Evidence for a polarity in the distribution of proteins from the cytoskeleton in Torpedo marmorata electrocytes.

Abstract

Abstract. The subcellular distribution of the 43,000-D protein (43 kD or u~) and of some major cytoskeletal proteins was investigated in Torpedo marmorata electrocytes by immunocytochemical methods (immunofluorescence and immunogold at the electron microscope level) on frozen-fixed sections and homogenates of electric tissue. A monoclonal antibody directed against the 43-kD protein (Nghirm, H. O., J. Cartaud, C. Dubreuil, C. Kordeli, G. Buttin, and J. P. Changeux, 1983, Proc. Natl. Acad. Sci. USA, 80:6403-6407), selectively labeled the postsynaptic membrane on its cytoplasmic face. Staining by anti-actin and anti-desmin antibodies appeared evenly distributed within the cytoplasm: anti-desmin antibodies being associated with the network of intermediate-sized filaments that spans the electrocyte, and anti-actin antibodies making scattered clusters throughout the cytoplasm without preferential labeling of the postsynaptic membrane. On the other hand, a dense coating by anti-actin antibodies became apparent on the postsynaptic membrane in homogenates of electric tissue pointing to the possible artifactual redistribution of a soluble cytoplasmic actin pool.Anti-fodrin and anti-ankyrin antibodies selectively labeled the non-innervated membrane of the cell. F actin was also detected in this membrane. Filamin and vinculin, two actin-binding proteins recently localized at the rat neuromuscular junction (Bloch, R.J., and Z. W. Hall, 1983, J. CellBiol., 97:217-223), were detected in the electrocyte by the immunoblot technique but not by immunocytochemistry.The data are interpreted in terms of the functional polarity of the electrocyte and of the selective interaction of the cytoskeleton with the innervated and noninnervated domains of the plasma membrane.T HE postsynaptic membrane of the neuromuscular junction and of the electromotor synapse corresponds to a local differentiation of the plasma membrane characterized by an accumulation of the nicotinic acetylcholine receptor (Ach-R).t This membrane specialization persists for a long period after denervation (reviewed in references 10 and 18), indicating that physical constraints maintain the Ach-R molecules in place and, in particular, prevent against their lateral diffusion. Interactions of the Ach-R with extrinsic components from the extracellular matrix (13, 55) and/or with the cytoskeleton (17, 48, 67) have been postulated to contribute to this differentiation process. cell--the non-innervated face--is specialized in the regeneration of the electrochemical gradient (40). The Ach-R is present exclusively on the innervated membrane, and Na ÷ K ÷ ATPase accumulates on the non-innervated one. These membranes thus constitute fully differentiated and stable domains of the plasma membrane. From this standpoint the diskshaped electrocyte shows a striking functional and structural polarity ( 18, 21).Postsynaptic membrane fractions purified from Torpedo comprise essentially the intrinsic Ach-R polypeptides plus few extrinsic components of apparent molecular mass 43 kD (63, 64) na...

Desmin and Actin Filaments in Membrane-Cytoskeletal Preparations of the Electric Tissue of Electrophorus electricus, L.

“…To study this association of cytoskeletal filaments with organelles and membranes, we prepared cytoskeleton-enriched membrane fractions, according to KORDELI et al (1986). Figure 3A shows dense arrangements of cytoskeletal filaments.…”

“…The primary antibody was omitted in control experiments. Membrane fractions, including cytoskeletal elements derived from main electric organ, were prepared as described by HEUSER and SALPETER (1979) with modifications introduced by KORDELI et al (1986). Pieces of electric tissue were gently homogenized in a loose-fitting glass Potter homogenizer, and rotated at 800 rpm in membrane-cytoskeleton buffer (MCB; 20 mM Tris-HCI, 100 mM KCI, 5 mM MgC13, 3 mM EGTA, 1 mM EDTA, 200,uM PMSF, 400 mM benzamidine, lOU/ml aprotinin, 1 jig/ml pepstatin A, pH 7.4).…”

A protein antigenically related to nuclear lamin B mediates the association of intermediate filaments with desmosomes.

“…However, it may not be relevant to other cells where the IF network is organized differently. For example, in the electrocyte of Torpedo electric tissue, a flat syncytium that receives a dense cholinergic irmervation on one of its surfaces, the desmin meshwork is anchored to the two opposite plasma membranes and is not organized around nuclei (Kordeli et al, 1986(Kordeli et al, , 1987. Biochemical and immunocytochemical investigations have demonstrated that the plasma membranes of the innervated and the noninnervated sides of the cell, as well as the cortical skeleton associated with them, have a different composition.…”

“…Biochemical and immunocytochemical investigations have demonstrated that the plasma membranes of the innervated and the noninnervated sides of the cell, as well as the cortical skeleton associated with them, have a different composition. We have recently shown that desmin filaments are inserted to the innervated membrane via a lamin B-related protein of Mr "054 kD, and to the noninnervated membrane via ankyrin (Kordeli et al, 1986;Cartaud et al, 1989). The postsynaptic membrane in rat muscle is also labeled with anti-lamin B antibodies (Cartaud et al, 1989).…”

“…Cryostat sections (4-5 #m) of bovine muzzle epithelium were obtained according to Kordeli et al (1986). Serum F (1:100-1:200 dilution) was used as first antibody and revealed with rhodarnine-conjngated goat anti-human IgG (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD).…”