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To clarify the role of profilins in cells, fusion proteins constructed with green fluorescent protein (GFP) should be extremely helpful. As profilins are considerably smaller than the GFP fusion partner (14-17 kDa compared with 27 kDa, respectively), we characterized the fusion proteins in vitro, to ascertain their biological function. We fused mouse profilin I and II to either the C-terminus or N-terminus of GFP. These fusion proteins were expressed in Escherichia coli and affinity-purified on polyproline-Sepharose. Interaction with vasodilator-stimulated phosphoprotein, a proline-rich ligand of profilin, was investigated by ELISA, as was binding to PtdIns(4,5)P2. The affinity for actin was quantitatively determined in polymerization assays. Our results show that fusion of GFP to the C-terminus of profilin I abolishes polyproline binding. In contrast, the other fusion proteins bound to polyproline-Sepharose and VASP. Binding to PtdIns(4,5)P2 was not significantly altered. Furthermore, fusion of either isoform with GFP did not decrease the affinity for actin. In localization studies with mammalian cells, all fusion proteins showed the localization expected for profilin in areas of high actin dynamics, such as leading lamellae and ruffles induced by epidermal growth factor. However, with regard to our in vitro data, we suspect that only a minor fraction of profilin I carrying the GFP at the C-terminus can target these sites. Therefore, other constructs should be preferred for further in vivo studies.

The microfilament protein alpha-actinin exists as a dimer. The N-terminal regions of both polypeptides, arranged in antiparallel orientation, comprise the actin-binding regions, while the C-terminal, larger parts consist of four spectrin-like repeats that interact to form a rod-like structure. To elucidate the fine structure of smooth-muscle alpha-actinin, we used energy-filtered transmission electron microscopy in conjunction with negative staining. Survey pictures of the protein purified from chicken gizzard revealed discrete, elongated particles whose length and width varied with the ionic strength of the buffer. It was determined to to 29.3 nm x 4.8 nm in 0.05 M KCl and 32.6 nm x 4.4 nm in 0.15 M KCl. Both ends of the molecule displayed hook-like structures consisting of globular domains, which were highly variable in their orientation with respect to the long axis of the molecule. Their location at the ends of the molecule, and the finding that these hooks were missing from particles obtained by thermolysin digestion indicated that they probably correspond to the N-terminal actin-binding regions. The rod-like center of the molecule revealed discrete globular masses which probably comprise the spectrin-like repeats. Their arrangement was compatible with the interpretation that three spectrin repeats of each polypeptide chain can form pairs with the respective sequences of the other chain. The rod-like 53-kDa fragment obtained after thermolysin digestion largely retained this structural organization but appeared wider (22.5 nm x 5.9 nm). Our results help to clarify previous discrepancies on the quatenary organization of alpha-actinin and suggest that effective actin-binding and cross-linking of alpha-actinin is based on the high flexibility of the terminal hooks.

Vinculin, a structural protein of animal cells, is critically involved in the assembly of microfilament/plasma membrane junctions at cell contacts. To understand its role in organizing the distal portions of microfilaments into specific, morphologically distinct structures at these sites in more detail, we characterized its interaction with filamentous actin and with itself by means of in vitro assays. Using recombinant proteins comprising different parts of the vinculin tail fused to the maltose-binding protein of Escherichia coli, we show in sedimentation assays that this part of vinculin harbors two discrete sites that can bind to actin independently. They reside within amino acid residues 893-985 and 1016-1066 of the 1066-residue polypeptide chain. However, both sites are necessary to cross-link or bundle actin filaments, as demonstrated by low shear viscometry. Crosslinking and bundling are alternatives determined by the molar ratio of fusion protein to F-actin. Both actin-binding sequences are capable of oligomer formation, as shown in chemical-cross-linking and dot-overlay assays. These data allow us to propose a possible role for vinculin in organizing the distal ends of microfilaments at the plasma membrane into the point-like structure characteristic for cell-matrix contacts.

Talin is a multidomain cytoskeletal protein containing discrete binding sites for acidic phospholipids, beta-integrin, actin and vinculin. Hence, it is thought to link microfilaments to the cytoplasmic membrane in cell-matrix adhesion sites, and this should critically depend on talin structure. To obtain more information on the latter, we used energy-filtered transmission electron microscopy of negatively stained talin purified from chicken smooth muscle. We show that in buffers of physiological ionic strength, talin adopts an elongated shape (56 +/- 7 nm in length), consisting of a series of globular masses. While these compact elements, arranged like beads on a string, were of rather uniform dimensions (3.8 nm in diameter), their center-to-center spacings varied, indicating the flexibility of the connecting strands. The ends of the elongated molecules frequently formed loops. The images obtained are consistent with the assumption that, under the conditions used, the majority of the talin molecules are monomeric. A minor fraction appeared as dimers, composed of two chains only partially intertwined, thus giving rise to Y-shaped particles. Electron micrographs revealed that the biochemically defined 50-kDa N-terminal talin head domain is composed of two globular subunits, while chemical cross-linking provided evidence that the C-terminal 220-kDa fragment is solely responsible for dimerization. These results imply that in the dimeric molecules, the polypeptide chains are arranged in parallel, in contrast to what has been described for human-platelet talin. In buffers of low ionic strength (0.02 M instead of 0.15 M KCl), the molecules collapsed into a compact shape. By showing the high flexibility and versatility of its morphology, our data favour the concept of talin as an important resilient link in microfilament-plasma-membrane attachment.

The mode of interaction of birch and bovine profilins with actin was compared using a number of techniques. Birch profilin was purified from pollen or as a recombinant protein from Escherichia coli, using poly(L-proline) affinity chromatography and a monoclonal antibody for the identification of the isolated product. On two-dimensional gels, the genuine and recombinant proteins were identical in molecular mass and isoelectric point and revealed that birch profilin, in contrast to the basic profilins found in mammals, is an acidic protein, analogous to maize profilins. Bovine profilin was obtained from calf thymus. In viscometric assays, the birch protein was seen to modulate actin filament formation analogous to animal profilin. Birch profilin increased the critical concentration required for muscle and brain actin polymerization in a concentration-dependent manner, supporting the notion of the formation of a heterologous complex between the plant protein and animal actin. The effect was Mg(2+)-sensitive, as had been described for homologous complexes. The dissociation constants obtained for the plant/vertebrate and the vertebrate/vertebrate system were both in the micromolar range. The affinity of birch profilin for muscle actin was slightly lower than that for nonmuscle (brain) actin. A binary complex of birch profilin and skeletal muscle actin could be isolated by gel chromatography. Cross-linking experiments with actin, birch profilin, the G-actin binding peptide thymosin beta 4 and gelsolin segment 1, the N-terminal fragment of an actin capping protein, showed that profilin competed with thymosin beta 4, but had no effect on segment 1 binding to actin. These data indicate that the actin-binding domains in plant and animal profilins are functionally highly conserved, although the overall sequence similarity is less than 25%.

The secretion of matrix-degrading proteinases and protein components involved in the production of cytotoxic metabolites is an important step in the sequence of defense reactions executed by polymorphonuclear leukocytes (PMNL) in response to stimulation. In the present report, we have analyzed degranulation of PMNL stimulated either with soluble synthetic peptides fLeu-Phe (fMet, formylmethionyl), or fAhx-Leu-Phe-Ahx-Tyr-Phe (Ahx, aminohexyl) which trigger chemotaxis and degranulation, or with opsonized zymosan which induces phagocytosis. The release of elastase, myeloperoxidase and lactoferrin-containing granules was not at all or only slightly (less than 6%) induced either by fAhx-Leu-Phe-Ahx-Tyr-Leu or by zymosan particles. In contrast, type-I collagenase and gelatinase were secreted in significant amounts after treatment with these agents. The disruption of microfilaments by cytochalasin B and subsequent stimulation of PMNL with the formyl-peptide led to the secretion of elastase, myeloperoxidase and lactoferrin, and enhanced the release of gelatinase. Disruption of microtubules by incubation with colcemid resulted in inhibition of fAhx-Leu-Phe-Ahx-Thyr-Leu and fAhx-Leu-Phe-Ahx-Tyr-Leu/cytochalasin-B-induced granule release. Furthermore, we found different patterns of enzyme distribution after fractionation by centrifugation: most (greater than 90%) type-I collagenase and gelatinase was measured in the supernatant whereas 60-90% of elastase, myeloperoxidase and lactoferrin had partitioned into the cytoskeleton-containing pellet. Our results suggest that the two main types of secretory vesicles identified in PMNL (specific and azurophilic granules) consist of subpopulations. The differential association of the various types of granules with cytoskeletal elements may serve to control their sequential discharge.

We have investigated the effect of caldesmon, a Ca2+/calmodulin-regulated actin-binding protein, on the complex between profilin and G-actin (profilactin). We found that smooth muscle caldesmon dissociates this complex rapidly and induces the polymerization of the released actin. Native profilactin (e.g. the complex isolated from calf thymus) proved more resistant to the attack of caldesmon than a heterologous complex reconstituted from calf thymus profilin and skeletal muscle actin. The mode of caldesmon-induced profilactin dissociation was similar to that described for Mg2+, and 2 mM MgCl2 potentiated the caldesmon effect. Since both caldesmon and profilin have been found enriched in ruffling membranes of animal cells, our in vitro findings may be relevant to the regulation of actin filaments in living cells.

Immunological techniques have been used to study the rate of synthesis and intracellular transport of myosin in the slime mould Physarum polycephalum. Quantitative precipitation of myosin in homogenates of Physarum was achieved using an antimyosin antibody produced in rabbit in response to purified Physarum myosin. Dodecylsulphate-gel electrophoresis revealed that about 50% of the precipitated material is myosin. The rates of synthesis of total cellular protein and myosin were measured over the mitotic cycle. Both were found to increase exponentially or linearly between two successive nuclear divisions. Similarly, no difference in the proportion of myosin-synthesising polysomes, assayed by precipitation with antimyosin serum, could be detected between the S phase and G2 phase of the mitotic cycle. Myosin makes up nearly 2% of total plasmodial proteins. Its transport into the nucleus occurs predominantly during the G2 phase.

Phosphofructokinase and gelsolin-like proteins coexist in many muscle and non-muscle tissues. They are both actin-binding proteins, and some of their biochemical parameters are remarkably similar. In a previous report [Füchtbauer, A., Jockusch, B. M., Leberer, E. & Pette, D. (1986) Proc. Natl Acad. Sci. USA 83, 9502-9506] it was shown that phosphofructokinase preparations contained actin-filament-severin activities characteristic for gelsolin. Therefore, we investigated a possible relationship between these proteins with respect to their actin-binding properties. Immunoblotting experiments with specific and non-cross-reacting antibodies to both proteins revealed two distinct polypeptides with slightly different molecular mass in SDS-PAGE of crude extracts from rabbit skeletal muscle, indicating that phosphofructokinase and gelsolin are not identical. An actin-filament-severing activity as well as the component detected by anti-gelsolin were found to copurify with phosphofructokinase during its preparation. However, the presumptive gelsolin was completely eliminated after a heat-denaturation step leaving the phosphofructokinase activity unaffected. Purified phosphofructokinase had no effects on the polymer state of preformed actin filaments. Unlike gelsolin, phosphofructokinase did not promote nucleation of actin polymerization but delayed the nucleation step. We therefore conclude that phosphofructokinase and gelsolin are functionally and structurally distinct proteins.