A Cell-free System to Study Regulation of Focal Adhesions and of the Connected Actin Cytoskeleton

Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca²⁺ concentrations induce quasi-reversible diffusion of β1 integrins out of focal adhesions, whereas low Ca²⁺ concentrations induce irreversible recruitment of β1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated β1 receptors show that the cytoplasmic portion of β1 is required for low Ca²⁺-induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified α-actinin colocalizes and redistributes with β1 receptors on ventral plasma membranes depleted of actin, implicating binding of α-actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.     

Contrasted Microcolinearity and Gene Evolution Within a Homoeologous Region of Wheat and Barley Species

We study here the evolution of genes located in the same physical locus using the recently sequenced Ha locus in seven wheat genomes in diploid, tetraploid, and hexaploid species and compared them with barley and rice orthologous regions. We investigated both the conservation of microcolinearity and the molecular evolution of genes, including coding and noncoding sequences. Microcolinearity is restricted to two groups of genes (Unknown gene-2, VAMP, BGGP, Gsp-1, and Unknown gene-8 surrounded by several copies of ATPase), almost conserved in rice and barley, but in a different relative position. Highly conserved genes between wheat and rice run along with genes harboring different copy numbers and highly variable sequences between close wheat genomes. The coding sequence evolution appeared to be submitted to heterogeneous selective pressure and intronic sequences analysis revealed that the molecular clock hypothesis is violated in most cases.     

Isolation and characterization of "Reprotoxin", a novel protein complex from Daboia russelii snake venom

In snake venoms, non-covalent protein-protein interaction leads to protein complexes with synergistic and, at times, distinct pharmacological activities. Here we describe a new protein complex containing phospholipaseA(2) (PLA(2)), protease, and a trypsin inhibitor. It is isolated from the venom of Daboia russelii by gel permeation chromatography, on a Sephadex G-75 column. This 44.6kDa complex exhibits only phospholipase A(2) activity. In the presence of 8M urea it is well resolved into protease (29.1kDa), PLA(2) (13kDa), and trypsin inhibitor (6.5kDa) peaks. The complex showed an LD(50) of 5.06mg/kg body weight in mice. It inhibited the frequency of spontaneous release of neurotransmitter in hippocampal neurons. It also caused peritoneal bleeding, and edema in the mouse foot pads. Interestingly, the complex caused degeneration of both the germ cells and the mouse Leydig cells of mouse testis. A significant reduction in both the diameter of the seminiferous tubules and height of the seminiferous epithelia were observed following intraperitoneal injection of the sub-lethal dose (3mg/kg body weight). This effect of the toxin is supported by the increase in the activities of acid and alkaline phosphatases and the nitric oxide content in the testes, and a decrease in the ATPase activity. Because of its potent organ atrophic effects on the reproductive organs, the toxin is named "Reprotoxin". This is the first report demonstrating toxicity to the reproductive system by a toxin isolated from snake venom.     

Plant invasion is associated with higher plant–soil nutrient concentrations in nutrient‐poor environments

Plant invasion is an emerging driver of global change worldwide. We aimed to disentangle its impacts on plant–soil nutrient concentrations. We conducted a meta‐analysis of 215 peer‐reviewed articles and 1233 observations. Invasive plant species had globally higher N and P concentrations in photosynthetic tissues but not in foliar litter, in comparison with their native competitors. Invasive plants were also associated with higher soil C and N stocks and N, P, and K availabilities. The differences in N and P concentrations in photosynthetic tissues and in soil total C and N, soil N, P, and K availabilities between invasive and native species decreased when the environment was richer in nutrient resources. The results thus suggested higher nutrient resorption efficiencies in invasive than in native species in nutrient‐poor environments. There were differences in soil total N concentrations but not in total P concentrations, indicating that the differences associated to invasive plants were related with biological processes, not with geochemical processes. The results suggest that invasiveness is not only a driver of changes in ecosystem species composition but that it is also associated with significant changes in plant–soil elemental composition and stoichiometry.     

Acceptability and suitability of Spodoptera exigua (Hübner) for Cotesia icipe Fernandez‐Triana & Fiaboe on amaranth

The beet armyworm Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) is a polyphagous insect that is distributed worldwide and was recently reported as an important pest on African indigenous vegetables. Cotesia icipe Fernandez‐Triana & Fiaboe (Hymenoptera: Braconidae) is a recently described parasitoid, reported from various Afrotropical countries. This work investigated the performance of C. icipe on S. exigua infesting Amaranthus dubius Mart. ex Thell. under laboratory conditions. Cotesia icipe was aggressive on the host and successfully oviposited on S. exigua with 70% of parasitoid females ovipositing after 2 hr of exposure. Parasitoid densities significantly affected the parasitism rate and the nonreproductive larval mortality. Parasitism rate was 9.7 ± 0.8% and 59.5 ± 3.1% for a single and cohort of five females released, respectively, when offered 50 host larvae. The cohort female release resulted in significantly higher larval nonreproductive mortality than the single release. However, there was no significant difference between parasitoid release densities in regard to pupal nonreproductive mortality. The larval and pupal mortalities in the presence of C. icipe were significantly higher than the natural mortalities at both parasitoid release densities. The parasitoid sex ratio was female‐biased for the cohort females but balanced when a single female was released. The hind tibia and forewing lengths were not affected by the density of female parasitoids but there were variations according to sex. The implication of these findings on the potential use of C. icipe for biological control of S. exigua in amaranth production systems is discussed.     

TMEM41B and VMP1 are scramblases and regulate the distribution of cholesterol and phosphatidylserine

TMEM41B and VMP1 are integral membrane proteins of the endoplasmic reticulum (ER) and regulate the formation of autophagosomes, lipid droplets (LDs), and lipoproteins. Recently, TMEM41B was identified as a crucial host factor for infection by all coronaviruses and flaviviruses. The molecular function of TMEM41B and VMP1, which belong to a large evolutionarily conserved family, remains elusive. Here, we show that TMEM41B and VMP1 are phospholipid scramblases whose deficiency impairs the normal cellular distribution of cholesterol and phosphatidylserine. Their mechanism of action on LD formation is likely to be different from that of seipin. Their role in maintaining cellular phosphatidylserine and cholesterol homeostasis may partially explain their requirement for viral infection. Our results suggest that the proper sorting and distribution of cellular lipids are essential for organelle biogenesis and viral infection.     

Identification and characterization of acetyl-CoA carboxylase gene cluster in <Emphasis Type="Italic">Streptomyces toxytricini</Emphasis>

The gene locus for acetyl-CoA carboxylase (ACC) involved in the primary metabolism was identified from the genomic library of Streptomyces toxytricini which produces a lipase inhibitor lipstatin. The 7.4 kb cloned gene was comprised of 5 ORFs including accD1, accA1, hmgL, fadST1, and stsF. In order to confirm the biochemical characteristics of AccA1, the gene was overexpressed in Escherichia coli cells, and the recombinant protein was purified through Ni²⁺ affinity chromatography. Because most of the expressed AccAl was biotinylated by host E. coli BirA in the presence of D-biotin, the non-biotinylated apo-AccA1 was purified after gene induction without D-biotin, followed by exclusion of holo-AccA1 using streptavidin beads. The separated apo-AccA1 was post-translationally biotinylated by S. toxytricini biotin apo-protein ligase (BPL) in a time- and enzyme-dependent manner. This result supports that this gene cluster of S. toxytricini encodes the functional ACC enzyme subunits to be biotinylated.