Abstract. Vinculin, a major structural component of vertebrate cell-cell and cell-matrix adherens junctions, has been found to interact with several other junetional components. In this report, we have identified and characterized a binding site for filamentous actin. These results included studies with gizzard vinculin, its proteolytic head and tail fragments, and recombinant proteins containing various gizzard vinculin sequences fused to the maltose binding protein (MBP) of Escherichia coli.In cosedimentation assays, only the vinculin tail sequence mediated a direct interaction with actin illaments. The binding was saturable, with a dissociation constant value in the micromolar range. Experiments with deletion clones localized the actin-binding domain to a region confined by residues 893--1016 in the 170-residue-long carboxyterminal segment, while the proline-rich hinge connecting the globular head to the rodlike tail was not required for this interaction.In fixed and permeabilized cells (cell models), as well as after microinjeetion, proteins containing the actin-binding domain specifically decorated stress fibers and the cortical network of fibroblasts and epithelial cells, as well as of brush border type microvilli. These results corroborated the sedimentation experiments.Our data support and extend previous work showing that vinculin binds directly to actin filaments. They are consistent with a model suggesting that in adhesive cells, the NH2-terminal head piece of vinculin directs this molecule to the focal contact sites, while its tail segment causes bundling of the actin filament ends into the characteristic spear tip-shaped structures. "~ 7"INCULIN is an important component of junctional complexes. Its ll6-kD polypeptide chain, folded I' into a large NH2-terminal head and an extended rodlike tail (7,8,24,25) was localized at the cytoplasmic face of both, cell-matrix and cell-cell junctions in vertebrates and invertebrates. It is indispensible for correct cell attachment and mobility, as shown by several independent lines of evidence. Focal adhesions of cultured fibroblasts are effectively disrupted upon microinjection of monoclonal antibodies directed against several epitopes in the vinculin head (34), and interference with the correct level of vinculin expression leads to drastic alterations in cellular morphology, adhesiveness and motility (10-12). Mutations in the single vinculin gene are incompatible with normal development in the nematode (2). In vitro, the protein, as isolated from chicken smooth muscle, interacts with several other junctional components, i.e., talin, ot-actinin, paxillin, tensin, and with itself, and also with acidic phospholipids (see refer- ences 14, 16, 23, 26, and 27). While talin, ~actinin and tensin are all believed to bind to act.in as well, for vinculin, the corresponding experimental data (17-19, 29, 34) have been controversely discussed (26,35,36). In particular, the purity of vinculin preparations used to show binding to filamentous actin was questioned, and contamin...

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*+-sensitive, as had been described for homologous complexes. The dissociation constants obtained for the planthertebrate and the vertebratehertebrate 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 p4 and gelsolin segment 1, the N-terminal fragment of an actin capping protein, showed that profilin competed with thymosin p4, 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%.Profilins are small (14-17 kDa), ubiquitous proteins probably involved in the regulation of actin dynamics (cf.

Human Chr 2p13-14 and homologous regions on mouse Chrs 6 and 11 have been subjects of previous studies because they comprise the loci for several neuromuscular diseases. Here we report on high-resolution mapping of 55 STS and EST loci on human Chr 2p13.3 and of 47 markers on the corresponding region on proximal mouse Chr. 11. The maps comprise several known genes, MEIS1/Meis1, RAB1a/Rab1a, MDH1/Mor2, OTX1/Otx1, and REL on human 2p13.3 and mouse Chr 11, respectively, as well as the wobbler (wr) critical region of the mouse. Whereas a perfect correspondence was found in most of the 4-Mb region, a small rearrangement was discovered around the OTX1/Otx1 locus. The detailed STS and EST transcript maps of these regions and a further narrowing down of the mouse wr critical region to the interval between D11Mit79 and D11Mit19 allow for the selection of positional candidate genes for wr, and the exclusion of others.

No abstract

Despite rapid progress in the physical characterization of murine and human genomes, little molecular information is available on certain regions, e.g., proximal mouse chromosome 11 (Chr 11) and human chromosome 2p (Chr 2p). We have localized the wobbler spinal atrophy gene wr to proximal mouse Chr 11, tightly linked to Rab1, a gene coding for a small GTP-binding protein, and Glnsps1, an intronless pseudogene of the glutamine synthetase gene. We have now used these markers to construct a 1.3-Mb yeast artificial chromosome (YAC) contig of the Rab1 region on mouse Chr 11. Four YAC clones isolated from two independent YAC libraries were characterized by rare-cutting analysis, fluorescence in situ hybridization (FISH), and sequence-tagged site (STS) isolation and mapping. Rab1 and Glns-ps1 were found to be only 200 kb apart. A potential CpG island near a methylated NarI site and a trapped exon, ETG1.1, were found between these loci, and a new STS, AHY1.1, was found over 250 kb from Rab1. Two overlapping YACs were identified that contained a 150-kb region of human Chr 2p, comprising the RAB1 locus, AHY1.1, and the human homologue of ETG1.1, indicating a high degree of conservation of this region in the two species. We mapped AHY1.1 and thus human RAB1 on Chr 2p13.4-p14 using somatic cell hybrids and a radiation hybrid panel, thus extending a known region of conserved synteny between mouse Chr 11 and human Chr 2p. Recently, the gene LMGMD2B for a human recessive neuromuscular disease, limb girdle muscular dystrophy type 2B, has been mapped to 2p13-p16. The conservation between the mouse Rab1 and human RAB1 regions will be helpful in identifying candidate genes for the wobbler spinal muscular atrophy and in clarifying a possible relationship between wr and LMGMD2B.