Expression of the EspB protein of enteropathogenic Escherichia coli within HeLa cells affects stress fibers and cellular morphology.

The EspB protein of enteropathogenic Escherichia coli (EPEC) is essential for the signaling events that lead to the accumulation of actin beneath intimately attached bacteria, a process that is known as the attaching and effacing effect. EspB is targeted to the host cell cytoplasm by a type III secretion apparatus. To determine the effect of intracellular EspB on the host cell cytoskeleton, we transfected HeLa cells with a plasmid containing the espB gene under the control of an inducible eukaryotic promoter. A HeLa cell clone that expressed espB mRNA and EspB protein after induction was selected for further study. The expression of EspB in these cells caused a dramatic change in cell morphology and a marked reduction in actin stress fibers. Cells expressing EspB were significantly impaired in their ability to support invasion by EPEC and Salmonella typhimurium. However, the expression of EspB within host cells could not compensate for the lack of EspB expression by an espB mutant strain of EPEC to restore attaching and effacing activity. These studies suggest that EspB is a cytoskeletal toxin that is translocated to the host cell cytoplasm, where it causes a redistribution of actin.

The EspB protein of enteropathogenic Escherichia coli is targeted to the cytoplasm of infected HeLa cells.

The EspB protein of enteropathogenic Escherichia coli (EPEC) is exported via a type III secretion apparatus. EspB is critical for signaling the host cell and for the development of the attaching and effacing lesion characteristic of EPEC infection. We used cellular fractionation and confocal laser scanning microscopy to determine the cellular location of EspB during infection of HeLa cells. Both methods indicated that EspB is targeted to the cytoplasm of infected cells. Using mutants, we found that EspB targeting to the host cell cytoplasm requires the type III secretion apparatus and the secreted proteins EspA and EspD, but not intimin. These results provide insights into the function of the type III secretion apparatus of EPEC and the functions of the Esp proteins.

Intercellular Ca2+ waves in rat heart muscle.

1. Confocal laser scanning microscopy was used to visualize intercellular transmission of Ca2+ waves in intact rat ventricular trabeculae micro-injected with the calcium indicator fluo-3. 2. Ca2+ waves usually failed to be transmitted from cell to cell. At identified individual end-to-end cell contacts, successful transmission interspersed with failure, which sometimes occurred despite an apparent small spritz of Ca2+ between cells. The probability of cell to cell transmission (Ptran) was 0.13. 3. Ca2+ waves arose preferentially near junctions of connected cells, where connexin-43 was found, but randomly in enzymatically disconnected heart cells. 4. beta-Adrenergic stimulation significantly increased Ptran (to 0.22) and heptanol, an uncoupler of gap junction channels, significantly decreased it (to 0.045). 5. In regions of high [Ca2+]i due to damage, wave frequency decreased markedly with each cell-cell junction passed. 6. The Ca2+ permeability of cardiac gap junctions may be regulated, and the low ability of cardiac gap junctions to transmit Ca2+ may help control the spread of Ca2+ from damaged regions.

Cytoskeleton-membrane interactions at the postsynaptic density of Xenopus neuromuscular junctions.

Acetylcholine receptors of mature muscle fibres are concentrated in the postsynaptic membrane by mechanisms that are not yet understood. As one possibility, receptors might be anchored to cytoskeletal elements in the postsynaptic density that is located beneath the membrane where receptors are concentrated. To address this possibility, we examined the cytoskeleton at the postsynaptic density and determined the organization of cytoskeletal filaments relative to clustered acetylcholine receptors (AChR). Xenopus nerve-muscle co-cultures were sheared to expose the cytoplasmic membrane surface, then quick-frozen, deep-etched, and rotary-replicated. Areas with a high concentration of AChR had aggregates of particles protruding from the cytoplasmic surface of the membrane, with actin microfilaments attached to and cross-linking the aggregates. Microfilaments contacted only a few of the particles in an aggregate. These findings suggest that short-range interactions may bind individual AChR into small aggregates, while microfilaments tie these aggregates together at the nerve-muscle junction.

Microfilament-membrane interactions in Xenopus myocytes.

We used quick-freeze, deep-etch, rotary-replication transmission electron microscopy to determine at molecular resolution the organization of microfilaments at the cytoplasmic surface of the sarcolemma of Xenopus myocytes. We demonstrate that actin microfilaments interact with the sarcolemma in two distinct ways. In one, which resembled focal contacts in Xenopus fibroblasts [Samuelsson et al., 1993: J. Cell Biol. 122:485-496], bundles of microfilaments approached the sarcolemma at sites containing aggregates of membrane-associated particles. Immunogold cytochemistry showed that these particle aggregates contained vinculin, talin and beta 1-integrin. In the second, which covered most of the cytoplasmic surface of the sarcolemma, individual actin microfilaments formed an extensive, lattice-like array. Particle aggregates associated with this array of actin microfilaments also labeled with antibodies to vinculin, talin and beta 1-integrin. The unique, lattice-like association of actin microfilaments with the membrane in Xenopus myocytes suggests that the organization of actin filaments over most of the sarcolemma is distinct from focal contacts, mediating widespread associations of the actin cytoskeleton with the cytoplasmic membrane face.

Association of acetylcholine receptors with peripheral membrane proteins: evidence from antibody-induced coaggregation.

Acetylcholine receptors (AChR) are associated with several peripheral membrane proteins that are concentrated on the cytoplasmic face of the plasma membrane at the neuromuscular junction, and at aggregates of AChR that form in vitro. We tested the linkage among these proteins by inducing microaggregation of AChR, then determining if a given peripheral membrane protein accumulated with the receptors in microaggregates. In most experiments, we used isolated membrane fragments that are rich in AChR and accessible to antibodies against intracellular antigens. We showed that the 43 kD receptor-associated protein always aggregated with AChR, whether microaggregation was driven by antibodies to the 43 kD protein, or to the receptor itself. Antibodies to the 58 kD receptor-associated protein also always aggregated the 58 kD protein with the receptor. Our results are consistent with a model for AChR-rich membrane in which the 43 kD and 58 kD proteins are both closely associated with the AChR. When we induced microaggregation in intact muscle cells with anti-AChR antibodies, our results were less definitive. The 43 kD receptor-associated protein microaggregated with AChR, but the 58 kD protein was not especially enriched at AChR microaggregates. We discuss the advantages of using isolated AChR-rich membrane fragments to study the association of AChR with peripheral membrane proteins.

Clustered acetylcholine receptors have two levels of organization in Xenopus muscle cells.

We studied the organization of acetylcholine receptor (AChR) clusters by shearing cultured Xenopus muscle cells with a stream of buffer, and preparing rotary replicas of the exposed cytoplasmic surface of the sarcolemma. AChR clusters contained numerous particles that protruded from the sarcolemma and formed an irregular array composed of discrete aggregates. AChR were located within these particle aggregates, as shown by comparison of the replicas to labeling by fluorescent alpha-bungarotoxin, and by immunogold cytochemistry with antibodies specific for the receptor. The aggregates were cross-linked by a dense network of 7 nm filaments that replicated with the banded pattern characteristic of actin microfilaments. The organization of receptors into the small aggregates was independent of the organization of these aggregates into clusters, as alkaline extraction removed the microfilament network and disrupted the irregular array of particle aggregates, but did not disperse individual receptors from the aggregates. We conclude that two levels of interactions organize AChR clusters in Xenopus muscle cells: short-range interactions that assemble individual AChR into small aggregates, and long-range interactions, perhaps mediated by actin microfilaments, that anchor the aggregates into larger clusters.

Structures linking microfilament bundles to the membrane at focal contacts.

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 beta 1-integrin, vinculin, and talin, but they appear to contain less alpha-actinin and filamin. We also identified a second, smaller class of aggregates of membrane particles that contained beta 1-integrin but not vinculin or talin and that were not associated with actin microfilaments. Our results indicate that vinculin, talin, and beta 1-integrin are assembled into distinctive structures that mediate multiple lateral interactions between microfilaments and the membrane at focal contacts.

Presynaptic localization of sodium/calcium exchangers in neuromuscular preparations.

Calcium ions play a critical role in neurotransmitter release. The cytosolic Ca2+ concentration ([Ca2+]cyt) at nerve terminals must therefore be carefully controlled. Several different mechanisms, including a plasmalemmal Na/Ca exchanger, are involved in regulating [Ca2+]cyt. We employed immunofluorescence microscopy with polyclonal antiserum raised against dog cardiac sarcolemmal Na/Ca exchanger to determine the distribution of the exchanger in vertebrate neuromuscular preparations. Our data indicate that the Na/Ca exchanger is concentrated at the neuromuscular junctions of the rat diaphragm. The exchanger is also present in the nonjunctional sarcolemma, but at a much lower concentration than in the junctional regions. Denervation markedly lowers the concentration of the exchanger in the junctional regions; this implies that the Na/Ca exchanger is concentrated in the presynaptic nerve terminals. In Xenopus laevis nerve and muscle cell cocultures, high concentrations of the exchanger are observed along the neurites as well as at the nerve terminals. The high concentrations of Na/Ca exchanger at presynaptic nerve terminals in vertebrate neuromuscular preparations suggest that the exchanger may participate in the Ca-dependent regulation of neurotransmitter release. The Na/Ca exchanger is also abundant in developing neurites and growth cones, where it may also be important for Ca2+ homeostasis.

Quick-freeze, deep-etch replication of cells in monolayers.

We have made several technical improvements for quick-freeze, deep-etch replication of monolayers of cells grown on, or attached to, glass coverslips. Cells studied include muscle cells of rat and Xenopus cultured on glass coverslips, and erythrocytes attached to coverslips coated with poly-L-lysine. We describe methods for identifying particular areas of cultures, e.g., clusters of acetylcholine receptors on muscle cells, by light microscopy and then relocating these areas after replication. For good preservation of structure by quick-freezing, it is necessary to ensure that the surface to be frozen is covered by a minimum depth of water (less than 10 microns). Insufficient or excess water left on the sample during freezing causes recognizable artifacts in its replica. We describe two ways to control the water table--one by improving visual control of water removal, the other by blowing excess water off the sample surface with a jet of nitrogen applied during its descent to the freezing block. Finally, we describe a new specimen holder that allows us to etch and replicate six samples at once with good thermal contact between the stage and samples.

Formaldehyde-amine fixatives for immunocytochemistry of cultured Xenopus myocytes.

Although formaldehyde is commonly used in immunocytochemical studies, this fixative can cause distortions in cell structure. We tested the possibility that adducts of formaldehyde and primary amines could be used as improved fixatives for immunolabeling studies of cultured cells. A variety of primary amines were reacted with formaldehyde and applied to cultured Xenopus muscle cells, after which the cultures were labeled for immunofluorescence. Amine-formaldehyde fixatives improved structural preservation of the myocytes as compared with formaldehyde alone. The extent of improvement depended on the amine tested; the best results were obtained using cyclohexylamine. Immunofluorescence localization of a variety of antigens was better in myocytes fixed with cyclohexylamine-formaldehyde than in cells fixed with formaldehyde alone. In addition, the fixative provided good ultrastructural preservation of cytoskeletal structures and permitted immunogold labeling for alpha-actinin by use of pre-embedding labeling techniques.

Membrane-related specializations associated with acetylcholine receptor aggregates induced by electric fields.

The localization of membrane-associated specializations (basal lamina and cytoplasmic density) at sites of acetylcholine receptor (AChR) aggregation is consistent with an involvement of these structures in receptor stabilization. We investigated the occurrence of these specializations in association with AChR aggregates that develop at the cathode-facing edge of Xenopus muscle cells during exposure to a DC electric field. The cultures were labeled with a fluorescent conjugate of alpha-bungarotoxin and the receptor distribution on selected cells was determined before and after exposure to the field. In thin sections taken from the same cells, the cathode-facing edge was characterized by plaques of basal lamina and cytoplasmic density co-extensive with sarcolemma of increased density. In sections cut in a plane similar to the fluorescence image, it was possible to demonstrate that the specializations were concentrated at areas of field-induced AChR aggregation, and at receptor clusters existing on control cells. This finding further indicates that these structures participate in AChR stabilization, and that the mechanisms involved in AChR aggregation that result from field exposure and nerve contact may be similar.

Changes in cell shape and actin distribution induced by constant electric fields.

The development of motility in cultured cells is usually associated with a polarization of the cell shape. In particular, the leading edge of the cell is extended into a lamella which acts as a locus for the elaboration of cell processes and for the formation of cell-substrate contacts and, at the opposite end, retraction fibres often extend beyond the trailing edge of the cell. The alignment of microfilament bundles (stress fibres) along the direction of migration and the presence of a band of actin at the leading edge of the cell suggest an involvement of this protein in the motile process. The direction of growth and orientation of various cell types in tissue culture can be influenced by externally applied d.c. electric fields but the effect of the field on cellular motile activities is unknown. Here we describe a galvanotropic response of cultured Xenopus epithelial cells. At a field strength of 5 V cm-1 these cells elongate perpendicularly with respect to the field. The anodal side of the cell retracts and both the ends and cathodal edge become active in the extension of ruffling lamellipodia. In parallel with the change in the cell axis, stress fibres are oriented perpendicularly to the field, and a band of actin is associated with the lamellae at the cathodal edge and at the ends of the cell.