Molecular Characterization of Caveolin-induced Membrane Curvature

The generation of caveolae involves insertion of the cholesterol-binding integral membrane protein caveolin-1 (Cav1) into the membrane, however, the precise molecular mechanisms are as yet unknown. We have speculated that insertion of the caveolin scaffolding domain (CSD), a conserved amphipathic region implicated in interactions with signaling proteins, is crucial for caveola formation. We now define the core membrane-juxtaposed region of Cav1 and show that the oligomerization domain and CSD are protected by tight association with the membrane in both mature mammalian caveolae and a model prokaryotic system for caveola biogenesis. Cryoelectron tomography reveals the core membrane-juxtaposed domain to be sufficient to maintain oligomerization as defined by polyhedral distortion of the caveolar membrane. Through mutagenesis we demonstrate the importance of the membrane association of the oligomerization domain/CSD for defined caveola biogenesis and furthermore, highlight the functional significance of the intramembrane domain and the CSD for defined caveolin-induced membrane deformation. Finally, we define the core structural domain of Cav1, constituting only 66 amino acids and of great potential to nanoengineering applications, which is required for caveolin-induced vesicle formation in a bacterial system. These results have significant implications for understanding the role of Cav1 in caveola formation and in regulating cellular signaling events.     

PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function

Caveolae are abundant cell-surface organelles involved in lipid regulation and endocytosis. We used comparative proteomics to identify PTRF (also called Cav-p60, Cavin) as a putative caveolar coat protein. PTRF-Cavin selectively associates with mature caveolae at the plasma membrane but not Golgi-localized caveolin. In prostate cancer PC3 cells, and during development of zebrafish notochord, lack of PTRF-Cavin expression correlates with lack of caveolae, and caveolin resides on flat plasma membrane. Expression of PTRF-Cavin in PC3 cells is sufficient to cause formation of caveolae. Knockdown of PTRF-Cavin reduces caveolae density, both in mammalian cells and in the zebrafish. Caveolin remains on the plasma membrane in PTRF-Cavin knockdown cells but exhibits increased lateral mobility and accelerated lysosomal degradation. We conclude that PTRF-Cavin is required for caveola formation and sequestration of mobile caveolin into immobile caveolae.     

Microcirculation in the upper trapezius muscle during sustained shoulder load in healthy women an endurance study using percutaneous laser-Doppler flowmetry and surface electromyography

Microcirculation in the upper portion of the trapezius muscle was measured percutaneously in a group of 16 healthy women of different ages by continuous laser-Doppler flowmetry (LDF) in relation to electromyography (EMG) during an endurance test. During the measurements the subject kept her arms straight and elevated at 45° in the scapular plane and held a 1-kg load in each hand as long as possible. This was followed by rest with the arms hanging and carrying no load. The 10-min recording period comprised 1-min initial rest followed by the endurance test and then recovery. Signal processing was done by computer on line. The LDF and root-mean-square (rms) EMG signals were normalized. Spectrum analyses of EMG mean power frequency (MPF) were performed. The amount of load produced was on average 2,267 (SD 939) N · m · s, i.e. shoulder torque × time expressed as Newton meter seconds, and the endurance time was 4.3 (SD 1.20) min. The rms-EMG as well as the LDF increased significantly during endurance, both when related to endurance time and to amount of load. The MPF showed no significant changes. The mean total increase in muscle blood flow was 175% of that recorded in the initial rest period. The average increase per each 10 s of contraction was 2.9%. Maximum was reached during the 1st min of recovery followed by a fall to the base level that was reached within 77 s on average. The amount of load produced and the blood flow increase was smaller than that found in a separate study of men, indicating a lower functional capacity. This may be of importance for the development of neck-shoulder disability in women.     

Purification and partial characterization of canine S100A12

Canine S100A12 (cS100A12) is a calcium-binding protein of the S100 superfamily of EF-hand proteins, and its expression is restricted to neutrophils and monocytes. Interaction of S100A12 with the receptor for advanced glycation end products (RAGE) has been suggested to play a central role in inflammation. Moreover, S100A12 has been shown to represent a sensitive and specific marker for gastrointestinal inflammation in humans. Only human, porcine, bovine, and rabbit S100A12 have been purified to date, and an immunoassay for the quantification of S100A12 is available only for humans. Therefore, the aim of this study was to develop a protocol for the purification of S100A12 and to partially characterize this protein in the dog (Canis lupus familiaris) as a prelude to the development of an immunologic method for its detection and quantification in canine serum and fecal specimens. Leukocytes were isolated from canine whole blood by dextran sedimentation, and canine S100A12 was extracted from the cytosol fraction of these cells. Further purification of cS100A12 comprised of ammonium sulfate precipitation, hydrophobic interaction chromatography, and strong cation- and anion-exchange column chromatography. Canine S100A12 was successfully purified from canine whole blood. The relative molecular mass of the protein was estimated at 10,379.5 and isoelectric focusing revealed an isoelectric point of 6.0. The approximate specific absorbance of cS100A12 at 280 nm was determined to be 1.78 for a 1 mg/ml solution. The N-terminal AA sequence of the first 15 residues of cS100A12 was Thr-Lys-Leu-Glu-Asp-His-X-Glu-Gly-Ile-Val-Asp-Val-Phe-His, and revealed 100% identity with the predicted protein sequence available through the canine genome project. Sequence homology for the 14 N-terminal residues identified for cS100A12 with those of feline, bovine, porcine, and human S100A12 was 78.6%. We conclude that canine S100A12 can be successfully purified from canine whole blood using the described methods.     

Strong intraspecific variation in genetic diversity and genetic differentiation in Daphnia magna: the effects of population turnover and population size

Theory predicts that genetic diversity and genetic differentiation may strongly vary among populations of the same species depending on population turnover and local population sizes. Yet, despite the importance of these predictions for evolutionary and conservation issues, empirical studies comparing high‐turnover and low‐turnover populations of the same species are scarce. In this study, we used Daphnia magna, a freshwater crustacean, as a model organism for such a comparison. In the southern/central part of its range, D. magna inhabits medium‐sized, stable ponds, whereas in the north, it occurs in small rock pools with strong population turnover. We found that these northern populations have a significantly lower genetic diversity and higher genetic differentiation compared to the southern/central populations. Total genetic diversity across populations was only about half and average within‐population diversity only about a third of that in southern/central populations. Moreover, an average southern population contains more genetic diversity than the whole metapopulation system in the north. We based our analyses both on silent sites and microsatellites. The similarity of our results despite the contrasting mutation rates of these markers suggests that the differences are caused by contemporary rather than by historical processes. Our findings show that variation in population turnover and population size may have a major impact on the genetic diversity and differentiation of populations, and hence may lead to differences in evolutionary processes like local adaptation, hybrid vigour and breeding system evolution in different parts of a species range.     

Constitutive formation of caveolae in a bacterium

Caveolin plays an essential role in the formation of characteristic surface pits, caveolae, which cover the surface of many animal cells. The fundamental principles of caveola formation are only slowly emerging. Here we show that caveolin expression in a prokaryotic host lacking any intracellular membrane system drives the formation of cytoplasmic vesicles containing polymeric caveolin. Vesicle formation is induced by expression of wild-type caveolins, but not caveolin mutants defective in caveola formation in mammalian systems. In addition, cryoelectron tomography shows that the induced membrane domains are equivalent in size and caveolin density to native caveolae and reveals a possible polyhedral arrangement of caveolin oligomers. The caveolin-induced vesicles or heterologous caveolae (h-caveolae) form by budding in from the cytoplasmic membrane, generating a membrane domain with distinct lipid composition. Periplasmic solutes are encapsulated in the budding h-caveola, and purified h-caveolae can be tailored to be targeted to specific cells of interest.     

Systematic discovery of pseudomonad genetic factors involved in sensitivity to tailocins

Tailocins are bactericidal protein complexes produced by a wide variety of bacteria that kill closely related strains and may play a role in microbial community structure. Thanks to their high specificity, tailocins have been proposed as precision antibacterial agents for therapeutic applications. Compared to tailed phages, with whom they share an evolutionary and morphological relationship, bacterially produced tailocins kill their host upon production but producing strains display resistance to self-intoxication. Though lipopolysaccharide (LPS) has been shown to act as a receptor for tailocins, the breadth of factors involved in tailocin sensitivity, and the mechanisms behind resistance to self-intoxication, remain unclear. Here, we employed genome-wide screens in four non-model pseudomonads to identify mutants with altered fitness in the presence of tailocins produced by closely related pseudomonads. Our mutant screens identified O-antigen composition and display as most important in defining sensitivity to our tailocins. In addition, the screens suggest LPS thinning as a mechanism by which resistant strains can become more sensitive to tailocins. We validate many of these novel findings, and extend these observations of tailocin sensitivity to 130 genome-sequenced pseudomonads. This work offers insights into tailocin–bacteria interactions, informing the potential use of tailocins in microbiome manipulation and antibacterial therapy.Subject terms: Functional genomics, Microbial ecology, Bacterial genetics, Soil microbiology