Immunofluorescence localization at the mammalian neuromuscular junction of the Mr 43,000 protein of Torpedo postsynaptic membranes.

Froehner, Stanley C.; Gulbrandsen, Vivian; Hyman, Carolyn; Jeng, Arco Y.; Neubig, Richard R.; Cohen, Jonathan B.

doi:10.1073/pnas.78.8.5230

Cited by 133 publications

(101 citation statements)

References 33 publications

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“…-/-muscles Rapsyn is a cytoplasmic protein colocalized with AChRs in the postsynaptic muscle membrane (Froehner et al, 1981). Synaptic localization of rapsyn occurs at the initial stages of neuromuscular synaptogenesis (Noakes et al, 1993), and deletion of rapsyn leads to a total absence of AChR clustering (Gautam et al, 1995).…”

Section: Abnormal Localization Of Rapsyn In γmentioning

confidence: 99%

Essential roles of the acetylcholine receptor γ-subunit in neuromuscular synaptic patterning

et al. 2008

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Formation of the vertebrate neuromuscular junction (NMJ) takes place in a stereotypic pattern in which nerves terminate at select sarcolemmal sites often localized to the central region of the muscle fibers. Several lines of evidence indicate that the muscle fibers may initiate postsynaptic differentiation independent of the ingrowing nerves. For example, nascent acetylcholine receptors (AChRs) are pre-patterned at select regions of the muscle during the initial stage of neuromuscular synaptogenesis. It is not clear how these pre-patterned AChR clusters are assembled, and to what extent they contribute to pre-and post-synaptic differentiation during development. Here, we show that genetic deletion of the AChR γ-subunit gene in mice leads to an absence of pre-patterned AChR clusters during initial stages of neuromuscular synaptogenesis. The absence of pre-patterned AChR clusters was associated with excessive nerve branching, increased motoneuron survival, as well as aberrant distribution of acetylcholinesterase (AChE) and rapsyn. However, clustering of muscle specific kinase (MuSK) proceeded normally in the γ-null muscles. AChR clusters emerged at later stages owing to the expression of the AChR epsilon-subunit, but these delayed AChR clusters were broadly distributed and appeared at lower level compared with the wild-type muscles. Interestingly, despite the abnormal pattern, synaptic vesicle proteins were progressively accumulated at individual nerve terminals, and neuromuscular synapses were ultimately established in γ-null muscles. These results demonstrate that the γ-subunit is required for the formation of pre-patterned AChR clusters, which in turn play an essential role in determining the subsequent pattern of neuromuscular synaptogenesis.

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Section: Abnormal Localization Of Rapsyn In γmentioning

confidence: 99%

Essential roles of the acetylcholine receptor γ-subunit in neuromuscular synaptic patterning

et al. 2008

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“…The fact that this polypeptide can be extracted from the membrane by alkali (9) suggests that it is not an integral membrane protein and does not span the membrane. Recent immunofluorescence experiments using an antiserum directed against the Mr 43,000 protein indicate that this component is restricted to the innervated membrane of the Torpedo electrocyte (23). Therefore, the Mr 43,000 protein is probably a peripheral membrane protein located on the intracellular surface of the postsynaptic membrane .…”

Section: Discussionmentioning

confidence: 99%

“…Such a location is interesting in light of reports that extraction of membranes at pH 11, which removes the Mr 43,000 protein, increases the rotational mobility (19,29) and the thermal sensitivity (22) of the AcChR . An immunologically related component of rat muscle endplates also appears to be intracellular (23,47) . In immunofluorescence experiments, antibodies against the Torpedo Mr 43,000 protein do not label neuromuscular junctions when incubated with intact muscles, while anti-AcChR antibodies do .…”

Section: Discussionmentioning

confidence: 99%

“…Although the Mr 43,000 protein is not necessary for permeability control, its removal from the membranes is associated with an increase in rotational mobility of the AcChR (18, 19) and of its sensitivity to proteases (20,21) and thermal denaturation (22). Antibodies directed against the Mr 43,000 protein bind to antigenic determinants concentrated at the innervated surface of the Torpedo electrocyte and also at the vertebrate neuromuscular junction (23). These results suggest that the Mr 43,000 protein has a structural function in nicotinic postsynaptic membranes.…”

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Nicotinic postsynaptic membranes from Torpedo: sidedness, permeability to macromolecules, and topography of major polypeptides.

John

Froehner

Goodenough

et al. 1982

The Journal of Cell Biology

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Experiments were conducted to examine the topographic arrangement of the polypeptides of the acetylcholine receptor (AcChR) and the nonreceptor M, 43,000 protein in postsynaptic membranes isolated from Torpedo electric organ. When examined by electron microscopy, >85% of vesicles were not permeable to ferritin or lactoperoxidase (LPO) . Exposure to saponin was identified as a suitable procedure to permeabilize the vesicles to macromolecules with minimal alteration of vesicle size or ultrastructure . The sidedness of vesicles was examined morphologically and biochemically. Comparison of the distribution of intramembrane particles on freeze-fractured vesicles and the distribution found in situ indicated that >85% of the vesicles were extracellular-side out. Vesicles labeled with a-bungarotoxin (a-BgTx) were reacted with antibodies against a-BgTx or against purified AcChR of Torpedo. Bound antibodies were detected by the use of ferritin-conjugated goat anti-rabbit antibody and were located on the outside of >99% of labeled vesicles . Similar results were obtained for normal vesicles or vesicles exposed to saponin. Quantification of the amount of [3 H]-a-BgTx bound to vesicles before and after they were made permeable with saponin indicated that <5% of aBgTx binding sites were cryptic in normal vesicles . It was concluded that >95% of postsynaptic membranes were oriented extracellular-side out.LPO-catalyzed radioiodinations were performed on normal and saponin-treated vesicles and on vesicles from which the Mr (relative molecular mass) 43,000 protein had been removed by alkaline extraction . In normal vesicles, polypeptides of the AcChR were iodinated while the Mr 43,000 protein was not. In vesicles made permeable with saponin, the pattern of labeling of AcChR polypeptides was unchanged, but the M, 43,000 protein was heavily iodinated. The relative iodination of AcChR polypeptides was unchanged in membranes equilibrated with agonist or with a-BgTx or after alkaline-extraction. It was concluded that the M, 43,000 protein is present on the intracellular surface of the postsynaptic membrane and that AcChR polypeptides are exposed on the extracellular surface.Analysis ofthe mechanism of permeability control by nicotinic cholinergic receptors is facilitated by the isolation ofthe plasma membrane of the innervated Torpedo electrocyte surface, which is highly enriched in nicotinic receptors (for a review, see reference 1). The nicotinic receptor (AcChR), which constitutes -r50% of the protein in the purified postsynaptic membrane, is composed of polypeptides of apparent molecular weights

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“…Rapsyn is best known for its role in clustering and maintaining a high density and number of AChR at synaptic sites (Froehner et al, 1981;Noakes et al, 1993;Gautam et al, 1995). For instance, in mice deficient in rapsyn, AChRs and other specific synaptic proteins fail to cluster on the muscle postsynaptic domain and mice die immediately after birth.…”

Section: Introductionmentioning

confidence: 99%

Failure of lysosome clustering and positioning in the juxtanuclear region in cells deficient in rapsyn

Aittaleb

Chen

Akaaboune

2015

Journal of Cell Science

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Rapsyn, a scaffold protein, is required for the clustering of acetylcholine receptors (AChRs) at contacts between motor neurons and differentiating muscle cells. Rapsyn is also expressed in cells that do not express AChRs. However, its function in these cells remains unknown. Here, we show that rapsyn plays an AChRindependent role in organizing the distribution and mobility of lysosomes. In cells devoid of AChRs, rapsyn selectively induces the clustering of lysosomes at high density in the juxtanuclear region without affecting the distribution of other intracellular organelles. However, when the same cells overexpress AChRs, rapsyn is recruited away from lysosomes to colocalize with AChR clusters on the cell surface. In rapsyn-deficient (Rapsn −/− ) myoblasts or cells overexpressing rapsyn mutants, lysosomes are scattered within the cell and highly dynamic. The increased mobility of lysosomes in Rapsn −/− cells is associated with a significant increase in lysosomal exocytosis, as evidenced by increased release of lysosomal enzymes and plasma membrane damage when cells were challenged with the bacterial pore-forming toxin streptolysin-O. These findings uncover a new link between rapsyn, lysosome positioning, exocytosis and plasma membrane integrity.

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Immunofluorescence localization at the mammalian neuromuscular junction of the Mr 43,000 protein of Torpedo postsynaptic membranes.

Cited by 133 publications

References 33 publications

Essential roles of the acetylcholine receptor γ-subunit in neuromuscular synaptic patterning

Essential roles of the acetylcholine receptor γ-subunit in neuromuscular synaptic patterning

Nicotinic postsynaptic membranes from Torpedo: sidedness, permeability to macromolecules, and topography of major polypeptides.

Failure of lysosome clustering and positioning in the juxtanuclear region in cells deficient in rapsyn

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