Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane.

Heuser, John E.; Salpeter, Shelley R.

doi:10.1083/jcb.82.1.150

Cited by 431 publications

(279 citation statements)

References 45 publications

(59 reference statements)

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“…No filaments decorated with the anti-actin m Ab were observed. Such a picture is consistent with the absence of cytoplasmic actin filaments in Torpedo electrocytes previously reported by Heuser and Salpeter using a deep-etching replication technique (32).…”

Section: Subcellular Localization Of Desmin In Agreement Withsupporting

confidence: 77%

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

Kordeli¹,

Cartaud²,

Nghiêm³

et al. 1986

The Journal of Cell Biology

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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...

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Section: Subcellular Localization Of Desmin In Agreement Withsupporting

confidence: 77%

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

Kordeli¹,

Cartaud²,

Nghiêm³

et al. 1986

The Journal of Cell Biology

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“…1 B and C) resembles the close packed arrangement of dimers of receptors observed in electric organ synapses and in tubular vesicles of isolated postsynaptic membranes (15,16). Based on the strength of the densities, which reflects the level of reproducibility, most receptors are consistently surrounded by three to five neighbors in defined positions.…”

Section: Resultsmentioning

confidence: 99%

Structure and superorganization of acetylcholine receptor–rapsyn complexes

Zuber

Unwin

2013

Proc. Natl. Acad. Sci. U.S.A.

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The scaffolding protein at the neuromuscular junction, rapsyn, enables clustering of nicotinic acetylcholine receptors in high concentration and is critical for muscle function. Patients with insufficient receptor clustering suffer from muscle weakness. However, the detailed organization of the receptor-rapsyn network is poorly understood: it is unclear whether rapsyn first forms a wide meshwork to which receptors can subsequently dock or whether it only forms short bridges linking receptors together to make a large cluster. Furthermore, the number of rapsyn-binding sites per receptor (a heteropentamer) has been controversial. Here, we show by cryoelectron tomography and subtomogram averaging of Torpedo postsynaptic membrane that receptors are connected by up to three rapsyn bridges, the minimum number required to form a 2D network. Half of the receptors belong to rapsyn-connected groups comprising between two and fourteen receptors. Our results provide a structural basis for explaining the stability and low diffusion of receptors within clusters.cryoelectron microscopy | neurotransmitter receptor clustering | synapse | ligand-gated ion channel | tetratricopeptide repeat T he cytoplasmic protein rapsyn, also known as the 43K protein, interacts with nicotinic acetylcholine receptors in the postsynaptic membrane at the neuromuscular junction, forming clusters where receptors are present in high concentration. Such clustering is necessary for efficient neurotransmission and, hence, muscle functioning. Rapsyn-deficient mice fail to form clusters, show severe muscle weakness and breathing difficulties, and die within hours after birth (1). Furthermore, many patients suffering from congenital myasthenic syndrome are found to have mutations in rapsyn (2).Rapsyn is a 43-kDa protein containing a myristoylation site necessary for submembrane localization, seven tetratricopeptide repeats (TRPs), a putative coiled-coil domain, and a RING-H2 domain (3). Two TPRs are necessary for rapsyn self-association, whereas association with receptor depends on the predicted coiled-coil (3, 4). Rapsyn is also linked through the RING-H2 domain to dystroglycan, a protein that indirectly binds actin cytoskeleton (5). Recombinant rapsyn expressed in heterologous systems spontaneously aggregates underneath the plasma membrane (3), suggesting that rapsyn might first form a wide scaffold to which receptors can subsequently dock (6). On the other hand, rapsyn and the receptor are cotransported in membrane vesicles toward the plasma membrane, and the receptor is necessary for rapsyn clustering in C2 myotubes, raising the possibility that rapsyn only forms short bridges linking receptors together to form a large cluster (7-9). Furthermore, the number of rapsyn-binding sites per receptor (a heteropentamer) has been variously estimated to be between one and five (6, 10, 11). Therefore, the molecular details and organization of the receptor-rapsyn network are poorly understood.To address these issues, we applied cryoelectron tomography, combined with...

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“…Quick-freeze, deep-etch (QF-DE) was performed as described previously (17,24) according to Heuser and Salpeter (12). Detergent-resistant cytoskeletons were washedonce with PEMand centrifuged at 12000 rpm for 5 min.…”

Section: Methodsmentioning

confidence: 99%

“…However, the resolution of traditional thin-section electron microscopy prevented a moredetailed structural analysis of the cytomatrix. Quick-freeze, deep-etch (QF-DE) electron microscopy is an excellent technique for observing the ultrastructure of cytoskeletons (12,13 have been shown to be extensively crosslinked by microtubule-associated proteins (17,21). This technique has also been applied to sea urchin eggs.…”

mentioning

confidence: 99%

Ultrastructure of Detergent-Resistant Cytoskeletons in the Noncortical Domain of Sea Urchin Eggs as Revealed by the Quick-Freeze Deep-Etch Technique.

Hisanaga

Endo

Hirokawa³

et al. 1992

Cell Struct. Funct.

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ABSTRACT. The ultrastructure of detergent-resistant cytoskeletons in the noncortical cytoplasm of sea urchin eggs was studied by quick-freeze, deep-etch electron microscopy. Twodifferent cytoskeletal organizations were identified in the detergent-treated sea urchin eggs. They were distinguished by the presence or the absence of long actin filaments and probably correspond to the cortex and the noncortical cytoplasm, respectively. The non-cortical cytoplasm was composed of a complex network (designated here as the ground network) of filaments 6 to13 nm in diameter, that interconnected aggregates of small globular materials, yolk granules and a meshwork of uniform filaments (8-9 nm in diameter). The 6 to 13 nm filaments comprising the ground network were branched and associated with filaments of the same or other sizes, resulting in the formation of an extremely complexnetwork. The meshworkof 8-9 nmfilaments was homogeneousin composition and constitutes a novel structure which has not been previously described. The 8-9 nmfilaments were connected to one another at their ends, forming a meshwork of polygons. Meshworks, ranging up to 3 /urn in diameter, were distributed throughout the non-cortical cytoplasm of the egg. Similar cytoplasmic structures were also observed in fertilized eggs.

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Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane.

Cited by 431 publications

References 45 publications

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

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

Structure and superorganization of acetylcholine receptor–rapsyn complexes

Ultrastructure of Detergent-Resistant Cytoskeletons in the Noncortical Domain of Sea Urchin Eggs as Revealed by the Quick-Freeze Deep-Etch Technique.

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