Kathrin Schlüter

Phosphorylation of the Vasodilator-stimulated Phosphoprotein Regulates Its Interaction with Actin

The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases in platelets and other cardiovascular cells. It promotes actin nucleation and binds to actin filaments in vitro and associates with stress fibers in cells. The VASP-actin interaction is salt-sensitive, arguing for electrostatic interactions. Hence, phosphorylation may significantly alter the actin binding properties of VASP. This hypothesis was investigated by analyzing complex formation of recombinant murine VASP with actin after phosphorylation with cAMP-dependent kinase in different assays. cAMP-dependent kinase phosphorylation had a negative effect on both actin nucleation and VASP interaction with actin filaments, with the actin nucleating capacity being more affected than actin filament binding and bundling. Replacing VASP residues known to be phosphorylated in vivo by acidic residues to mimic phosphorylation had similar although less dramatic effects on VASP-actin interactions. In contrast, phosphorylation had no significant effect on VASP oligomerization or its interaction with its known ligands profilin, vinculin, and zyxin. When overexpressing VASP mutants in eukaryotic cells, they all showed targeting to focal contacts and stress fibers. Our results imply that VASP phosphorylation may act as an immediate negative regulator of actin dynamics.

DIFFERENTIAL COLOCALIZATION OF PROFILIN WITH MICROFILAMENTS IN PTK2 CELLS

Profilins are thought to be involved in the control of actin dynamics in eukaryotic cells. In accordance with this concept, profilin was found to be colocalized with the cortical microfilament webs in leading lamellae of locomoting and spreading fibroblasts. However, so far, there is little information on the distribution of profilin in other cell types. In this study, we report on the colocalization of profilin with various microfilament suprastructures in the epithelial cell line PtK2. This cell line, which is derived from rat kangaroo, contains a profilin sharing an N-terminal epitope with bovine and human profilin I, as seen by immunoblotting with monoclonal antibodies. By using immunofluorescence in conjunction with conventional fluorescence microscopy and confocal laser-scanning microscopy, we found profilin in ruffling areas of the peripheral lamellae and nascent stress fibers of spreading cells, whereas the peripheral belts of stationary cells growing in epithelioid sheets lacked profilin staining. In these cells, profilin was primarily distributed in a fine reticular or vesicular network that was not related to the microfilament system. Conspicuously low levels of profilins was not related to the contractile ring of mitotic cells. This was found for different fixation protocols and antibodies of the IgG and IgM type, respectively, indicating that lack of staining of the cleavage furrow was not due to antibody penetration problems. Depending on the fixation protocol, the nuclear matrix appeared strongly positive or negative for profilin. Cells microinjected with birch pollen profilin and labeled with a birch profilin-specific monoclonal antibody corroborated the results obtained with the endogeneous protein: The injected profilin was targeted to the cortical web and to nascent stress fibers of spreading cells but not to the cleavage ring of mitotic cells. These results suggest that high concentrations of a profilin I homologue are preferentially located with those microfilament suprastructures in PtK2 cells that are subject to rapid modulation by external signals.

Birch pollen profilin: structural organization and interaction with poly-(l-proline) peptides as revealed by NMR

The secondary structure of birch pollen profilin, a potent human allergen, was elucidated by multidimensional nuclear magnetic resonance (NMR), as a prerequisite to study the interaction of this profilin with ligands for its poly‐(l‐proline) (PLP)‐binding site. The chemical shifts of the 15N‐labeled backbone amide groups were used to monitor complex formation with various PLP peptides. Titration with deca‐l‐proline (P10) yielded a K D of 0.2 mM. P8 was the shortest PLP to provoke a significant reaction. (GP5)3G bound significantly, confirming the interaction between profilins and the protein VASP containing this motif. Birch profilin interacted also with GP6GP5, found in the cyclase‐associated protein (CAP), a suspected profilin ligand.

An Open-Loop Control Approach for Magnetic Shape Memory Actuators Considering Temperature Variations

Magnetic shape memory alloys (MSMA) offer remarkable potentials for actuation purposes because of a large achievable strain and a short response time. But, apart from these advantages, MSMA show a hysteretic behavior between the input and output quantities. Hysteretic phenomena represent an important challenge for the design of control systems for MSMA-based actuators. Furthermore, this hysteretic behavior is sensitive to temperature variations, a situation that arises in many applications. To face the problem of increasing/decreasing temperature during operation, an open-loop control approach considering temperature variations is presented in this paper. For this purpose, an actuator prototype is characterized with particular emphasis on temperature influence concerning the input-output behavior. The presence of a time-varying nonlinearity is addressed by means of a set of hysteresis models and relative compensators to improve the positioning performance of the actuator system. Subsequently, the obtained models are integrated in the control loop and tested experimentally. Finally, the results achieved with the introduced control concept are presented.

An alpha-actinin-profilin chimaera with two alternatively operating actin-binding sites

Studying the mode of interaction between actin and actin-binding proteins, we constructed a chimaeric protein consisting of the sequence for bovine profilin I (P), to which the sequence for the actin-binding domain of Dictyostelium discoideum alpha-actinin (alphaA1-2) was fused N-terminally. The resulting hybrid clone was expressed in Escherichia coli, and the chimaeric protein, alphaA1-2P, purified by affinity chromatography on poly-(L-proline) (PLP) columns and identified using specific antibodies. High resolution electron microscopy demonstrated that this protein consists of two discrete subdomains. In biochemical, viscometric and electron microscopic analyses, we showed that both modules in this molecule are biologically active. The chimaera binds to poly-(L-proline) and inhibits the polymerization of G-actin in KCl, which is consistent with the assumption that the profilin part is intact. Inhibition of actin polymerization in KCl was stronger than that of the parental profilin, and the Kd value of its interaction with rabbit skeletal muscle actin, as determined by falling ball viscometry, was smaller (mean value 0.5 x 10(-6) M, as compared to 1.9 x 10(-6) M for bovine profilin). In 2mM MgCl2, the actin polymerized rapidly, consistent with the interpretation that under these conditions the chimaera, like profilin, is less efficient as an actin-sequestering agent. In the presence of alphaA1-2P, the resulting filaments were decorated with particles projecting from the filament axis. We conclude that under these conditions the alphaA1-2 domain of alphaA1-2P is preferentially active, attaching the chimaeric particles laterally to the filaments. Hence, the parental modules combined in alphaA1-2P permit this molecule to switch from a G-actin- to an F-actin-binding form.

Development of a Miniaturized Clamping Device Driven by Magnetic Shape Memory Alloys

This article deals with the potential of magnetic shape memory (MSM) actuators for the use in micro applications and in particular in clamping devices. The most common actuator concepts are introduced briefly, including the so called push-push concept. Subsequently, an overview of clamping technology is given and the advantages of mechanical clamping are pointed out. It is proposed to use the push-push actuation concept for the development of an energy-efficient clamping device driven by magnetic shape memory alloys. Hence, the design of the MSM clamping device and the dimensioning of its magnetic circuits are presented. Finally, the characteristics of the device are experimentally examined and the results are summarized.Copyright

Membrane-Microfilament Attachment Sites: the Art of Contact Formation

In animals, embryogenesis and wound healing require a finely tuned coupling of signal reception with the intracellular cytoskeleton. In particular, tissue formation is specified by the differentiation of discrete cellular contact sites between cells or between cells and their substratum. These contacts are morphologically distinct structures, but are also highly dynamic, and their reversible assembly is controlled by intra- as well as by extracellular factors. Regarding the cytoplasmic site of these contacts, the data from numerous studies imply that there is a catalogue of structural proteins linking the distal portions of the microfilaments to the integral components of the plasma membrane. The interaction of these structural proteins with each other is probably modulated by posttranslational modification and by accessory proteins mediating between the cytoskeletal elements and the signaling pathways. Thus, forming these contacts must be a highly complex event. Only a detailed analysis of each of the protein modules involved in such interactions, their posttranslational modification, and their “cross talk” will provide the basis for the understanding of the entire process. Table 1 gives a summary of the major proteins found in adhesive con¬tacts of the cell-substratum type (focal contacts) and their classification according to their proposed function, as described in numerous articles (reviewed in Burridge et al. 1988; Geiger and Ginsberg 1991; Turner and Burridge 1991; Critchley et al. 1991; Luna and Hitt 1992; Schaller and Parsons 1993). Classical examples of structural components are, of course, actin itself, α actinin, and vinculin. In addition, talin and radixin, two proteins with sequence homology to band 4.1, an actin-membrane-link- ing protein of the red blood cell, fall into this category.