Membrane binding properties of IRSp53-missing in metastasis domain (IMD) protein.

The 53-kDa insulin receptor substrate protein (IRSp53) organizes the actin cytoskeleton in response to stimulation of small GTPases, promoting the formation of cell protrusions such as filopodia and lamellipodia. IMD is the N-terminal 250 amino acid domain (IRSp53/MIM Homology Domain) of IRSp53 (also called I-BAR), which can bind to negatively charged lipid molecules. Overexpression of IMD induces filopodia formation in cells and purified IMD assembles finger-like protrusions in reconstituted lipid membranes. IMD was shown by several groups to bundle actin filaments, but other groups showed that it also binds to membranes. IMD binds to negatively charged lipid molecules with preference to clusters of PI(4,5)P2. Here, we performed a range of different in vitro fluorescence experiments to determine the binding properties of the IMD to phospholipids. We used different constructs of large unilamellar vesicles (LUVETs), containing neutral or negatively charged phospholipids. We found that IMD has a stronger binding interaction with negatively charged PI(4,5)P2 or PS lipids than PS/PC or neutral PC lipids. The equilibrium dissociation constant for the IMD-lipid interaction falls into the 78-170µM range for all the lipids tested. The solvent accessibility of the fluorescence labels on the IMD during its binding to lipids is also reduced as the lipids become more negatively charged. Actin affects the IMD-lipid interaction, depending on its polymerization state. Monomeric actin partially disrupts the binding, while filamentous actin can further stabilize the IMD-lipid interaction.

N-WASP coordinates the delivery and F-actin-mediated capture of MT1-MMP at invasive pseudopods.

Metastasizing tumor cells use matrix metalloproteases, such as the transmembrane collagenase MT1-MMP, together with actin-based protrusions, to break through extracellular matrix barriers and migrate in dense matrix. Here we show that the actin nucleation-promoting protein N-WASP (Neural Wiskott-Aldrich syndrome protein) is up-regulated in breast cancer, and has a pivotal role in mediating the assembly of elongated pseudopodia that are instrumental in matrix degradation. Although a role for N-WASP in invadopodia was known, we now show how N-WASP regulates invasive protrusion in 3D matrices. In actively invading cells, N-WASP promoted trafficking of MT1-MMP into invasive pseudopodia, primarily from late endosomes, from which it was delivered to the plasma membrane. Upon MT1-MMP's arrival at the plasma membrane in pseudopodia, N-WASP stabilized MT1-MMP via direct tethering of its cytoplasmic tail to F-actin. Thus, N-WASP is crucial for extension of invasive pseudopods into which MT1-MMP traffics and for providing the correct cytoskeletal framework to couple matrix remodeling with protrusive invasion.

Bacterial and host determinants of MAL activation upon EPEC infection: the roles of Tir, ABRA, and FLRT3.

Infection of host cells by pathogenic microbes triggers signal transduction pathways leading to a multitude of host cell responses including actin cytoskeletal re-arrangements and transcriptional programs. The diarrheagenic pathogens Enteropathogenic E. coli (EPEC) and the related Enterohemorrhagic E. coli (EHEC) subvert the host-cell actin cytoskeleton to form attaching and effacing lesions on the surface of intestinal epithelial cells by injecting effector proteins via a type III secretion system. Here we use a MAL translocation assay to establish the effect of bacterial pathogens on host cell signaling to transcription factor activation. MAL is a cofactor of Serum response factor (SRF), a transcription factor with important roles in the regulation of the actin cytoskeleton. We show that EPEC induces nuclear accumulation of MAL-GFP. The translocated intimin receptor is essential for this process and phosphorylation of Tyrosine residues 454 and 474 is important. Using an expression screen we identify FLRT3, C22orf28 and TESK1 as novel activators of SRF. Importantly we demonstrate that ABRA (actin-binding Rho-activating protein, also known as STARS) is necessary for EPEC-induced nuclear accumulation of MAL and the novel SRF activator FLRT3, is a component of this pathway. We further demonstrate that ABRA is important for structural maintenance of EPEC pedestals. Our results uncover novel components in pathogen-activated cytoskeleton signalling to MAL activation.

Myopathy-causing actin mutations promote defects in serum-response factor signalling.

Mutations in the gene encoding skeletal muscle alpha-actin (ACTA1) account for approx. 20% of patients with the muscular disorder nemaline myopathy. Nemaline myopathy is a muscular wasting disease similar to muscular dystrophy, but distinguished by deposits of actin and actin-associated proteins near the z-line of the sarcomere. Approx. one-third of the over 140 myopathy actin mutations have been characterized either biochemically or in cultured cells to determine their effects on the actin cytoskeleton. However, the actin defects causing myopathy are likely to be heterogeneous, with only a few common trends observed among the actin mutants, such as reduced polymerization capacity or an inability to fold properly. Notably, the transcriptional programme regulated by serum-response factor, which is instrumental in muscle development and maintenance, is directly controlled by the balance of actin assembly and disassembly in cells. In the present study, we explored the impact of myopathy mutations in actin on the control of the transcriptional response by serum-response factor and found that the majority of mutants examined have altered serum-response factor signalling. We propose that altered serum-response factor signalling could be a major factor in actin-based nemaline myopathy, and that this area could be exploited to develop therapies for sufferers.

Identification of the insulin-responsive tyrosine phosphorylation sites on IRSp53.

The 53-kDa insulin receptor substrate protein (IRSp53) is part of a regulatory network that organises the actin cytoskeleton in response to stimulation by small GTPases, promoting formation of actin-rich cell protrusions such as filopodia and lamellipodia. It had been established earlier that IRSp53 is tyrosine phosphorylated in response to stimulation of the insulin and insulin-related growth factor receptors, but the consequences of tyrosine phosphorylation for IRSp53 function are unknown. Here, we have used a variety of IRSp53 truncation and point mutants to identify insulin-responsive tyrosine phosphorylation sites on IRSp53. We have found that the C-terminal half of IRSp53 (residues 251-521) undergoes tyrosine phosphorylation in response to insulin stimulation of the insulin beta receptor or epidermal growth factor stimulation via the epidermal growth factor receptor, and that the key residue for insulin receptor-mediated phosphorylation is tyrosine 310, located in a region between the N-terminal IRSp53/MIM homology domain (IMD, residue 1-250) and the central SH3 domain (residues 374-438) that is predicted to be natively unstructured. Mutation of tyrosine 310 to phenylalanine or glutamic acid abrogates the phosphorylation in response to insulin stimulation, but not in response to stimulation of the epidermal growth factor receptor. The N-terminal IMD, which mediates dimerisation of IRSp53, is required for efficient tyrosine phosphorylation downstream of either the insulin or epidermal growth factor receptor stimulation, yet does not appear to include a tyrosine-phosphorylated site itself. Thus, we have identified tyrosine 310 as a primary site of tyrosine phosphorylation in response to insulin signalling and we have shown that although IRSp53 is tyrosine phosphorylated in response to epidermal growth factor receptor signalling, tyrosine 310 is not crucial. Furthermore, the tyrosine phosphorylation status does not appear to affect the cell morphology and production of filopod-like structures upon expression of IRSp53.

A simple model for the cooperative stabilisation of actin filaments by phalloidin and jasplakinolide.

The stabilisation of magnesium actin filaments by phalloidin and jasplakinolide was studied using the method of differential scanning calorimetry. The results showed that actin could adapt three conformations in the presence of drugs. One conformation was adapted in direct interaction with the drug, while another conformation was identical to that observed in the absence of drugs. A third conformation was induced through allosteric inter-protomer interactions. The effect of both drugs propagated cooperatively along the actin filaments. The number of the cooperative units determined by using a quantitative model was larger for jasplakinolide (15 actin protomers) than for phalloidin (7 protomers).

The effect of phalloidin and jasplakinolide on the flexibility and thermal stability of actin filaments.

In this work the effect of phalloidin and jasplakinolide on the dynamic properties and thermal stability of actin filaments was studied. Temperature dependent fluorescence resonance energy transfer measurements showed that filaments of Ca-actin became more rigid in the presence of phalloidin or jasplakinolide. Differential scanning calorimetric data implied that the stiffer filaments also had greater thermal stability in the presence of phalloidin or jasplakinolide. The fluorescence and calorimetric measurements provided evidences that the extent of stabilization by jasplakinolide was greater than that by phalloidin.

Chiral separation of thiazide diuretics by HPLC on Chiralcel OD-RH, Chiralcel OJ-R and Chirobiotic-T phases.

This contribution deals with comparative studies on the chiral separation of thiazide diuretics using cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD-RH), cellulose tris(4-methylbenzoate) (Chiralcel OJ-R) and teicoplanin (Chirobiotic T) phases. All columns showed good chiral recognition ability for this class of compounds. Out of seven compounds investigated, six were resolved with baseline resolution with at least one of the three columns.

Mapping of stereoselective recognition sites on human serum transferrin by capillary electrophoresis and molecular modelling.

Stereoselective recognition of chiral compounds can be used for mapping of surface interaction sites on proteins. Iron-free human serum transferrin is a suitable chiral selector in capillary electrophoresis used in native form in solution. Separation of optical isomers of tryptophan-methylester, tryptophan-ethylester and tryptophan-butylester and various drugs were studied in capillary zone electrophoresis applying a distinct transferrin zone prior to sample injection. Changes in the electrophoretic patterns (i.e., in the migration properties) of the molecules reflected the possible interactions with the protein. The tryptophan derivatives and eight drugs possessed stereoselective interactions, seven drugs showed interactions without appreciable chiral separation, and the others did not present any direct complexation with the protein molecules. Molecular modelling was performed to characterize the binding areas at the iron binding site of iron-free transferrin. The docking of tryptophan derivatives on transferrin showed that the R-enantiomers possess a stronger complexation with transferrin, whereas the S-enantiomers are bound by weaker interactions, which is in excellent agreement with the capillary electrophoresis results, where the R-enantiomers were always retarded stronger by transferrin. A ranking of drugs by the lipo score parameter of the docking shows an accordance with the stereoselective interactions by the protein.