This article outlines the present knowledge of the architecture, molecular composition, and dynamics of focal contacts of adhesive animal cells. These structures, developed at the plasma membrane at sites where cells touch their substratum, are essential for cellular attachment in tissue formation during embryogenesis and wound healing. In tissue culture, they are particularly prominent and thus amenable to detailed investigation. Focal contacts consist of a cytoplasmic face, comprising cytoskeletal elements, a transmembrane connecting region, and a extracellular face composed of proteins of the extracellular matrix. The molecular anatomy of the numerous proteins involved, the basis for classifying them as structural or regulatory components, and their in vitro interactions are described. Based on this information, current models on the dynamics of their assembly and of possible regulatory mechanisms involving a variety of signal transduction pathways are discussed.

In epithelial cells, α-, β-, and γ-catenin are involved in linking the peripheral microfilament belt to the transmembrane protein E-cadherin. α-Catenin exhibits sequence homologies over three regions to vinculin, another adherens junction protein. While vinculin is found in cell–matrix and cell–cell contacts, α-catenin is restricted to the latter. To elucidate, whether vinculin is part of the cell–cell junctional complex, we investigated complex formation and intracellular targeting of vinculin and α-catenin. We show that α-catenin colocalizes at cell–cell contacts with endogenous vinculin and also with the transfected vinculin head domain forming immunoprecipitable complexes. In vitro, the vinculin NH2-terminal head binds to α-catenin, as seen by immunoprecipitation, dot overlay, cosedimentation, and surface plasmon resonance measurements. The K d of the complex was determined to 2–4 × 10−7 M. As seen by overlays and affinity mass spectrometry, the COOH-terminal region of α-catenin is involved in this interaction.Complex formation of vinculin and α-catenin was challenged in transfected cells. In PtK2 cells, intact α-catenin and α-catenin1-670, harboring the β-catenin– binding site, were directed to cell–cell contacts. In contrast, α-catenin697–906 fragments were recruited to cell–cell contacts, focal adhesions, and stress fibers. Our results imply that in vivo α-catenin, like vinculin, is tightly regulated in its ligand binding activity.

. Yeast mutants of cell cycle gene cdc481 arrest as large budded cells with microtubules spreading aberrantly throughout the cytoplasm from a single spindle plaque . The gene was cloned and disruption proved it to be essential . The CDC48 sequence encodes a protein of 92 kD that has an internal duplication of 200 amino acids and includes a nucleotide binding consensus sequence. Vertebrate VCP has a 70% identity over the entire length of the protein . Yeast Secl8p and mammalian N-ethylmaleimide-sensitive fusion protein, which are involved in intracellular transport, yeast Paslp,

By screening a yeast two-hybrid library with COOH-terminal fragments of vinculin/metavinculin as the bait, we identified a new protein termed raver1. Raver1 is an 80-kD multidomain protein and widely expressed but to varying amounts in different cell lines. In situ and in vitro, raver1 forms complexes with the microfilament-associated proteins vinculin, metavinculin, and α-actinin and colocalizes with vinculin/metavinculin and α-actinin at microfilament attachment sites, such as cell–cell and cell matrix contacts of epithelial cells and fibroblasts, respectively, and in costameres of skeletal muscle. The NH2-terminal part of raver1 contains three RNA recognition motifs with homology to members of the heterogeneous nuclear RNP (hnRNP) family. Raver1 colocalizes with polypyrimidine tract binding protein (PTB)/hnRNPI, a protein involved in RNA splicing of microfilament proteins, in the perinucleolar compartment and forms complexes with PTB/hnRNPI. Hence, raver1 is a dual compartment protein, which is consistent with the presence of nuclear location signal and nuclear export sequence motifs in its sequence. During muscle differentiation, raver1 migrates from the nucleus to the costamere. We propose that raver1 may coordinate RNA processing and targeting as required for microfilament anchoring in specific adhesion sites.

Focal contact assembly involves interaction between VASP and vinculin, which is enhanced by PIP2-induced vinculin activation and oligomerization. Given that vinculin and VASP both bind to F-actin, vinculin-VASP complexes might bundle the distal ends of actin filaments in focal contacts. We propose that PIP2-dependent signalling modulates microfilament organization at cellular adhesion sites by regulating vinculin-VASP complexes.

VASP (vasodilator-stimulated pbospboprotein), a protein associated with microfilaments at cellular contact sites, has been identified as a ligand for proftin and zyxin, two proteins also involved in microfilament dynamics and organization at these regions. Here, we report that VASP also directly binds to vlnculin, another component of adherens junctions. Competition experiments with a vinculin-derived peptide showed that a proline-rich motif, located in the hinge region that connects vinculin's head and tail domains, is involved in VASP binding. The same motif is present in zyxin but the interactions of VASP with vinculln and zyxin differ in detail. Hence, this motif may be recognized by VASP in different ways when presented in distinct cellular sites.

Protein sequencing shows that porcine brain tubulin retains the N‐terminal sequences of α and β tubulin after a mild treatment with subtilisin, C‐terminal peptides released by subtilisin were purified and characterized by automated Edman degradation and mass spectrometry. We confirm the polyglutamylation of α tubulin on glutamic acid residue 445 reported by others and show in addition that class 11 β tubulin, the major β tubulin isotype of adult brain, is also polyglutamylated. The substitution is restricted to glutamic acid residue 435. Thus all major tubulin isotypes of adult brain are subjected to polyglutamylation.

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 pointlike structure characteristic for cell-matrix contacts.