Concanavalin-A-induced transmembrane linkage of concanavalin A surface receptors to intracellular myosin-containing filaments.

Abstract

With normal rat kidney cells in monolayer culture, we have studied the distribution on the cell surface of receptors for concanavalin A, and the distribution of the smooth muscle myosin-like protein inside the same cell, using specific fluorescence microscopic methods. The concanavalin A receptors were initially uniformly dispersed over the cell surface, but 20 min after the addition of concanavalin A at 370, the receptors showed a variety of nonuniform surface distributions, including extended parallel linear arrays. These arrays of receptors were found to be superimposed on the linear arrays of the intracellular myosin-containing filaments, indicating that a transmembrane linkage of the receptors and the filaments had occurred. This linkage required a lateral redistribution of concanavalin A receptors, since it did not occur with succinylated concanavalin A, but was subsequently induced if the cells that had been reacted with succinylated concanavalin A were then treated with antibodies to concanavalin A. The redistributions of concanavalin A receptors on the surfaces of these normal rat kidney cells, however, were much less extensive than the patching that was induced on the surfaces of the same cells infected with, and transformed by, Rous sarcoma virus. The concept of transmembrane control-that cell surface molecules can interact with structural elements in the cell interior-has been the subject of much experimentation and discussion (for reviews, see refs. 173). Almost all of the experimental evidence supporting this concept, however, is indirect, much of it involving inferences drawn from the effects produced by drugs such as colchicine and cytochalasin B on the lateral mobility of cell surface receptors. In this paper, direct evidence is provided for such transmembrane interactions in a line of normal rat kidney (NRK) cells in monolayer culture. We have studied the distribution on the cell surface of receptors for concanavalin A (Con A) and of the smooth muscle myosin-like protein inside the cell (4-6). These distributions were determined simultaneously on the same cell by fluorescence microscopy, using fluorescein-conjugated Con A(FI-Con A) to detect the Con A receptors, and an indirect rhodamine immunofluorescence method for the myosin. Initially the Fl-Con A was found to be uniformly dispersed over the cell surface, while the intracellular myosin was organized into extended filamentous structures. After 20 min at 370, however, the FlCon A showed a variety of nonuniform distributions, ranging from a mottled pattern to extended parallel linear arrays. In the latter case, the linear arrays of Con A receptors were superimposable on the linear arrays of the intracellular myosin-containing filaments, strongly suggesting that the receptors and the filaments had become physically linked. These observations are relevant to our recently published comparative study (7) (7).Protein Modification. Fl-Con A was prepared as described (7). Succinylated Con A (Suc-Con A) was a single-stage modified product (9) ...

Immunolocalization of the cellular src protein in interphase and mitotic NIH c-src overexpresser cells.

“…Initially, the fluorescence covered the cell surface uniformly with no discernible structure, in accordance with the earlier report of Ash and Singer (1976, Fig. 5 a).…”

“…The labeling of the ConA receptors was effected following the procedure of Ash and Singer (1976). The coverslips maintained in 35-ram dishes on which cultured ceils were grown, rinsed three to four times with serum-free DME, and incubated with 1 ml of the same medium containing 50 t~g of either unlabeled ConA or F-ConA for various times at 370C.…”

“…After 20 rain of exposure of normal rat kidney or WI38 human cells to Con A or to antibodies directed against different integral proteins, the binding of these reagents to their respective receptors at the cell surface has been shown to induce these receptors to redistribute to a limited extent into patches situated at the cell surface (Ash and Singer, 1976;Ash et al, 1977). By contrast, the binding of a multivalent ligand to its specific receptors on the surface of many cell in suspension, including lymphocytes, results first in a clustering of the receptors in small patches at the cell surface and, then, aggregation of the patches into a single cap (reviewed in Ash et al, 1977).…”

“…The induction of patching and capping of cell surface molecules by labeled lectins in a variety of cells, followed or not by endocytosis of the lectin-receptor complexes, is the model system that has been the most extensively used to examine the possible mechanism of initiation and processing of endocytosis (Ash and Singer, 1976;Albertini and Anderson, 1977). The second part of this article describes experiments in which the NIH (pM c-src/focus) B cells and control NIH 3T3 cells were exposed, for various times to unlabeled or fluorescently labeled Con A and the fate of the labeled lectin was compared with that of pp60 ~-src.…”

Influence of the Cytoskeleton on the Expression of a Mouse Hepatitis Virus (MHV-3) in Peritoneal Macrophages: Acute and Persistent Infection

“…In chick fibroblasts infected with Newcastle disease virus (NDV), this is indicated by both an alteration of the membrane-operated uptake of metabolic precursors and an altered response to the agglutinating effect of plant lectins (Rott et al, 1975;Reeve et al, 1975). The existence of functional associations between surface macromolecules and cytokinetic elements (Ash & Singer, 1976;Sundquist & Ehrnst, 1976;Toh & Hard, 1977;Schreiner et al, 1977;Koch & Smith, 1978;Flanagan & Koch, 1978;Thom et al, 1979;Hoesli et al, 1980) may also be of importance in the evolution of some virus infections as it has been observed that translation of vesicular stomatitis virus (VSV) requires ribosomal association with the cytoskeletal framework (Cervera et al, 1981) and that macromolecular changes at the cell exterior can affect the organization of microfilament structures (Mallucci & Wells, 1976;Wells & Mallucci, 1978). On the other hand, since cytoskeletal elements can play a role in the control and display of cell surface macromolecular components (De Petris, 1974;Nicolson, 1975;Yahara & Edelman, 1975;Sundquist & Ehrnst, 1976;Edelman, 1976), a primary alteration of the cytoskeleton may lead to structural and functional changes of the surface membrane, which may in turn alter the assembly and release of membrane-bound (enveloped) viruses and modify the cytopathic effect that they cause.…”