CSF-1 and PI 3-kinase regulate podosome distribution and assembly in macrophages

Podosomes are actin-rich adhesive foci found in several cell types, including macrophages. They have a core containing actin and actin-binding proteins and a peripheral ring of integrins and associated proteins. We show that podosomes are abundant in polarized mouse bone marrow-derived macrophages (BMM) and are found primarily in lamellae. We investigated the effects of CSF-1, which induces membrane ruffling, cell spreading, and subsequent polarization and migration, on podosome formation. CSF-1 induces a transient increase in podosome number and enhances the formation of circular arrays of podosomes. Conversely, CSF-1 withdrawal leads to a reduction in podosomes and a decrease in polarized cells. The PI 3-kinase inhibitor LY294002 induces loss of podosomes together with rapid retraction of lamellae and loss of polarity. Our results indicate that CSF-1 acts via PI 3-kinase to enhance podosome assembly and that this is linked to macrophage polarization.     

Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking

Rho GTPases are well known to regulate actin dynamics. They activate two types of actin nucleators, WASP/WAVE proteins and Diaphanous-related formins (DRFs), which induce different types of actin organization. Their ability to interact with membranes allows them to target actin polymerization to discrete sites on the plasma membrane and to intracellular membrane compartments and thereby induce membrane protrusions or regulate vesicle movement. Most studies have concentrated on just three of the 22 mammalian Rho proteins, RhoA, Rac1 and Cdc42. However, recent research indicates that several other members of the Rho family, including Rif, RhoD, TC10 and Wrch1, and also related Rho-of-plants proteins (ROPs) in plants, stimulate actin polymerization and affect plasma membrane protrusion and/or vesicular traffic.     

Fibulin 5 Forms a Compact Dimer in Physiological Solutions

Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)-rich extracellular matrix protein that is essential for the formation of elastic tissues. Missense mutations in fibulin 5 cause the elastin disorder cutis laxa and have been associated with age-related macular degeneration, a leading cause of blindness. We investigated the structure, hydrodynamics, and oligomerization of fibulin 5 using small angle x-ray scattering, EM, light scattering, circular dichroism, and sedimentation. Compact structures for the monomer were determined by small angle x-ray scattering and EM, and are supported by close agreement between the theoretical sedimentation of the structures and the experimental sedimentation of the monomer in solution. EM showed that monomers associate around a central cavity to form a dimer. Light scattering and equilibrium sedimentation demonstrated that the equilibrium between the monomer and the dimer is dependent upon NaCl and Ca²⁺ concentrations and that the dimer is dominant under physiological conditions. The dimerization of fragments containing just the cbEGF domains suggests that intermolecular interactions between cbEGFs cause dimerization of fibulin 5. It is possible that fibulin 5 functions as a dimer during elastinogenesis or that dimerization may provide a method for limiting interactions with binding partners such as tropoelastin.Fibulins are a family of seven extracellular matrix glycoproteins, some of which associate with elastic fibers and basement membranes (1, 2). They are involved in the assembly, organization, and stabilization of macromolecular complexes (3). Fibulins contain arrays of cbEGF²-like domains and a fibulin-type C-terminal (Fc) module (4). Fibulins 3–5 have a modified N-terminal cbEGF domain, followed by five cbEGF domains (4).Fibulin 5 (supplemental Fig. S1) is highly expressed in developing arteries with a low expression in adult vessels that is up-regulated following vascular injury and in atherosclerosis (5, 6). Expression has been detected in other elastin-rich tissues, including aorta, skin, uterus, lung, heart, ovary, and colon (5, 6). The extensibility of such tissues is provided by elastic fibers (7), and aging is associated with a loss of elasticity (8). Fibulin 5 is essential for elastinogenesis. The fibulin 5 knock-out mouse exhibits disorganized elastic fibers resulting in severe elastinopathies, with loose skin, vascular abnormalities, and emphysematous lungs. Similar changes are seen in an aged phenotype (9, 10). Mutations in fibulin 5 lead to the elastin disorder cutis laxa (11–13) and have been associated with age-related macular degeneration (14, 15).It has been shown that fibulin 5 binds elastic fibers (16) and interacts with tropoelastin (10), fibrillin 1 (17), lysyl oxidase-like protein 1 (18), -2, and -4 (19), latent transforming growth factor-β-binding protein 2 (19), emilin 1 (20), apolipoprotein (a) (21), and superoxide dismutase (22). Through an RGD motif fibulin 5 interacts with integrins (6, 9, 23).The assembly of elastic fibers is a complex hierarchical process. A model proposes that fibulin 5 associates with microfibrils via interactions with fibrillin 1; tropoelastin molecules bind fibulin 5 and coacervate, and lysyl oxidase-like protein 1 enzymes cross-link tropoelastin to form mature elastin (7, 16). Data that support this model indicate that fibulin 5 potentially increases the coacervation of tropoelastin, enhancing elastic fiber formation (24). However, other data suggest that fibulin 5 slows the maturation of elastin assemblies (25).Rotary-shadowing EM has suggested that fibulin 5 exists as a short rod with a globular domain at one end (26). We used size-exclusion column multiangle laser light scattering (SEC-MALLS), small angle x-ray scattering (SAXS), EM single particle analysis, analytical ultracentrifugation (AUC), CD, and isoelectric focusing to investigate the structures of fibulin 5 in monomeric and dimeric form, and the equilibrium between the two forms.     

Haemoglobin concentration and body condition of nestling Great Tits Parus major : a comparison of first and second broods in two contrasting seasons

The physiological condition of nestling altricial birds depends on the quantity and quality of food delivered to them by parents. One indicator of the condition of Great Tit Parus major nestlings is the haemoglobin concentration in their blood. The present study demonstrates the influence of weather conditions (temperature and rainfall) on nestling haemoglobin concentrations during two consecutive breeding seasons in two different habitat types (parkland vs. woodland) in the city of Łódź in Central Poland. This influence probably results from the effects of weather on the trophic base of the Tits. Dry, hot weather strongly affected bush and herbal foliage later in the breeding season (mid-June to mid-July) in 2006, presumably by interfering with the development of herbivorous arthropod populations. This in turn caused food shortages for second broods of Great Tits, which resulted in nestlings having low haemoglobin levels. In the following year, temperature was on average lower, and rainfall was regular but not very heavy. These conditions enabled the development of arthropod assemblages, and the trophic base for birds was much richer. Haemoglobin concentrations in the blood of nestlings from second broods were significantly higher than those of first broods and, unexpectedly, second-brood nestlings in 2007 were on average in better physiological state than first-brood nestlings in 2006 in both habitats. The relationship between haemoglobin concentration, brood category and year was very similar to that for nestling body mass. However, it was independent of both body mass and brood size. In some years and under certain conditions, second broods can be more successful than first broods.     

Evolution of enhanced reproduction in the hybrid-derived invasive, California wild radish (Raphanus sativus)

Evolution is receiving increased attention as a potentially important factor in invasions. For example, hybridization may have stimulated the evolution of invasiveness in several well-known plant pests. However, the mechanism for success of such hybrid-derived lineages remains unknown in the majority of the cases studied. Here we ask whether increased reproductive success (in terms of maternal fitness) has evolved in an invasive lineage with confirmed hybrid ancestry. We compare the relative fitness of the non-native, hybrid-derived California wild radish (Raphanus sativus) to that of its two progenitor species in field experiments at different sites and in different years. We found that California wild radish has high survivorship and produces more fruits per plant and more seeds per plant than either of its progenitors in several environments. Furthermore, populations of California wild radish display a strong genotype-by-environment interaction, indicating that maintenance of genetic and phenotypic diversity between populations may be responsible for the weed’s ability to invade a wide breadth of California habitats. Our results suggest that hybridization may contribute the evolution of enhanced invasiveness and, also, that by limiting the introduction and subsequent hybridization of congeners, we may be able to prevent the evolution of new invasive lineages.     

Major Role of Epidermal Growth Factor Receptor and Src Kinases in Promoting Oxidative Stress-dependent Loss of Adhesion and Apoptosis in Epithelial Cells

A growing body of evidence suggests that reactive oxygen species are critical components of cell signaling pathways, in particular regulating protein phosphorylation events. Here, we show that oxidative stress in response to hydrogen peroxide treatment of human epithelial cells induces robust tyrosine phosphorylation on multiple proteins. Using an anti-phosphotyrosine purification and liquid chromatography-tandem mass spectrometry approach, we have identified many of these H₂O₂-induced tyrosine-phosphorylated proteins. Importantly, we show that epidermal growth factor receptor (EGFR) and Src are the primary upstream kinases mediating these events through their redox activation. The finding that many of the identified proteins have functions in cell adhesion, cell-cell junctions, and the actin cytoskeleton prompted us to examine stress-induced changes in adhesion. Immunofluorescence analysis showed that H₂O₂ alters cell adhesion structures and the actin cytoskeleton causing loss of adhesion and apoptosis. Remarkably, these cellular changes could be attenuated by inhibition of EGFR and Src, identifying these kinases as targets to block oxidative damage. In summary, our data demonstrate that EGFR and Src together play a central role in oxidative stress-induced phosphorylation, which in turn results in loss of adhesion, morphological changes, and cell damage in epithelial cells. These data also provide a general model for redox signaling in other cell systems.     

Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains

During inflammation, leukocytes bind to the adhesion receptors ICAM-1 and VCAM-1 on the endothelial surface before undergoing transendothelial migration, also called diapedesis. ICAM-1 is also involved in transendothelial migration, independently of its role in adhesion, but the molecular basis of this function is poorly understood. Here we demonstrate that, following clustering, apical ICAM-1 translocated to caveolin-rich membrane domains close to the ends of actin stress fibres. In these F-actin-rich areas, ICAM-1 was internalized and transcytosed to the basal plasma membrane through caveolae. Human T-lymphocytes extended pseudopodia into endothelial cells in caveolin- and F-actin-enriched areas, induced local translocation of ICAM-1 and caveolin-1 to the endothelial basal membrane and transmigrated through transcellular passages formed by a ring of F-actin and caveolae. Reduction of caveolin-1 levels using RNA interference (RNAi) specifically decreased lymphocyte transcellular transmigration. We propose that the translocation of ICAM-1 to caveola- and F-actin-rich domains links the sequential steps of lymphocyte adhesion and transendothelial migration and facilitates lymphocyte migration through endothelial cells from capillaries into surrounding tissue.     

High affinity HERG K(+) channel blockade by the antiarrhythmic agent dronedarone: resistance to mutations of the S6 residues Y652 and F656

Pharmacological inhibition of human-ether-a-go-go-related gene (HERG) K(+) channels by structurally and therapeutically diverse drugs is associated with the 'acquired' form of long QT syndrome and with potentially lethal cardiac arrhythmias. Two aromatic amino-acid residues (Y652 and F656) on the inner (S6) helices are considered to be key constituents of a high affinity drug binding site within the HERG channel pore cavity. Using wild-type (WT) and mutant HERG channels expressed in mammalian cell lines, we have investigated HERG channel current (I(HERG)) blockade at 37+/-1 degrees C by dronedarone (DRONED), a non-iodinated analogue of the Class III antiarrhythmic agent amiodarone (AMIOD). Under our conditions WT I(HERG) tails, measured at -40 mV following activating pulses to +30 mV, were blocked with IC(50) values of approximately 59 and 70 nM for DRONED and AMIOD, respectively. I(HERG) inhibition by DRONED was contingent upon channel gating, with block developing rapidly on membrane depolarization, but with no preference for activated over inactivated channels. High external [K(+)] (94 mM) reduced the potency of I(HERG) inhibition by both DRONED and AMIOD. Strikingly, mutagenesis to alanine of the S6 residue F656 (F656A) failed to eliminate blockade by both DRONED and AMIOD, whilst Y652A had comparatively little effect on DRONED but some effect on AMIOD. These findings demonstrate that high affinity drug blockade of I(HERG) can occur without a strong dependence on the Y652 and F656 aromatic amino-acid residues.     

A molecular dynamics method for calculating molecular volume changes appropriate for biomolecular simulation

Photothermal methods permit measurement of molecular volume changes of solvated molecules over nanosecond timescales. Such experiments are an important tool in investigating complex biophysical phenomena including identifying transient species in solution. Developing a microscopic understanding of the origin of volume changes in the condensed phase is needed to complement the experimental measurements. A molecular dynamics (MD) method exploiting available simulation methodology is demonstrated here that both mimics experimental measurements and provides microscopic resolution to the thermodynamic measurements. To calculate thermodynamic volume changes over time, isothermal-isobaric (NPT) MD is performed on a solution for a chosen length of time and the volume of the system is thus established. A further simulation is then performed by "plucking" out a solute molecule of interest to determine the volume of the system in its absence. The difference between these volumes is the thermodynamic volume of the solute molecule. NPT MD allows the volume of the system to fluctuate over time and this results in a statistical uncertainty in volumes that are calculated. It is found in the systems investigated here that simulations lasting a few nanoseconds can discern volume changes of approximately 1.0 ml/mole. This precision is comparable to that achieved empirically, making the experimental and theoretical techniques synergistic. The technique is demonstrated here on model systems including neat water, both charged and neutral aqueous methane, and an aqueous beta-sheet peptide.