Cyclase-associated protein is essential for the functioning of the endo-lysosomal system and provides a link to the actin cytoskeleton

Data from mutant analysis in yeast and Dictyostelium indicate a role for the cyclase-associated protein (CAP) in endocytosis and vesicle transport. We have used genetic and biochemical approaches to identify novel interacting partners of Dictyostelium CAP to help explain its molecular interactions in these processes. Cyclase-associated protein associates and interacts with subunits of the highly conserved vacuolar H(+)-ATPase (V-ATPase) and co-localizes to some extent with the V-ATPase. Furthermore, CAP is essential for maintaining the structural organization, integrity and functioning of the endo-lysosomal system, as distribution and morphology of V-ATPase- and Nramp1-decorated membranes were disturbed in a CAP mutant (CAP bsr) accompanied by an increased endosomal pH. Moreover, concanamycin A (CMA), a specific inhibitor of the V-ATPase, had a more severe effect on CAP bsr than on wild-type cells, and the mutant did not show adaptation to the drug. Also, the distribution of green fluorescent protein-CAP was affected upon CMA treatment in the wildtype and recovered after adaptation. Distribution of the V-ATPase in CAP bsr was drastically altered upon hypo-osmotic shock, and growth was slower and reached lower saturation densities in the mutant under hyper-osmotic conditions. Taken together, our data unravel a link of CAP with the actin cytoskeleton and endocytosis and suggest that CAP is an essential component of the endo-lysosomal system in Dictyostelium.     

Genetic population structure as indirect measure of dispersal ability in a Lake Tanganyika cichlid

Diversification and speciation processes are influenced by intrinsic (ecological specialization, dispersal) and extrinsic (habitat structure and instability) factors, but the effect of ecological characteristics on dispersal is difficult to assess. This study uses mitochondrial control region sequences to investigate the population structure and demographic history of the endemic Lake Tanganyika cichlid Neolamprologus caudopunctatus with a preference for the rock-sand interface along two stretches of continuous, rocky shoreline, and across a sandy bay representing a potential dispersal barrier. Populations along uninterrupted habitat were not differentiated; whereas, the sandy bay separated two reciprocally monophyletic clades. The split between the two clades between 170,000 and 260,000 years BP coincides with a period of rising water level following a major lowstand, and indicates that clades remained isolated throughout subsequent lake level fluctuations. Low long-term effective population sizes were inferred from modest genetic diversity estimates, and may be due to recent population expansions starting from small population sizes 45,000–60,000 years BP. Comparisons with available data from specialized rock-dwelling species of the␣same area suggest that habitat structure and lake level fluctuations determine phylogeographic patterns on large scales, while fine-scale population structure and demography are modulated by species-specific ecologies.     

Biochemical engineering of the N-acyl side chain of sialic acid leads to increased calcium influx from intracellular compartments and promotes differentiation of HL60 cells

Sialylation of glycoconjugates is essential for mammalian cells. Sialic acid is synthesized in the cytosol from N-acetylmannosamine by several consecutive steps. Using N-propanoylmannosamine, a novel precursor of sialic acid, we are able to incorporate unnatural sialic acids with a prolonged N-acyl side chain (e.g., N-propanoylneuraminic acid) into glycoconjugates taking advance of the cellular sialylation machinery. Here, we report that unnatural sialylation of HL60-cells leads to an increased release of intracellular calcium after application of thapsigargin, an inhibitor of SERCA Ca2+-ATPases. Furthermore, this increased intracellular calcium concentration leads to an increased adhesion to fibronectin. Finally, we observed an increase of the lectin galectin-3, a marker of monocytic differentiation of HL60-cells.     

Extracellular, circulating proteasomes and ubiquitin - incidence and relevance

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation and is also deeply involved in the regulation of most basic cellular processes. Its proteolytic core, the 20S proteasome, has found to be attached also to the cell plasma membrane and certain observations are interpreted as to suggest that they may be released into the extracellular medium, e.g. in the alveolar lining fluid, epididymal fluid and possibly during the acrosome reaction. Proteasomes have also been detected in normal human blood plasma and designated circulating proteasomes; these have a comparatively low specific activity, a distinct pattern of subtypes and their exact origin is still enigmatic. In patients suffering from autoimmune diseases, malignant myeloproliferative syndromes, multiple myeloma, acute and chronic lymphatic leukaemia, solid tumour, sepsis or trauma, respectively, the concentration of circulating proteasomes has been found to be elevated, to correlate with the disease state and has even prognostic significance. Similarly, ubiquitin has been discovered as a normal component of human blood and seminal plasma and in ovarian follicular fluid. Increased concentrations were measured in diverse pathological situations, not only in blood plasma but also in cerebrospinal fluid, where it may have neuroprotective effects. As defective spermatozoa are covered with ubiquitin in the epididymal fluid, extracellular ubiquitination is proposed to be a mechanism for quality control in spermatogenesis. Growing evidence exists also for a participation of extracellular proteasomes and ubiquitin in the fertilization process.     

Single-cell enabled comparative genomics of a deep ocean SAR11 bathytype

Bacterioplankton of the SAR11 clade are the most abundant microorganisms in marine systems, usually representing 25% or more of the total bacterial cells in seawater worldwide. SAR11 is divided into subclades with distinct spatiotemporal distributions (ecotypes), some of which appear to be specific to deep water. Here we examine the genomic basis for deep ocean distribution of one SAR11 bathytype (depth-specific ecotype), subclade Ic. Four single-cell Ic genomes, with estimated completeness of 55%–86%, were isolated from 770 m at station ALOHA and compared with eight SAR11 surface genomes and metagenomic datasets. Subclade Ic genomes dominated metagenomic fragment recruitment below the euphotic zone. They had similar COG distributions, high local synteny and shared a large number (69%) of orthologous clusters with SAR11 surface genomes, yet were distinct at the 16S rRNA gene and amino-acid level, and formed a separate, monophyletic group in phylogenetic trees. Subclade Ic genomes were enriched in genes associated with membrane/cell wall/envelope biosynthesis and showed evidence of unique phage defenses. The majority of subclade Ic-specfic genes were hypothetical, and some were highly abundant in deep ocean metagenomic data, potentially masking mechanisms for niche differentiation. However, the evidence suggests these organisms have a similar metabolism to their surface counterparts, and that subclade Ic adaptations to the deep ocean do not involve large variations in gene content, but rather more subtle differences previously observed deep ocean genomic data, like preferential amino-acid substitutions, larger coding regions among SAR11 clade orthologs, larger intergenic regions and larger estimated average genome size.     

Random amplified polymorphic DNA markers reveal genetic variation in the symbiotic fungus of leaf-cutting ants

RAPD markers were used to examine the degree of genetic variation within the putatively asexual basidiomycete fungus (Lepiotaceae: provisionally named Leucoagaricus gongylophorus) associated with the leaf-cutting ant species Atta cephalotes. We analyzed fungal isolates from ant nests in two geographically distant sites, two isolates from Panama and five isolates from Trinidad. Ten decamer primers were used to amplify total DNA from these seven fungal isolates, and RAPD banding patterns were compared. Genetic similarity among isolates was determined by pair-wise comparisons of the shared number of DNA bands on an agarose gel. There was considerable genetic variation among isolates of the symbiotic fungus even within sites. Pairs of fungal isolates from the two different sites shared an average of only 36% of the bands in their RAPD profiles, while pairs from the within sites shared an average of 72% of the bands. RAPD markers may be useful for further investigation of the genetic structure of the fungal symbiont within species of leaf-cutting ants.     

An insertion element of the extremely thermophilic archaeon Sulfolobus solfataricus transposes into the endogenous β-galactosidase gene

Three phenotypically stable mutants of the extremely thermophilic archaeon Sulfolobus solfataricus have been isolated by screening for β-galactosidase negative colonies on plates with X-Gal (5-bromo-4-chloro-3-indolyl-(3-d-galactopyranoside). From one of these mutants an insertion element, designated ISC1217, was isolated and characterized. Sequence analysis of ISC1217 and of the regions adjacent to the insertion site in the β-galactosidase gene revealed features typical of a transposable element: ISC1217 contained terminal inverted repeats and was flanked by a direct repeat of 6 bp. The 1147 by sequence contained an open reading frame encoding a putative protein of 354 amino acid residues and, overlapping this, two smaller open reading frames on the opposite strand. There were approximately 8 copies of the insertion element in the S. solfataricus genome. ISC1217 did not cross-hybridize with DNA of other Sulfolobus species. All three independently isolated β-galactosidase mutants of S. solfataricus arose by transposition of ISC1217 or a related element.