Clustered acetylcholine receptors have two levels of organization in <i>Xenopus</i> muscle cells

Luther, Paul W.; Samuelsson, Steven J.; Bloch, Robert J.; Pumplin, David W.

doi:10.1002/cm.970280209

Cited by 6 publications

(2 citation statements)

References 39 publications

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“…We were thus concerned whether the cortical cytoskeleton is retained in our preparation. In a similar preparation of sheared membranes from Xenopus myocytes, the actin microfilament network organization is still retained [20]. To demonstrate the existence of cytoskeleton in our preparation we labelled actin with FITC-conjugated phalloidin.…”

Section: Detection Of Microfilamentsmentioning

confidence: 94%

Imaging plasma membrane proteins in large membrane patches of Xenopus oocytes

Singer-Lahat

Dascal

Mittelman

et al. 2000

Pflugers Arch - Eur J Physiol

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We describe the preparation of a Xenopus oocyte plasma membrane patch attached to a cover-slip with its intracellular face exposed to the bath solution. The proteins attached to the plasma membrane were visualized by confocal microscopy after fluorescence labelling. Since cortical microfilament elements were detected in these plasma membrane preparations we termed the patches plasma membrane-cortex patches. The way these patches are formed and the low concentration of proteins needed for cytochemical detection make the membrane-cortex patches similar to electrophysiological membrane patches and therefore allow the cytochemical study of ion channels to be correlated with electrophysiological experiments. Furthermore, the described patch is similar to manually isolated plasma membranes used for biochemical analysis by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Cytochemical analysis of membrane-cortex patches also enables the detection of the two-dimensional pattern of organization of membrane proteins (clustered or non-clustered forms). In addition, patch preparations enable cytochemical study of the relative localization of membrane proteins. The methodology enables integration of electrophysiological, biochemical and cytochemical studies of ion channels, giving a comprehensive perspective on ion channel function.

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Section: Detection Of Microfilamentsmentioning

confidence: 94%

Imaging plasma membrane proteins in large membrane patches of Xenopus oocytes

Singer-Lahat

Dascal

Mittelman

et al. 2000

Pflugers Arch - Eur J Physiol

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“…Earlier studies have suggested that microclusters and clusters represent distinct stages of neural factor-induced AChR aggregation, with microclusters serving as the precursors of full-size clusters in cultured muscle cells (41)(42)(43)(44), as well as in developing neuromuscular synapses (2). The dispersal of clusters in response to pharmacological agents (protein kinase C activators and phosphoprotein phosphatase inhibitors) similarly proceeds via microclusters as intermediates (43,45).…”

Section: Inhibitors Of the Rho Pathway Impair Agrin-induced Achrmentioning

confidence: 99%

Cooperative Regulation by Rac and Rho of Agrin-induced Acetylcholine Receptor Clustering in Muscle Cells

Weston¹,

Gordon²,

Teressa³

et al. 2003

Journal of Biological Chemistry

117

104

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During embryonic development, innervation induces the anatomical and biochemical specialization of a defined region of the muscle cell membrane immediately under the motor nerve ending. A prominent aspect of this specialization is the accumulation of high densities of nicotinic acetylcholine receptors (AChR) 1 at these sites (1, 2). The aggregation of AChR and other synaptic components is mediated by agrin, a heparansulfated proteoglycan that is synthesized by motor neurons and secreted into the synaptic cleft (3, 4). The recruitment of AChR into clusters in postsynaptic membranes ensures high efficiency synaptic transmission at neuromuscular junctions.Agrin-induced redistribution of surface AChR involves the co-clustering of multiple associated proteins, several of which have been identified to date (2, 5). These include the musclespecific receptor tyrosine kinase (MuSK) (6), the linker protein rapsyn (7), and the scaffolding proteins dystroglycan and utrophin (8). The clustering of MuSK upon its activation by agrin, the formation of AChR complexes with rapsyn, as well as the aggregation of these complexes and their stabilization upon the formation of dystroglycan-utrophin scaffolds appear to be sequential events that are to some extent independently regulated (9 -11). Although the signaling mechanisms that couple agrin activation of MuSK to the clustering of postsynaptic components are incompletely characterized, there is recent evidence for the participation of Src tyrosine kinases (12, 13), the Rho GTPases Rac and Cdc42 (14), and Dishevelled, a component of the Wnt signaling pathway (15).Focal changes in the peripheral actin-based cytoskeleton are thought to underlie the aggregation of AChR at neuromuscular junctions (16 -18). The monomeric G proteins Rac and Rho function to link extracellular signals to dynamic changes in actin cytoskeleton organization leading to the assembly of lamellipodia and actin-myosin filaments, respectively (19 -22). Rac activation induces actin polymerization at the plasma membrane, causing the appearance of lamellipodia with resultant stimulation of cell spreading and motility (23,24). Rho exerts the opposite effect by stimulating actin stress fiber appearance and focal adhesion complex formation to promote cell adhesion and contractility (25,26). As several recent studies have shown, Rac and Rho are mutually inhibitory in several cell types, and the balance between their antagonistic activities is responsible for the dynamic changes in cell morphology, adhesion, and motility (27,28). In other systems Rac serves as an upstream activator of Rho (29).We have recently shown that agrin triggers the activation of Rac and Cdc42 and that this activation is necessary but not sufficient for formation of full size AChR clusters (14). In this study we present evidence that Rho plays a crucial role in agrin-initiated signaling that is complementary to the contribution of Rac/Cdc42 and that together these Rho family GTPases serve to couple signaling initiated by extracellular agrin to the for...

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