from lipid rafts and requires cholesterol for its egress from and entry into cells. Viral accessory protein Nef plays a major role in this process. In this study, it not only increased the biosynthesis of lipid rafts and viral particles with newly synthesized cholesterol, but also enriched them. Furthermore, via the consensus cholesterol recognition motif at its C terminus, Nef bound cholesterol. When this sequence was mutated, Nef became unable to transport newly synthesized cholesterol into lipid rafts and viral particles. Interestingly, although its levels in lipid rafts were not affected, this mutant Nef protein was poorly incorporated into viral particles, and viral infectivity decreased dramatically. Thus, Nef also transports newly synthesized cholesterol to the site of viral budding. As such, it provides essential building blocks for the formation of viruses that replicate optimally in the host.T he negative effector (Nef) protein from human and simian immunodeficiency viruses is a membrane-associated myristoylated protein that measures 27-35 kDa (1-3). It is critical for high levels of viremia and the progression to AIDS in infected humans (4) and monkeys (5). This phenotype has been correlated with increased viral infectivity in vitro, which provides a convenient assay to study its effects in cultured cells (6, 7). This infectivity enhancement can be dependent on or independent of CD4 that serves as the receptor for viral entry. In the former case, Nef decreases the expression of CD4 on the cell surface, thereby increasing the incorporation of viral envelope (Env) proteins into virions (8). In the latter case, Nef still increases viral infectivity significantly (9, 10). This enhancement cannot be complemented by the expression of Nef in target cells. Although no differences were identified in major structural components and morphology between wild-type and mutant virions that lack Nef, ⌬Nef viruses displayed less efficient reverse transcription in target cells. Because Nef is expressed abundantly at the earliest stages of the viral replicative cycle (11), Nef could affect viral morphogenesis and budding to increase the fitness of the virus and facilitate its entry into recipient cells.Lipid rafts, also known as detergent-resistant membranes (DRMs), are microdomains in the plasma membrane that are enriched in sphingolipids, cholesterol, and a subset of cellular proteins (12, 13). Two major pathways contribute to cholesterol homeostasis in mammalian cells (14). Most exogenous cholesterol, which originates from low-density lipoproteins, is internalized via coated pits and distributed to intracellular pools. In addition, cells can synthesize cholesterol in their endoplasmic reticulum when the uptake of exogenous cholesterol is blocked. The newly synthesized cholesterol is then transported into the Golgi apparatus and distributed to various intracellular pools. Because cellular cholesterol is compartmentalized, some sites (including DRMs) are enriched in this newly synthesized lipid (15,16).Previous...

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By activating NAK via small GTPases and their downstream effectors, Nef interacts with regulatory pathways required for cell growth, cytoskeletal rearrangement and endocytosis. Thus, NAK could participate in the budding of new virions, the modification of viral proteins and the increased endocytosis of surface molecules such as CD4. Moreover, blocking the activity of these GTPases could lead to new therapeutic interventions against AIDS.

Lipid rafts, also known as detergent-resistant membranes (DRM), are microdomains in the plasma membrane enriched in sphingolipids and cholesterol (reviewed in [1, 2]). Human immunodeficiency virus 1 (HIV) buds via lipid rafts [3, 4]. However, the targeting of viral structural components to DRM and its consequences for viral replication are not understood. Moreover, the negative factor Nef from HIV increases viral infectivity (reviewed in [5, 6]). With no apparent differences in structural components and morphology between wild-type and DeltaNef virons, the latter viruses display less efficient reverse transcription in target cells. As Nef is expressed abundantly early in the viral replicative cycle [7], we hypothesized that Nef could affect viral morphogenesis and budding to render viruses more infectious. In this report, we demonstrated first that Nef increases viral budding from lipid rafts. Second, in the presence of Nef, viral envelopes contain more ganglioside (GM1), which is a major component of lipid rafts. This finding correlated directly with the increased infectivity of HIV. Finally, the depletion of exogenous and endogenous cholesterol biochemically and genetically, which disrupted lipid rafts, decreased viral infectivity only in the presence of Nef. Importantly, HIV lacking the nef gene remained unaffected by these manipulations. We conclude that lipids in virions are essential for viral infectivity. Thus, HIV becomes more infectious when it buds from lipid rafts, and Nef plays a major role in this process.

Hortaea werneckii, ascomycetous yeast from the order Capnodiales, shows an exceptional adaptability to osmotically stressful conditions. To investigate this unusual phenotype we obtained a draft genomic sequence of a H. werneckii strain isolated from hypersaline water of solar saltern. Two of its most striking characteristics that may be associated with a halotolerant lifestyle are the large genetic redundancy and the expansion of genes encoding metal cation transporters. Although no sexual state of H. werneckii has yet been described, a mating locus with characteristics of heterothallic fungi was found. The total assembly size of the genome is 51.6 Mb, larger than most phylogenetically related fungi, coding for almost twice the usual number of predicted genes (23333). The genome appears to have experienced a relatively recent whole genome duplication, and contains two highly identical gene copies of almost every protein. This is consistent with some previous studies that reported increases in genomic DNA content triggered by exposure to salt stress. In hypersaline conditions transmembrane ion transport is of utmost importance. The analysis of predicted metal cation transporters showed that most types of transporters experienced several gene duplications at various points during their evolution. Consequently they are present in much higher numbers than expected. The resulting diversity of transporters presents interesting biotechnological opportunities for improvement of halotolerance of salt-sensitive species. The involvement of plasma P-type H+ ATPases in adaptation to different concentrations of salt was indicated by their salt dependent transcription. This was not the case with vacuolar H+ ATPases, which were transcribed constitutively. The availability of this genomic sequence is expected to promote the research of H. werneckii. Studying its extreme halotolerance will not only contribute to our understanding of life in hypersaline environments, but should also identify targets for improving the salt- and osmotolerance of economically important plants and microorganisms.