Clathrin-independent endocytosis: a unique platform for cell signaling and PM remodeling

There is increasing interest in endocytosis that occurs independently of clathrin coats and the fates of membrane proteins internalized by this mechanism. The appearance of clathrin-independent endocytic and membrane recycling pathways seems to vary with different cell types and cargo molecules. In this review we focus on studies that have been performed using HeLa and COS cells as model systems for understanding this membrane trafficking system. These endosomal membranes contain signaling molecules including H-Ras, Rac1, Arf6 and Rab proteins, and a lipid environment rich in cholesterol and PIP(2) providing a unique platform for cell signaling. Furthermore, activation of some of these signaling molecules (H-Ras, Rac and Arf6) can switch the constitutive form of clathrin-independent endocytosis into a stimulated one, associated with PM ruffling and macropinocytosis.     

Minimum data requirements for designing a set of marine protected areas, using commonly available abiotic and biotic datasets

Marine protected areas (MPAs) can be an effective tool for marine biodiversity conservation, yet decision-makers usually have limited and biased datasets with which to make decisions about where to locate MPAs. Using commonly available abiotic and biotic datasets, I asked how many datasets are necessary to achieve robust patterns of conservation importance. I applied a decision support tool for marine protected area design in two regions of British Columbia, Canada, and sequentially excluded the datasets with the most limited geographic distribution. I found that the reserve selection method was robust to some missing datasets. The removal of up to 15 of the most geographically limited datasets did not significantly change the geographic patterns of the importance of areas for conservation. Indeed, including abiotic datasets plus at least 12 biotic datasets resulted in a spatial pattern similar to including all available biotic datasets. It was best to combine abiotic and biotic datasets in order to ensure habitats and species were represented. Patterns of clustering differed according to whether I used one set alone or both combined. Biotic datasets served as better surrogates for abiotic datasets than vice versa, and both represented more biodiversity features than randomly selected reserves. These results should provide encouragement to decision-makers engaged in MPA planning with limited spatial data.     

Mediator-assisted simultaneous probing of cytosolic and mitochondrial redox activity in living cells

This work describes an electron transfer mediator-assisted amperometric flow injection method for assessing redox enzyme activity in different subcellular compartments of the phosphoglucose isomerase deletion mutant strain of Saccharomyces cerevisiae, EBY44. The method is demonstrated using the ferricyanide-menadione double mediator system to study the effect of dicoumarol, an inhibitor of cytosolic and mitochondrial oxidoreductases and an uncoupler of the electron transport chain. Evaluation of the role of NAD(P)H-producing pathways in mediating biological effects is facilitated by introducing either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pentose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric in vitro assays show no difference between the cofactors. Respiratory competent cells show cytosolic inhibition only when NADPH is produced, whereas production of NADH reveals uncoupling at low dicoumarol concentrations and inhibition of complexes III and IV at higher concentrations. Spectrophotometric assays only indicate the presence of cytosolic inhibition regardless of the reduced cofactor used. This article shows the applicability of the amperometric method and emphasizes the significance of determining biological effects of chemicals in living cells.     

The response of <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> CH34 to spaceflight in the international space station

The survival and behavior of Cupriavidus metallidurans strain CH34 were tested in space. In three spaceflight experiments, during three separate visits to the ‘International Space Station’ (ISS), strain CH34 was grown for 10–12 days at ambient temperature on mineral agar medium. Space- and earth-grown cells were compared post-flight by flow cytometry and using 2D-gel protein analysis. Pre-, in- and post-flight incubation conditions and experiment design had a significant impact on the survival and growth of CH34 in space. In the CH34 cells returning from spaceflight, 16 proteins were identified which were present in higher concentration in cells developed in spaceflight conditions. These proteins were involved in a specific response of CH34 to carbon limitation and oxidative stress, and included an acetone carboxylase subunit, fructose biphosphate aldolase, a DNA protection during starvation protein, chaperone protein, universal stress protein, and alkyl hydroperoxide reductase. The reproducible observation of the over-expression of these same proteins in multiple flight experiments, indicated that the CH34 cells could experience a substrate limitation and oxidative stress in spaceflight where cells and substrates are exposed to lower levels of gravity and higher doses of ionizing radiation. Bacterium C. metallidurans CH34 was able to grow normally under spaceflight conditions with very minor to no effects on cell physiology, but nevertheless specifically altered the expression of a few proteins in response to the environmental changes.     

Influence of class B scavenger receptors on cholesterol flux across the brush border membrane and intestinal absorption

To learn more about how the step of cholesterol uptake into the brush border membrane (BBM) of enterocytes influences overall cholesterol absorption, we measured cholesterol absorption 4 and 24 h after administration of an intragastric bolus of radioactive cholesterol in mice with scavenger receptor class B, type 1 (SR-BI) and/or cluster determinant 36 (CD36) deleted. The cholesterol absorption efficiency is unaltered by deletion of either one or both of the receptors. In vitro determinations of the cholesterol uptake specific activity of the BBM from the mice reveal that the scavenger receptors facilitate cholesterol uptake into the proximal BBM. It follows that cholesterol uptake into the BBM is not normally rate-limiting for the cholesterol absorption process in vivo; a subsequent step, such as NPC1L1-mediated transfer from the BBM into the interior of the enterocyte, is rate-limiting. The absorption of dietary cholesterol after 4 h in mice lacking SR-BI and/or CD36 and fed a high-fat/high-cholesterol diet is delayed to more distal regions of the small intestine. This effect probably arises because ATP binding cassette half transporters G5 and G8-mediated back flux of cholesterol from the BBM to the lumen of the small intestine limits absorption and causes the local cholesterol uptake facilitated by SR-BI and CD36 to become rate-limiting under this dietary condition.     

Genetic Analysis of a Novel Xylella fastidiosa Subspecies Found in the Southwestern United States

Xylella fastidiosa, the causal agent of several scorch diseases, is associated with leaf scorch symptoms in Chitalpa tashkentensis, a common ornamental landscape plant used throughout the southwestern United States. For a number of years, many chitalpa trees in southern New Mexico and Arizona exhibited leaf scorch symptoms, and the results from a regional survey show that chitalpa trees from New Mexico, Arizona, and California are frequently infected with X. fastidiosa. Phylogenetic analysis of multiple loci was used to compare the X. fastidiosa infecting chitalpa strains from New Mexico, Arizona, and trees imported into New Mexico nurseries with previously reported X. fastidiosa strains. Loci analyzed included the 16S ribosome, 16S-23S ribosomal intergenic spacer region, gyrase-B, simple sequence repeat sequences, X. fastidiosa-specific sequences, and the virulence-associated protein (VapD). This analysis indicates that the X. fastidiosa isolates associated with infected chitalpa trees in the Southwest are a highly related group that is distinct from the four previously defined taxons X. fastidiosa subsp. fastidiosa (piercei), X. fastidiosa subsp. multiplex, X. fastidiosa subsp. sandyi, and X. fastidiosa subsp. pauca. Therefore, the classification proposed for this new subspecies is X. fastidiosa subsp. tashke.Xylella fastidiosa is a gram-negative bacterium that multiplies within the xylem and causes serious disease problems in many diverse plant species. X. fastidiosa is considered a “new world” pathogen and is mainly found within North, Central, and South America (30). In many native plant species this bacterium exists as an apparently benign endophyte, while in other instances proliferation of X. fastidiosa within the xylem leads to disease typified by symptoms, including leaf scorch, chlorosis, stunting, branch dieback, inedible fruit, and eventually the death of the plant (4, 15). X. fastidiosa is transmitted by xylem-feeding insect vectors such as sharpshooters, leafhoppers, and spittle bugs (35). Diseases caused by X. fastidiosa include Pierce''s disease in grapes (7), citrus variegated chlorosis (CVC) (6), coffee leaf scorch (18), pecan leaf scorch (36), phony peach (41), plum leaf scald (32), and almond leaf scorch (25). X. fastidiosa has also been shown to be the causative agent of diseases found in landscape plants such as oleander leaf scorch (31), mulberry leaf scorch (14), and oak leaf scorch (3). In addition to the examples above proven through the completion of Koch''s postulates, X. fastidiosa is known to be associated with leaf scorch type diseases in several other ornamental landscape species including crape myrtle, olive, day lily, and southern magnolia (12).Chitalpa (Chitalpa tashkentensis Elias and Wisura) is an ornamental landscape plant that was developed for arid landscapes such as California, Arizona, Texas, and New Mexico. Chitalpa, originally bred in Russia and introduced into the United States in 1977, is an intergenic hybrid between desert willow (Chilopsis linearis Cav.) and Catalpa bignonioides Walt. (28). In the past, chitalpa trees across the Southwest were observed to display leaf scorch symptoms of unknown origin. X. fastidiosa was detected in many chitalpa trees that displayed leaf scorch symptoms in southern New Mexico (34). The first known occurrence of Pierce''s disease in New Mexico was reported in 2007, and the strains of X. fastidiosa found in infected New Mexico grapes were very similar to those present in chitalpa trees from the same area (33). The common use of chitalpa as a landscape plant in the Southwest coupled with the recent discovery that it can harbor X. fastidiosa strains similar to those associated with Pierce''s disease in New Mexico prompted a survey of chitalpa trees across the Southwest. The results of this survey show that chitalpa trees from New Mexico and Arizona are frequently infected with X. fastidiosa. Chitalpa plants imported into New Mexico nurseries from California were also found to contain similar strains of X. fastidiosa. A multilocus phylogenetic analysis was performed to further characterize these strains of X. fastidiosa. This analysis revealed that the X. fastidiosa isolates infecting chitalpa plants in New Mexico, Arizona, and imported into nurseries from California are highly related to each other and are distinct from the previously described subspecies fastidiosa (38).     

Evaluation of the Airborne Bacterial Population in the Periodically Confined Antarctic Base Concordia

The environmental airborne bacterial population in relation to human confinement was investigated over a period of 1 year in the Concordia Research Station, which is located on the Eastern Antarctic plateau. The unique location of the station makes it suitable for different research domains such as glaciology, atmospheric sciences, astronomy, etc. Furthermore, it is used as a test bed for long-duration spaceflights to study the physiologic and psychological adaptation to isolated environments. A total of 96 samples were collected at eight different locations in the station at regular intervals. The airborne bacterial contamination was for 90% of the samples lower than 10.0 × 10² colony-forming units per cubic meter of air (CFU/m³) and the total bacterial contamination increased over time during confinement but diminished after re-opening of the base. Viable airborne bacteria with different morphology were identified by biochemical analyses. The predominant microflora was identified as Staphylococcus sp. (24.9% of total) and Bacillus sp. (11.6% of total) and was associated with human activity, but also environmental species such as Sphingomonas paucimobilis (belonging to the α-Proteobacteria) could establish themselves in the airborne population. A few opportunistic pathogens (6%) were also identified. Rob Van Houdt and Patrick De Boever contributed equally.