Identification of a homologue for annexin VII (synexin) in Dictyostelium discoideum

Immunological and biochemical data have been used to show that the slime mold Dictyostelium discoideum expresses a Ca2+/phospholipid-binding protein related to vertebrate annexins. The Dictyostelium protein (apparent molecular mass 46 kDa) is recognized by an antibody directed against an annexin consensus peptide and exhibits the properties characteristic for annexins, i.e. it interacts in a Ca2(+)-dependent manner with negatively charged phospholipids. Limited proteolysis converts the 46-kDa protein into a 32-kDa derivative which retains the Ca2+/phospholipid-binding properties of the 46-kDa polypeptide. Partial protein sequence data identify the Dictyostelium protein as the typical annexin and indicate that the 46-kDa protein is an annexin VII (synexin) homologue. The identification of an annexin in a simple eucaryote should lead to the introduction of genetic approaches to analyze the physiological role of the annexins.     

The influence of adrenergic nerves of the response of blood vessels in the rabbit ear to 2--phenylalanine-8-lysine vasopressin (Octapressin).

The blood vessels in the rabbit ear have been used to investigate the effects of octapressin on vascular smooth muscle. Continuous administration of this drug resulted in tachphlatis. Cocaine enhanced the response of the ear to octapressin; however, this potentiation was not evident when phentolamine was concurrently added to the perfusate. Denervation and reserpinisation of the ear also eliminated the potentiation of octapressin's response by cocaine. The results suggest that there is a small, indirect sympathomimetic component involved in octapressin's action on the vasculature.     

Importance of phospholipid bilayer integrity in the analysis of protein–lipid interactions

The integrity of supported phospholipid bilayer membranes is of crucial importance for the investigation of lipid–protein interactions. Therefore we recorded the formation of supported membranes on SiO₂ and mica by quartz crystal microbalance and controlled the integrity by atomic force microscopy. This study aims to analyze how membrane defects affect protein–lipid interactions. The experiments focused on a lipid mixture of POPC/DOPC/Chol/POPS/PI(4,5)P₂ (37:20:20:20:3) and the binding of the peripheral membrane associated protein annexin A2. We found that formation of a continuous undisturbed bilayer is an indispensable precondition for a reliable determination and quantification of lipid–protein-interactions. If membrane defects were present, protein adsorption causes membrane disruption and lipid detachment on a support thus leading to false determination of binding constants. Our results obtained for PI(4,5)P₂ and cholesterol containing supported membranes yield new knowledge to construct functional surfaces that may cover nanoporous substrates, form free standing membranes or may be used for lab-on-a-chip applications.     

Structural basis of the Ca(2+)-dependent association between S100C (S100A11) and its target, the N-terminal part of annexin I

Background: S100C (S100A11) is a member of the S100 calcium-binding protein family, the function of which is not yet entirely clear, but may include cytoskeleton assembly and dynamics. S100 proteins consist of two EF-hand calcium-binding motifs, connected by a flexible loop. Like several other members of the family, S100C forms a homodimer. A number of S100 proteins form complexes with annexins, another family of calcium-binding proteins that also bind to phospholipids. Structural studies have been undertaken to understand the basis of these interactions. Results: We have solved the crystal structure of a complex of calcium-loaded S100C with a synthetic peptide that corresponds to the first 14 residues of the annexin I N terminus at 2.3 A resolution. We find a stoichiometry of one peptide per S100C monomer, the entire complex structure consisting of two peptides per S100C dimer. Each peptide, however, interacts with both monomers of the S100C dimer. The two S100C molecules of the dimer are linked by a disulphide bridge. The structure is surprisingly close to that of the p11-annexin II N-terminal peptide complex solved previously. We have performed competition experiments to try to understand the specificity of the S100-annexin interaction. Conclusions: By solving the structure of a second annexin N terminus-S100 protein complex, we confirmed a novel mode of interaction of S100 proteins with their target peptides; there is a one-to-one stoichiometry, where the dimeric structure of the S100 protein is, nevertheless, essential for complex formation. Our structure can provide a model for a Ca(2+)-regulated annexin I-S100C heterotetramer, possibly involved in crosslinking membrane surfaces or organising membranes during certain fusion events.     

Cooperative adsorption of ezrin on PIP2-containing membranes

By means of the quartz crystal microbalance (QCM) and scanning force microscopy (SFM), the adsorption of ezrin, a member of the ezrin/radixin/moesin protein family, on l-alpha-phosphatidylinositol-4,5-bisphosphate (PIP(2)) containing solid-supported membranes was investigated. An increase in the PIP(2) content in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes resulted in an increased amount of bound ezrin strongly supporting the crucial role of PIP(2) for ezrin recruitment to membranes. No ezrin adsorption to membranes composed of pure POPC was detected. To characterize the binding process in more detail, the kinetics and reversibility of ezrin adsorption were investigated by the QCM technique, showing that the protein remains partly bound after rinsing with pure buffer, which we suspected to be a result of lateral interactions between the proteins. SFM images revealed the formation of two-dimensional ezrin clusters on PIP(2)-doped POPC membranes. Time-elapsed SFM images show that the growth of protein domains occurs from a few nucleation sites. The QCM data in conjunction with the results obtained by SFM led us to propose that the binding process of ezrin occurs in a positive cooperative manner. When lateral interactions of the proteins on the membrane were taken into account, we were able to simulate the kinetics obtained from time-resolved QCM readouts by employing a model developed by Minton. On the basis of the kinetic analysis, we were also able to reconstruct the adsorption isotherm.     

Gene–Environment Interactions at Nucleotide Resolution

Interactions among genes and the environment are a common source of phenotypic variation. To characterize the interplay between genetics and the environment at single nucleotide resolution, we quantified the genetic and environmental interactions of four quantitative trait nucleotides (QTN) that govern yeast sporulation efficiency. We first constructed a panel of strains that together carry all 32 possible combinations of the 4 QTN genotypes in 2 distinct genetic backgrounds. We then measured the sporulation efficiencies of these 32 strains across 8 controlled environments. This dataset shows that variation in sporulation efficiency is shaped largely by genetic and environmental interactions. We find clear examples of QTN:environment, QTN: background, and environment:background interactions. However, we find no QTN:QTN interactions that occur consistently across the entire dataset. Instead, interactions between QTN only occur under specific combinations of environment and genetic background. Thus, what might appear to be a QTN:QTN interaction in one background and environment becomes a more complex QTN:QTN:environment:background interaction when we consider the entire dataset as a whole. As a result, the phenotypic impact of a set of QTN alleles cannot be predicted from genotype alone. Our results instead demonstrate that the effects of QTN and their interactions are inextricably linked both to genetic background and to environmental variation.     

LaeA control of velvet family regulatory proteins for light-dependent development and fungal cell-type specificity

VeA is the founding member of the velvet superfamily of fungal regulatory proteins. This protein is involved in light response and coordinates sexual reproduction and secondary metabolism in Aspergillus nidulans. In the dark, VeA bridges VelB and LaeA to form the VelB-VeA-LaeA (velvet) complex. The VeA-like protein VelB is another developmental regulator, and LaeA has been known as global regulator of secondary metabolism. In this study, we show that VelB forms a second light-regulated developmental complex together with VosA, another member of the velvet family, which represses asexual development. LaeA plays a key role, not only in secondary metabolism, but also in directing formation of the VelB-VosA and VelB-VeA-LaeA complexes. LaeA controls VeA modification and protein levels and possesses additional developmental functions. The laeA null mutant results in constitutive sexual differentiation, indicating that LaeA plays a pivotal role in inhibiting sexual development in response to light. Moreover, the absence of LaeA results in the formation of significantly smaller fruiting bodies. This is due to the lack of a specific globose cell type (Hülle cells), which nurse the young fruiting body during development. This suggests that LaeA controls Hülle cells. In summary, LaeA plays a dynamic role in fungal morphological and chemical development, and it controls expression, interactions, and modification of the velvet regulators.     

Activation of F-actin binding capacity of ezrin: synergism of PIP₂ interaction and phosphorylation

Ezrin is a membrane-cytoskeleton linker protein that can bind F-actin in its active conformation. Several means of regulation of ezrin's activity have been described including phosphorylation of Thr-567 and binding of L-α-phosphatidylinositol-4,5-bisphosphate (PIP₂). However, the relative contributions of these events toward activation of the protein and their potential interdependence are not known. We developed an assay based on solid-supported membranes, to which different ezrin mutants (ezrin T567A (inactive mutant), wild-type, and T567D (active pseudophosphorylated mutant)) were bound, that enabled us to analyze the influence of phosphorylation and PIP₂ binding on ezrin's activation state in vitro. The lipid bilayers employed contained either DOGS-NTA-Ni to bind the proteins via an N-terminal His-tag, or PIP₂, to which ezrin binds via specific binding sites located in the N-terminal region of the protein. Quantitative analysis of the binding behavior of all three proteins to the two different receptor lipids revealed that all three bind with high affinity and specificity to the two receptor lipids. Fluorescence microscopy on ezrin-decorated solid-supported membranes showed that, dependent on the mode of binding and the phosphorylation state, ezrin is capable of binding actin filaments. A clear synergism between phosphorylation and the receptor lipid PIP₂ was observed, suggesting a conformational switch from the dormant to the active, F-actin binding state by recognition of PIP₂, which is enhanced by the phosphorylation.