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A protein of Mr≈ 85 000 has been isolated and purified from pig stomach smooth muscle that modulates the polymer state of actin in a Ca2+‐dependent manner. When added either to preformed F‐actin filaments or to G‐actin, prior to polymerisation, the modulator induces the formation of shorter filaments. The average filament length in the presence of the modulator is directly dependent on its molar ratio to actin indicating a stoichiometric rather than a catalytic type of interaction. When mixed with G‐actin the modulator forms a stable complex with two actin monomers; this complex is presumed to act as a potent nucleus for actin polymerisation. The dynamics of the interaction with F‐actin suggests a direct severing of actin filaments by the modulator via a binding to intrafilamentous actins.

A protein of Mr≈ 85 000 has been isolated and purified from pig stomach smooth muscle that modulates the polymer state of actin in a Ca 2+-dependent manner. When added either to preformed F-actin filaments or to G-actin, prior to polymerisation, the modulator induces the formation of shorter filaments. The average filament length in the presence of the modulator is directly dependent on its molar ratio to actin indicating a stoichiometric rather than a catalytic type of interaction. When mixed with G-actin the modulator forms a stable complex with two actin monomers; this complex is presumed to act as a potent nucleus for actin polymerisation. The dynamics of the interaction with F-actin suggests a direct severing of actin filaments by the modulator via a binding to intrafilamentous actins.

In this article we discuss three aspects of cell contact formation: (a) the molecular architecture of the cytomatrix in cell-to-substrate focal contacts, (b) the dynamic properties of membrane- and microfilament-associated proteins in the contact areas, and (c) the involvement of microtubules in the coordinated and directed formation of new substrate contacts during cell locomotion. We show that different microfilament-associated proteins exhibit distinct patterns of association with focal contacts: some proteins are specifically associated with focal contacts (vinculin and talin); alpha-actinin is enriched in the contact areas but also is present along the stress fibers and in the lamellipodium; actin and filamin are detected throughout the contact areas but in apparently reduced amounts compared with the associated stress fibers; and tropomyosin, myosin, and spectrin are either absent from the endofacial surfaces of contact areas or are present in only very small amounts. Fluorescence photobleaching recovery analyses performed with living cells microinjected with fluorescently labeled actin, vinculin, and alpha-actinin indicate that each of these proteins maintains a dynamic equilibrium between a soluble cytoplasmic pool and a membrane-bound fraction. Correlation of the distribution of vinculin and tubulin in motile fibroblasts to local movements of the leading edge of the same cells indicates that free-end microtubules extend into actively ruffling areas along the lamellipodium and that new vinculin-containing contacts are preferentially formed in these protruding regions.

Gelsolin is a calcium-dependent actin severing and capping protein. Calcium ‘opens’ the molecule to make actin binding sites accessible, but removal of calcium from the medium does not necessarily fully reverse this process. The calcium sensitivity of actin monomer binding and actin filament severing is here shown to vary considerably with the source of gelsolin and conditions of preparation. Plasma gelsolin undergoes irreversible loss of calcium sensitivity when prepared in the presence of calcium ions. This is not due solely to effects of bound calcium, because purified human plasma gelsolin expressed in E. coliand stored in calcium shows no comparable loss of calcium sensitivity when prepared or stored in calcium. These results suggest the presence of factors in plasma which, in the presence of calcium, promote an irreversible structural change in gelsolin resulting in permanent loss of calcium sensitivity.

A gelsolin-like actin-modulating protein was isolated from rat skeletal muscle and characterized with respect to its interaction with actin. The protein, with a molecular mass of approx. 85 kDa, forms a stoichiometric complex with two actin molecules and is activated by micromolar concentrations of Ca2+. It effectively severs actin filaments and promotes nucleation of actin polymerization. The activity of this protein is detectable already in crude extracts by its capability to reduce the steady state viscosity of actin. Actin-modulating activities were determined in muscle extracts of rats kept under protein catabolic conditions, i.e. as generated by corticosterone treatment and starvation. In both cases we found a marked increase of modulator activity. The possibility is discussed that the increased activity of actin modulator indicates a fragmentation of actin filaments prior to the proteolytic degradation of actin.

An actin-modulating protein has been isolated from the obliquely striated body wall muscle of the earthwormLumbricus terrestris. The isolation procedure included extraction in the absence of Ca2+ with subsequent ammonium sulfate fractionation followed by ion exchange chromatography and gel filtration. The purified modulator preparation appears on SDS-PAGE as a doublet of bands with molecular weights of 43 and 45 kDa. Both separately isolated components exhibit the same actin-modulating properties and therefore probably represent two isoforms. Substoichiometric amounts of modulator sever actin filaments and effectively promote nucleation of actin polymerization, which results in the formation of short actin filaments. Both effets are completely dependent on the presence of Ca2+ and activation of the modulator occurs in a narrow range of free Ca2+ concentrations around 10-6 mmol l-1. The modulator increases the critical concentration for actin polymerization, indicating that it binds to the fast polymerizing end of the actin filaments. The modulator forms a stoichiometric complex with two actin molecules whereby its ability to sever actin filaments is lost.

Myosin was isolated and purified from plasmodia of the slime mould Physarum polycephalum by a new method. This method is based on actomyosin extraction at low ionic strength after extensive washing, followed by the selective precipitation of myosin at pH 6·1 under relaxing conditions. The yield of myosin was 3–5 times higher than reported for other methods. In contrast to earlier studies a remarkably strong tendency to filament formation was found for slime mould myosin, probably due to a better preservation of some structural properties during preparation. Conditions were worked out under which numerous filaments up to 4 μm in length can be produced. It was established that not only a gradual decrease of ionic strength may influence filament formation, but also pH, ATP concentration and the presence of divalent cations. Compared to the current filament models a difference exists in the structure of the filaments. No central bare zone can be found, and thus, they lack an apparent bipolarity. Along the entire filament there are lateral projections representing the head portion of myosin molecules. A clear periodicity with an axial repeat of about 14 nm was observed, indicating a highly ordered arrangement of these projections. In this paper it is shown for the first time that myosin from one of the primitive motile systems is able to form aggregates of high structural order, indicating that the contraction of non-muscular actomyosin systems is not necessarily effected with oligomeric or randomly aggregated myosin. The possible role of myosin aggregation in vivo and the similarity of filament structure to that recently reported for myosin from vertebrate smooth muscle and striated muscle are discussed.

We have identified an approximately 85-kD protein in chicken erythrocytes which is immunologically, structurally, and functionally related to the gelsolin found in many muscle and nonmuscle cell types. Cell fractionation reveals a Ca2+-dependent partitioning of gelsolin into the soluble cytoplasm and the membrane-associated cytoskeleton of differentiating or mature erythrocytes. Depending on either the presence of Ca2+ during cell lysis or on the preincubation of the intact cells with the Ca2+-ionophore A23187, up to 40% of the total cellular gelsolin is found associated with the membrane skeleton. Expression of gelsolin shows a strong negative regulation during erythroid differentiation. From quantitations of its steady-state molar ratio to actin, gelsolin is abundant in early progenitor cells as revealed from avian erythroblastosis virus- and S13 virus-transformed cells which are arrested at the colony forming unit erythroid (CFU-e) stage of erythroid development. In these cells, which have a rudimentary and unstable membrane skeleton, gelsolin remains quantitatively cytoplasmic, irrespective of the Ca2+ concentration. During chicken embryo development and maturation, the expression of gelsolin decreases by a factor of approximately 10(3) in erythroid cells. This down regulation is independent from that of actin, which is considerably less, and is observed also when S13-transformed erythroid progenitor cells are induced to differentiate under conditions where the actin content of these cells does not change. In mature erythrocytes of the adult the amount of gelsolin is low, and significantly less than required for potentially capping of all membrane-associated actin filaments. We suggest that the gelsolin in erythroid cells is involved in the assembly of the actin filaments present in the membrane skeleton, and that it may provide for a mechanism, by means of its severing action on actin filaments, to extend the meshwork of the spectrin-actin-based membrane skeleton in erythroid cells during erythropoiesis.