Structures linking microfilament bundles to the membrane at focal contacts

Abstract

Abstract. We used quick-freeze, deep-etch, rotary replication and immunogold cytochemistry to identify a new structure at focal contacts. In Xenopus fibroblasts, elongated aggregates of particles project from the membrane to contact bundles of actin microfilaments. Before terminating, a single bundle of microfilaments interacts with several aggregates that appear intermittently over a distance of several microns. Aggregates are enriched in proteins believed to mediate actin-membrane interactions at focal contacts, including B~-integrin, vinculin, and talin, but they appear to contain less t~-actinin and filamin. We also identified a second, smaller class of aggregates of membrane particles that contained/~-integrin but not vinculin or talin and that were not associated with actin microfilaments. Our results indicate that vinculin, talin, and ~-integrin are assembled into distinctive structures that mediate multiple lateral interactions between microfilaments and the membrane at focal contacts.F OCAL contacts are specialized structures where actin filaments converge and terminate at the plasma membrane (16,32,44,63,65). Interactions between the membrane and microfilaments involve several proteins that are concentrated at focal contacts, including integrin (e.g., references 17, 21, 22; reviewed in 1, 61) and cytoskeletal proteins such as vinculin (26), talin (13), fimbrin (9), tensin (71), paxillin (68), zyxin (5), and ot-actinin (40). Biochemical studies have suggested several ways in which these proteins could anchor microfilaments to the cytoplasmic surface of the plasma membrane at focal contacts. Early studies suggested that particular isoforms of integrin ("CSAT'; 47) bound talin, and that this complex then bound vinculin (35; reviewed in 15). Later studies showed that integrins could also bind tx-actinin (53). Thus, integrin could anchor actin filaments indirectly through the actin-binding protein, tx-actinin, as well as through talin and vinculin. If both of these mechanisms are important for actin-membrane interactions, one would expect all these proteins to be concentrated at focal contacts.Structural studies also suggest two models for microfilament-membrane interactions at focal contacts. Focal contacts are several microns long, and individual actin filaments course over this entire distance before terminating (8). These filaments could bind to the membrane laterally as they approach their termini. In this case, vinculin, talin, integrin, and other proteins of focal contacts would be expected to concentrate at many points along the filament length. Alter- natively, filaments could attach only at their termini. In this case, the proteins of focal contacts would be expected to occur only at the ends of microfilaments.Predictions of the relationship between actin microfilaments and the membrane, and of the distribution of proteins at focal contacts, can be tested by ultrastructural methods coupled with immunocytochemistry. We exposed the cytoplasmic surfaces of ventral membrane by mechanical shearing, the...

Analyzing focal adhesion structure by atomic force microscopy

“…Double immunogold labeling performed on ultrathin frozen sections from chick heart fibroblasts showed that vinculin is situated closer to the membrane than ␣-actinin in FAs (Chen and Singer, 1982) whereas in wet-cleaved chicken embryo fibroblasts, both talin and vinculin are present at high concentrations in a dense network close to the plasma membrane (Feltkamp et al, 1991). Rotary replication in combination with immunogold labeling of sheared Xenopus fibroblasts demonstrated that FA microfilaments are highly bundled and these bundles appear to be linked to the plasma membrane by laterally attached discrete protein aggregates containing vinculin, talin and ␤1-integrin (Samuelsson et al, 1993). In agreement, ␤1-integrin was found to localize in linearly arranged discrete clusters by immuno-EM experiments performed on wet-cleaved fibroblasts (Meijne et al, 1994).…”

“…pictures of rotary replicas show tight bundling of microfilaments within the FA plaque (Samuelsson et al, 1993). Thus, in FAs, microfilaments may be grouped into discrete functional units.…”

“…3H), which could represent integrin-containing aggregates observed by immuno-EM (Samuelsson, 1993), although no regularity in the spacing of these globular structures was found. The abundance of these globular structures varied (compare Fig.…”

Organization of point contacts in neuronal growth cones

“…Talin is a 270-kDa cytoskeletal protein that interacts with integrins (Luna and Hitt, 1992;Samuelsson et al, 1993). In the primary DRG cultures, talin was distributed in punctate clusters that were often more prominent in the growth cone central domain, and some immunoreactivity extended into filopodia (Fig.…”

Integrity of the network sarcoplasmic reticulum in skeletal muscle requires small ankyrin 1

“…Primary cultures of cultured FDB fibers treated with con-siRNA and sAnk1-siRNA were fixed overnight in 2% glutaraldehyde, 5 mg/ml tannic acid in 0.2 M cacodylate buffer, pH 7.2, and then stained further with uranyl acetate, embedded and processed for thin-section electron microscopy, as described (Lovering et al, 2011), with the exception that, after embedding, myofibers were separated from the glass coverslips with hydrofluoric acid (Pumplin et al, 1993). Myofibers were sampled randomly and representative images are shown.…”