Elongation factor 1beta is an actin-binding protein.

A 17 kDa polypeptide found in association with actin in cellular extracts of Dictyostelium discoideum was identified as a proteolytic fragment of eEF1beta. Antibody elicited against the 17 kDa protein reacted with a single 29 kDa polypeptide in Dictyostelium, indicating that the 17 kDa peptide arises from degradation of a larger precursor. The cDNA isolated from a Dictyostelium library using this antibody as a probe encodes Dictyostelium elongation factor 1beta. Amino acid degradation of the 17 kDa protein fragment confirmed the identity of the protein as eEF1beta. Direct interaction of eEF1beta with actin in vitro was further demonstrated in mixtures of actin with the 17 kDa protein fragment of Dictyostelium eEF1beta, recombinant preparations of Dictyostelium eEF1beta expressed in Escherichia coli, and the intact eEF1betagamma complex purified from wheat germ. Localization of eEF1beta in Dictyostelium by immunofluorescence microscopy reveals both diffuse cytoplasmic staining, and some concentration in the cortical and hyaline cytoplasm. The results support the existence of physical and functional interactions of the translation apparatus with the cytoskeleton, and suggest that eEF1beta may function in a dual role both to promote the elongation phase of protein synthesis, and to interact with cytoplasmic actin.

Severe developmental defects in Dictyostelium null mutants for actin-binding proteins.

The actin cytoskeleton is implicated in many cellular processes, such as cell adhesion, locomotion, contraction and cytokinesis, which are central to any development. The extent of polymerization, cross-linking, and bundling of actin is regulated by several actin-binding proteins. Knock-out mutations in these proteins have revealed in many cases only subtle, if any, defects in development, suggesting that the actin system is redundant, with multiple proteins sharing overlapping functions. The apparent redundancy may, however, reflect limitations of available laboratory assays in assessing the developmental role of a given protein. By using a novel assay, which reproduces conditions closer to the natural ones, we have re-examined the effects of disruption of many actin-binding proteins, and show here that deletion of alpha-actinin, interaptin, synexin, 34-kDa actin-bundling protein, and gelation factor affect to varying degrees the efficiency of Dictyostelium cells to complete development and form viable spores. No phenotypic defects were found in hisactophilin or comitin null mutants.

Evidence of intramolecular regulation of the Dictyostelium discoideum 34 000 Da F-actin-bundling protein.

Intramolecular interaction within the Ca(2+)-regulated 34 kDa actin-bundling protein from Dictyostelium discoideum was found to contribute to the regulation of its actin-binding activity. Recombinant N-terminally truncated proteins aa77-295, 124-295, and 139-295 bound actin at > or = 2:1 stoichiometry, which is 5-fold greater than the intact protein aa1-295 as assessed by cosedimentation with F-actin. These proteins also have enhanced cross-linking activity as assessed by viscometry and electron microscopy. All truncated 34 kDa proteins failed to bind (45)Ca(2+) on blots and displayed Ca(2+)-insensitive binding with actin, although most proteins possessed intact putative EF-hand Ca(2+)-binding motifs. An intramolecular interaction within the 34 kDa protein was inferred from direct demonstrations of domain-domain interaction among the truncated 34 kDa proteins both in the presence and absence of actin. The intramolecular interaction between interaction zone 1 (aa71-123) and interaction zone 2 (aa193-254) is proposed to maintain the N-terminal inhibitory region (aa1-76) in close proximity with the strong actin-binding site (aa193-254) in order to modulate the interaction of the intact protein with actin filaments.

Three actin cross-linking proteins, the 34 kDa actin-bundling protein, alpha-actinin and gelation factor (ABP-120), have both unique and redundant roles in the growth and development of Dictyostelium.

The contribution of three actin cross-linking proteins, alpha-actinin (alphaA), gelation factor (ABP-120), and the 34 kDa actin-bundling protein to cellular functions has been studied in three single mutant (alphaA-, 120-, and 34-) and three double mutant (alphaA-/120-, 34-/alphaA-, 34-/120-) strains of Dictyostelium generated by homologous recombination. Strains alphaA-/120- and 34-/alphaA- exhibited a reduced rate of pinocytosis, grew to lower saturation densities, and produced small cells in shaking cultures. All strains grew normally in bacterial suspensions and on agar plates with a bacterial lawn. Slow growth under conditions of reduced temperature and increased osmolarity was observed in single mutants 34- and alphaA-, respectively, as well as in some of the double mutant strains. Motility, chemotaxis, and development were largely unaltered in 34-/alphaA- and 34-/120- cells. However, 34-/alphaA- cells showed enhanced aggregation when starved in suspension. Moreover, morphogenesis was impaired in both double mutant strains and fruiting bodies of aberrant morphology were observed. These defects were reverted by re-expression of one of the lacking cross-linking proteins. The additive and synthetic phenotypes of these mutations indicate that actin cross-linking proteins serve both unique and overlapping functions in the actin cytoskeleton.

Three distinct F-actin binding sites in the Dictyostelium discoideum 34,000 dalton actin bundling protein.

The Dictyostelium 34 kDa protein is an actin bundling protein composed of 295 amino acids. However, the region(s) of the molecule that bind actin filaments is (are) unknown. Studies of the cosedimentation of 125I-34 kDa protein and F-actin show that the 34 kDa protein binds to F-actin with positive cooperativity and Hill coefficients of 1.9 and 3.0, for filaments 4.9 microm and 0.6 microm, respectively. The Hill coefficient is larger for short filaments that are more efficiently bundled than long filaments, suggesting that one of the binding sites is used in interfilament contacts or contributes to filament orientation within the bundle. Three distinct actin binding sites were identified using a synthetic peptide, protein truncations, and a novel epitope library screening method. The ability to bind actin was assessed by 125I-F-actin overlays under denaturing and nondenaturing conditions, cosedimentation, viscometry, and pyrene-labeled actin disassembly. The three actin binding domains were identified as amino acids 1-123, 193-254, and 279-295. The 62 amino acid domain (193-254) can cosediment with F-actin. The estimated Kapp obtained by the disassembly of pyrene-labeled actin was 0.11 microM and 2.7 microM for the amino acids 1-123 and 279-295, respectively. These results identify three distinct regions of the 34 kDa protein that may contribute to the positive cooperative formation of F-actin bundles.

Fibrillarin, a conserved pre-ribosomal RNA processing protein of Giardia.

The flagellated protozoan Giardia has been shown by 16S rRNA sequence analysis to be one of the most primitive of the eukaryotes. A gene encoding the protein fibrillarin, a pre-rRNA processing protein implicated in rRNA methylation and ribosome assembly, has been isolated. A genomic DNA fragment 1,240 base pairs long containing an open reading frame of 981 base pairs (327 amino acids) was sequenced. The deduced protein sequence of 35.3 kDa is similar to other known fibrillarin sequences. The Giardia sequence includes the amino terminal glycine/arginine rich domain characteristic of eukaryotic fibrillarins but is unique in having a large number of acidic residues in this domain. Phylogenetic analysis of the available fibrillarin sequences is consistent with the assignment of Giardia to a position close to the most primitive of the eukaryotes. A monoclonal antibody to yeast fibrillarin crossreacts with a 36 kDa polypeptide from Giardia on western blots and diffusely stains both nuclei of the organism by immunofluorescence microscopy. This result is consistent with the absence of well defined nucleoli in this organism. The evolutionary conservation of fibrillarin suggests an important function for this protein in ribosome biosynthesis, and this function appears to be maintained from the archaebacteria, which lack a nucleus, to Giardia, which contains a nucleus but lacks a prominent nucleolus, to higher mammals, which have both nucleus and nucleolus.

Overexpression, purification, and characterization of recombinant Dictyostelium discoideum calcium-regulated 34,000-dalton F-actin bundling protein from Escherichia coli.

The Dictyostelium discoideum 34-kDa protein is an F-actin bundling protein that demonstrates diverse distributions in the cell during cell shape changes and cell movement. The protein is expressed at a very low level in the amoeba, just 0.4% of the total cell protein. This presents a challenging problem when purifying sufficient protein for structural and biochemical studies. The purification procedure is lengthy and yields only a few milligrams of protein. An alternative protein expression system, that of the bacterial T7 expression system, was used to produce large quantities of recombinant 34-kDa protein (r34-kDa). The soluble r34-kDa protein constitutes up to a quarter of the total bacterial protein, and was purified to homogeneity by a modification of the purification procedure for the native D. discoideum 34-kDa protein (N34-kDa). The r34-kDa possesses all the same functional characteristics as the N34-kDa protein with respect to its interactions with F-actin in vitro: it bound to and cross-linked F-actin, mediated F-actin bundle formation, directly bound calcium, and demonstrated calcium-sensitive F-actin binding activities.

The structure, function, and assembly of actin filament bundles.

The cellular organization, function, and molecular composition of selected biological systems with prominent actin filament bundles are reviewed. An overall picture of the great variety of functions served by actin bundles emerges from this overview. A unifying theme is that the actin cross-linking proteins are conserved throughout the eukaryotic kingdom and yet assembled in a variety of combinations to produce actin bundles of differing functions. Mechanisms of actin bundle formation in vitro are considered illustrating the variety of physical and chemical driving forces in this exceedingly complex process. Our limited knowledge regarding the formation of actin filament bundles in vivo is contrasted with the elegant biophysical studies performed in vitro but nonetheless reveals that interactions with membranes, nucleation sites, and other organizational components must contribute to formation of actin bundles in vivo.

Actin-binding membrane proteins identified by F-actin blot overlays.

Actin and associated proteins at the cytoskeleton-plasma membrane interface stabilize the membrane bilayer, control cell shape, and delimit specialized membrane domains. To identify membrane proteins that bind directly to F-actin, we have developed a blot overlay assay with 125I-labeled F-actin. In the soil amoebae, Dictyostelium discoideum, the major proteins reactive in this assay are p30a, a 34-kD peripheral membrane protein that is concentrated in filopodia and at sites of cell-cell adhesion, and ponticulin, a 17-kD transmembrane glycoprotein required for efficient chemotaxis and for control of pseudopod dynamics. Proteins with apparent molecular masses of approximately 34- and approximately 17-kD also are observed on F-actin blot overlays of many mammalian cell lines. However, in mammalian cells, the most prominent F-actin binding proteins in this assay exhibit apparent molecular masses of 78-, 80-, 81-, approximately 120-, and 205-kD. Bovine neutrophils contain the 78-, 81-, and 205-kD proteins, all of which co-isolate with a plasma membrane-enriched fraction. We have previously identified the 78-, 80-, and 81-kD proteins as moesin, radixin, and ezrin, respectively. These proteins, which are members of the protein 4.1 superfamily, colocalize with actin in cell surface extensions and have been implicated in the protrusion of microvilli, filopodia, and membrane ruffles. The 205-kD protein (p205) appears to be absent from current databases, and its characteristics are still under investigation. We here report that the 120-kD protein is drebrin, a submembranous actin-binding protein originally identified as a developmentally regulated brain protein. Thus, it appears that F-actin blot overlays provide an efficient assay for simultaneous monitoring of a subset of F-actin binding proteins, including p30a, ponticulin, moesin, radixin, ezrin, p205, and drebrin.

Photosensory and thermosensory responses in Dictyostelium slugs are specifically impaired by absence of the F-actin cross-linking gelation factor (ABP-120).

Chemotactic aggregation of starving amoebae of Dictyostelium discoideum leads to formation of a motile, multicellular organism - the slug - whose anterior tip controls its phototactic and thermotactic behaviour. To determine whether proteins that regulate the in vitro assembly of actin are involved in these responses, we tested phototaxis and thermotaxis in mutant slugs in which the gene encoding one of five actin-binding proteins had been disrupted. Of the proteins tested - severin, alpha-actinin, fimbrin, the 34 kD actin-bundling protein and the F-actin cross-linking gelation factor (ABP-120) - only ABP-120 proved essential for normal phototaxis and thermotaxis in the multicellular slugs. The related human protein ABP-280 is required for protein phosphorylation cascades initiated by lysophosphatidic acid and tumor necrosis factor alpha. The repeating segments constituting the rod domains of ABP-120 and ABP-280 may be crucial for the function of both proteins in specific signal transduction pathways by mediating interactions with regulatory proteins.

Dictyostelium discoideum cells lacking the 34,000-dalton actin-binding protein can grow, locomote, and develop, but exhibit defects in regulation of cell structure and movement: a case of partial redundancy.

Cells lacking the Dictyostelium 34,000-D actin-bundling protein, a calcium-regulated actin cross-linking protein, were created to probe the function of this polypeptide in living cells. Gene replacement vectors were constructed by inserting either the UMP synthase or hygromycin resistance cassette into cloned 4-kb genomic DNA containing sequences encoding the 34-kD protein. After transformation and growth under appropriate selection, cells lacking the protein were analyzed by PCR analyses on genomic DNA, Northern blotting, and Western blotting. Cells lacking the 34-kD protein were obtained in strains derived from AX2 and AX3. Growth, pinocytosis, morphogenesis, and expression of developmentally regulated genes is normal in cells lacking the 34-kD protein. In chemotaxis studies, 34-kD- cells were able to locomote and orient normally, but showed an increased persistence of motility. The 34-kD- cells also lost bits of cytoplasm during locomotion. The 34-kD- cells exhibited either an excessive number of long and branched filopodia, or a decrease in filopodial length and an increase in the total number of filopodia per cell depending on the strain. Reexpression of the 34-kD protein in the AX2-derived strain led to a "rescue" of the defect in the persistence of motility and of the excess numbers of long and branched filopodia, demonstrating that these defects result from the absence of the 34-kD protein. We explain the results through a model of partial functional redundancy. Numerous other actin cross-linking proteins in Dictyostelium may be able to substitute for some functions of the 34-kD protein in the 34-kD cells. The observed phenotype is presumed to result from functions that cannot be adequately supplanted by a substitution of another actin cross-linking protein. We conclude that the 34-kD actin-bundling protein is not essential for growth, but plays an important role in dynamic control of cell shape and cytoplasmic structure.

Role of the Dictyostelium 30 kDa protein in actin bundle formation.

We have studied the formation of bundles in mixtures of actin with the Dictyostelium 30 kDa actin-bundling protein as a function of 30 kDa protein concentration, actin concentration, and filament length. The presence of the 30 kDa protein promotes formation of filament bundles at actin concentrations and filament lengths that are not spontaneously aligned into liquid crystalline domains in the absence of the 30 kDa protein. Bundle formation in the presence of the 30 kDa protein was observed over a broad range of actin filament lengths and concentrations. Bundling was filament length dependent, and short filaments were more efficiently bundled. Bundles formed at actin concentrations as low as 2 microM. The volume fraction of the bundled portion and concentrations of actin and the 30 kDa protein in the bundled portion were measured using a sedimentation assay. Bundles have concentrations of actin and 30 kDa protein that are 10-20 and 5-20 times, respectively, greater than that of the bulk solution. Computer modeling reveals that bundling of actin by a bundling protein increases both the mean length and the polydispersity of the length distribution, factors which lower the actin concentration required for spontaneous alignment within the bundle. We propose that entropy-driven spontaneous ordering may contribute to bundle formation in two ways. Bundling of actin creates longer aggregates with a more polydisperse length distribution in which actin aligns spontaneously within the bundle at very low concentrations. In addition, bundling creates locally high concentrations of actin within these aggregates that will spontaneously align, providing an additional driving force for bundle ordering.

Association of the Dictyostelium 30 kDa actin bundling protein with contact regions.

'Contact regions' are plasma membrane domains derived from areas of intercellular contact between aggregating Dictyostelium amebae (H.M. Ingalls et al. (1986). Proc. Nat. Acad. Sci. USA 83, 4779). Purified contact regions contain a prominent actin-binding protein with an M(r) of 34,000. Immunoblotting with monoclonal antibodies identifies this polypeptide as a 34,000 M(r) actin-bundling protein (known as 30 kDa protein), previously shown to be enriched in filopodia (M. Fechheimer (1987). J. Cell Biol. 104, 1539). About four times more 30 kDa protein by mass is associated with contact regions than is found in total plasma membranes isolated from aggregating cells. In agreement with these observations, immunostaining of the 30 kDa protein in aggregating cells reveals a prominent localization along the plasma membrane at sites of intercellular contact. By contrast, alpha-actinin does not appear to be significantly enriched at sites of cell to cell contact. Binding experiments using purified plasma membranes, actin and 30 kDa protein indicate that the 30 kDa protein is associated with the plasma membrane primarily through interactions with actin filaments. Calcium ions are known to decrease the interaction of actin with 30 kDa protein in solution. Surprisingly, membrane-associated complexes of actin and the 30 kDa protein are much less sensitive to dissociation by micromolar levels of free calcium ions than are complexes in solutions lacking membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence and localization of vinculin in Giardia.

A requisite element of pathogenicity in Giardia infections is the parasites' ability to adhere to the intestinal epithelial brush border. The presence of vinculin in Giardia was studied because this protein is known to link the cytoskeleton to the plasma membrane and is localized at adhesion foci in many cell-cell and cell-substrate contact sites. Actin, alpha-actinin, and vinculin were identified in Giardia by western blot analysis. Giardia trophozoites attached to glass substrates were examined by interference reflection microscopy (IRM) and immunofluorescence. The IRM defined the lateral crest, bare area, and overlap region of the ventral disk, as well as the ventrolateral flange and lateral shields as close contact areas between parasite and substrate. These close contact regions were then correlated with immunofluorescence localization of actin, alpha-actinin, and vinculin. Actin was seen in the lateral crest, while alpha-actinin was observed in the ventral disc periphery and lateral shields. Vinculin was viewed at the bare and overlap areas of the ventral disc and portions of the lateral crest, as well as the ventrolateral flange and lateral shields. The correspondence of close contact sites with vinculin localization suggests a role for vinculin in Giardia attachment and adherence.

Differential localization of alpha-actinin and the 30 kD actin-bundling protein in the cleavage furrow, phagocytic cup, and contractile vacuole of Dictyostelium discoideum.

Dictyostelium discoideum amoebae possess eight different actin crosslinking proteins. Immunofluorescence microscopy has been employed in this study to investigate the intracellular localization of two of these proteins, alpha-actinin and the 30 kD actin-bundling protein, to investigate whether they are redundant, or alternatively, make distinct contributions to cell structure and movement. The 30 kD protein is concentrated in the cleavage furrow of dividing cells, while enhanced staining for alpha-actinin is not apparent in this region. By contrast, alpha-actinin is concentrated around the contractile vacuole, while the 30 kD protein is not preferentially localized in the area of this organelle. Association of alpha-actinin with the contractile vacuole was confirmed by colocalization with calmodulin, a marker of this organelle. There are temporal differences in the localization of the 30 kD protein and alpha-actinin during phagocytosis. The 30 kD protein is localized in the phagocytic cup, but disassociates from phagosomes soon after internalization [Furukawa et al., 1992: Protoplasma 169: 18-27]. alpha-actinin enters the phagocytic cup after the 30 kD protein, and remains associated with the phagosome after the 30 kD protein has disassociated. These results support the hypothesis that alpha-actinin and the 30 kD protein play distinct roles in cell structure and movement in Dictyostelium.

Formation of liquid crystals from actin filaments.

Actin is cross-linked by actin-binding proteins in the cytoplasm to form either isotropic or highly oriented anisotropic structures. The inherent orientation among actin filaments could influence whether an isotropic or highly oriented anisotropic structure is formed. A highly oriented state can arise spontaneously through the formation of liquid crystals as predicted by polymer theory. In this study, the ability of filamentous actin to form liquid crystalline domains was detected using the anisotropic component of scattered light and by observation of birefringence. As liquid crystalline domains formed, the intensity of the anisotropic component of scattered light increased, and birefringent macroscopic oriented domains were directly observed. The formation of liquid crystalline domains was dependent on the concentration of actin filaments and on the average filament length controlled by varying the ratio of gelsolin to actin monomers. The concentration of actin filaments required to form liquid crystalline domains increased moderately as the average length was decreased. At a fixed actin concentration, orientation among the filaments attained a maximum value at a ratio of actin to gelsolin in the range from 1500 to 2000 and decreased as the ratio was increased or decreased from this range. The results are not well explained by theoretical treatments for liquid crystal formation by monodisperse, charged worm-like chains. Differences from the theoretical predictions for formation of liquid crystals are most likely due to the polydisperse filament length of actin. This phenomenon may have important effects on the structural and rheological properties of the cytoplasm in living cells.

A 27,000-D core of the Dictyostelium 34,000-D protein retains Ca(2+)-regulated actin cross-linking but lacks bundling activity.

Actin cross-linking proteins are important for formation of isotropic F-actin networks and anisotropic bundles of filaments in the cytoplasm of eucaryotic cells. A 34,000-D protein from the cellular slime mold Dictyostelium discoideum mediates formation of actin bundles in vitro, and is specifically incorporated into filopodia. The actin cross-linking activity of this protein is inhibited by the presence of micromolar calcium. A 27,000-D fragment obtained by digestion with alpha-chymotrypsin lacks the amino-terminal six amino acids and the carboxyl-terminal 7,000 D of the intact polypeptide. The 27,000-D fragment retains F-actin binding activity assessed by cosedimentation assays and by 125I-[F-actin] blot overlay technique, F-actin cross-linking activity as assessed by viscometry, and calcium binding activity. Ultrastructural analyses indicate that the 27,000-D fragment is deficient in the bundling activity characteristic of the intact 34,000-D protein. Actin filaments are aggregated into microdomains but not bundle in the presence of the 27,000-D fragment. A polarized light scattering assay was used to demonstrate that the 34,000-D protein increases the orientational correlation among F-actin filaments. The 27,000-D fragment does not increase the orientation of the actin filaments as assessed by this technique. A terminal segment(s) of the 34,000-D protein, lacking in the 27,000-D fragment, contributes significantly to the ability to cross-link actin filaments into bundles.

Inhibition of actin filament depolymerization by the Dictyostelium 30,000-D actin-bundling protein.

We have studied the effect of the Dictyostelium discoideum 30,000-D actin-bundling protein on the assembly and disassembly of pyrenyl-labeled actin in vitro. The results indicate that the protein is a potent inhibitor of the rate of actin depolymerization. The inhibition is rapid, dose dependent, and is observed at both ends of the filament. There is little effect of 30-kD protein on the initial rate of elongation from F-actin seeds or on the spontaneous nucleation of actin polymerization. We could detect little or no effect on the critical concentration. The novel feature of these results is that the filament ends are free for assembly but are significantly impaired in disassembly with little change in the critical concentration at steady state. The effects appear to be largely independent of the cross-linking of actin filaments by the 30-kD protein. Actin cross-linking proteins may not only cross-link actin filaments, but may also differentially protect filaments in cells from disassembly and promote the formation of localized filament arrays with enhanced stability.

Mechanisms responsible for F-actin stabilization after lysis of polymorphonuclear leukocytes.

While actin polymerization and depolymerization are both essential for cell movement, few studies have focused on actin depolymerization. In vivo, depolymerization can occur exceedingly rapidly and in a spatially defined manner: the F-actin in the lamellipodia depolymerizes in 30 s after chemoattractant removal (Cassimeris, L., H. McNeill, and S. H. Zigmond. 1990. J. Cell Biol. 110:1067-1075). To begin to understand the regulation of F-actin depolymerization, we have examined F-actin depolymerization in lysates of polymorphonuclear leukocytes (PMNs). Surprisingly, much of the cell F-actin, measured with a TRITC-phalloidin-binding assay, was stable after lysis in a physiological salt buffer (0.15 M KCl): approximately 50% of the F-actin did not depolymerize even after 18 h. This stable F-actin included lamellar F-actin which could still be visualized one hour after lysis by staining with TRITC-phalloidin and by EM. We investigated the basis for this stability. In lysates with cell concentrations greater than 10(7) cells/ml, sufficient globular actin (G-actin) was present to result in a net increase in F-actin. However, the F-actin stability was not solely because of the presence of free G-actin since addition of DNase I to the lysate did not increase the F-actin loss. Nor did it appear to be because of barbed end capping factors since cell lysates provided sites for barbed end polymerization of exogenous added actin. The stable F-actin existed in a macromolecular complex that pelleted at low gravitational forces. Increasing the salt concentration of the lysis buffer decreased the amount of F-actin that pelleted at low gravitational forces and increased the amount of F-actin that depolymerized. Various actin-binding and cross-linking proteins such as tropomyosin, alpha-actinin, and actin-binding protein pelleted with the stable F-actin. In addition, we found that alpha-actinin, a filament cross-linking protein, inhibited the rate of pyrenyl F-actin depolymerization. These results suggested that actin cross-linking proteins may contribute to the stability of cellular actin after lysis. The activity of crosslinkers may be regulated in vivo to allow rapid turnover of lamellipodia F-actin.