Cooperative effects of tropomyosin on the dynamics of the actin filament.

Tropomyosin (Tpm) plays an important role in regulating the organisation and functions of the actin cytoskeleton. Here, we describe a new approach to analyse the effects of Tpm on actin dynamics. Using F-actin proteolytically modified within the DNase-binding loop (ECP-actin), we show that Tpm binding almost completely suppresses the increased subunit exchange intrinsic for this F-actin.

Four paralog gelsolin genes are differentially expressed in the earthworm Lumbricus terrestris.

We have identified and characterized four distinct variants of the gelsolin-related protein (EWAM P1-P4) in the earthworm L. terrestris. All of these proteins biochemically qualify as gelsolins since they sever actin filaments in a calcium dependent manner.

The interaction of gelsolin with tropomyosin modulates actin dynamics.

We have investigated the interactions between the actin-binding proteins gelsolin and tropomyosin, with special respect to any effects on the functional properties of gelsolin. Limited proteolysis indicated that the loop connecting the gelsolin domains G3 and G4 is involved in tropomyosin binding. Under nonpolymerizing conditions, binding of tropomyosin neither prevented the formation of a 2: 1actin-gelsolin complex, nor did it affect the nucleating activity of gelsolin in actin polymerization, likely as a result of competitive displacement of tropomyosin from gelsolin.

Isoforms of gelsolin from lobster striated muscles differ in calcium-dependence.

Two distinct isoforms of the Ca-dependent actin filament severing protein gelsolin were identified in cross-striated muscles of the American lobster. The variants (termed LG1 and LG2) differ by an extension of 18 AA at the C-terminus of LG1, and by two substitutions at AA735 and AA736, the two C-terminal amino acids of LG2. Functional comparison of the isolated and purified proteins revealed gelsolin-typical properties for both with differences in Ca(2+)-sensitivity, LG2 being activated at significant lower Ca-concentration than LG1: Half maximal activation for both filament severing and G-actin binding was ∼4×10(-7)M Ca(2+) for LG2 vs.

Electron-microscopical localization of gelsolin in various crustacean muscles.

Gelsolin was localized by immunoelectron microscopy in fast and slow cross-striated muscles of the lobster Homarus americanus. When ultrathin sections of the muscles were labelled with anti-gelsolin and a gold-conjugated second antibody, 90% of all gold particles in the myoplasm were detected on myofibrils, preferentially in the I-band and AI-region of the sarcomeres. Both the region of the H-zone (lacking thin filaments) and the Z-disc contained no or little gold label.

Difference in polymerization and steady-state dynamics of free and gelsolin-capped filaments formed by alpha- and beta-isoactins.

The polymerization of scallop beta-like actin is significantly slower than that of skeletal muscle alpha-actin. To reveal which steps of polymerization contribute to this difference, we estimated the efficiency of nucleation of the two actins, the rates of filament elongation at spontaneous and gelsolin-nucleated polymerization and the turnover rates of the filament subunits at steady-state. Scallop actin nucleated nearly twice less efficient than rabbit actin.

Involvement of a gelsolin-related protein in spermatogenesis of the earthworm Lumbricus terrestris.

A gelsolin-related protein was isolated from seminal vesicles of the annelid Lumbricus terrestris. Compared with the isoforms of the gelsolin-related protein previously found in the muscle of the annelid body wall, the isolated protein was assigned to the first isoform (EWAM-P1) because of its electrophoretic mobility, chromatographic elution behaviour, immunological cross-reactivity and identical nucleotide sequence of segments obtained by reverse transcription/polymerase chain reaction. Immunofluorescence studies with smear preparations of developing male germ cells revealed characteristic changes of the local distribution of actin and EWAM-P1 during spermatogenesis.

Calcium-induced conformational changes in the C-terminal half of gelsolin stabilize its interaction with the actin monomer.

The basic mechanism for the nucleating effect of gelsolin on actin polymerization is the formation of a complex of gelsolin with two actin monomers. Probably due to changes in the C-terminal part of gelsolin, a stable ternary complex is only formed at [Ca(2+)] >10(-5) M [Khaitlina, S., and Hinssen, H.

Topological assignment of the N-terminal extension of plasma gelsolin to the gelsolin surface.

The actin-binding protein gelsolin is highly conserved in vertebrates and exists in two isoforms, a cytoplasmic and an extracellular variant, generated by alternative splicing. In mammals, these isoforms differ only by an N-terminal extension in plasma gelsolin, a short sequence of up to 25 amino acids. Cells and tissues may contain both variants, as plasma gelsolin is secreted by many cell types.

Damped elastic recoil of the titin spring in myofibrils of human myocardium.

The giant protein titin functions as a molecular spring in muscle and is responsible for most of the passive tension of myocardium. Because the titin spring is extended during diastolic stretch, it will recoil elastically during systole and potentially may influence the overall shortening behavior of cardiac muscle. Here, titin elastic recoil was quantified in single human heart myofibrils by using a high-speed charge-coupled device-line camera and a nanonewtonrange force sensor.

Regulation of sodium channel activity by capping of actin filaments.

Ion transport in various tissues can be regulated by the cortical actin cytoskeleton. Specifically, involvement of actin dynamics in the regulation of nonvoltage-gated sodium channels has been shown. Herein, inside-out patch clamp experiments were performed to study the effect of the heterodimeric actin capping protein CapZ on sodium channel regulation in leukemia K562 cells.

Nebulin is a thin filament protein of the cardiac muscle of the agnathans.

Nebulin is an integral protein of skeletal muscle thin filaments and probably acts as a ruler for the thin filament length. Cardiac muscles of higher vertebrates have been shown earlier to lack nebulin. Instead in human and chicken cardiac muscle the much smaller protein nebulette replaces nebulin.

The vasodilator-stimulated phosphoprotein promotes actin polymerisation through direct binding to monomeric actin.

The vasodilator-stimulated phosphoprotein (VASP) functions as a cellular regulator of actin dynamics. VASP may initialise actin polymerisation, suggesting a direct interaction with monomeric actin. The present study demonstrates that VASP directly binds to actin monomers and that complex formation depends on a conserved four amino acid motif in the EVH2 domain.

Ca-dependent binding of actin to gelsolin.

Ca(2+) of 0.3-1.0 microM induces both the exposure of tryptic cleavage sites within the gelsolin molecule inaccessible in the Ca-free conformation, and binding of one actin monomer to the N-terminal half of gelsolin.