scholarly journals Asynchronous assembly of the acetylcholine receptor and of the 43-kD nu1 protein in the postsynaptic membrane of developing Torpedo marmorata electrocyte.

1989 ◽  
Vol 108 (1) ◽  
pp. 127-139 ◽  
Author(s):  
E Kordeli ◽  
J Cartaud ◽  
H O Nghiêm ◽  
A Devillers-Thiéry ◽  
J P Changeux
Keyword(s):  
Plasma Membrane ◽  
Acetylcholine Receptor ◽  
Postsynaptic Membrane ◽  
Synaptic Membrane ◽  
Asymmetric Distribution ◽  
Subsequent Stage ◽  
Torpedo Marmorata ◽  
Intracellular Compartments ◽  
Membrane Compartments

The assembly of the nicotinic acetylcholine receptor (AchR) and the 43-kD protein (v1), the two major components of the post synaptic membrane of the electromotor synapse, was followed in Torpedo marmorata electrocyte during embryonic development by immunocytochemical methods. At the first developmental stage investigated (45-mm embryos), accumulation of AchR at the ventral pole of the newly formed electrocyte was observed within columns before innervation could be detected. No concomitant accumulation of 43-kD immunoreactivity in AchR-rich membrane domains was observed at this stage, but a transient asymmetric distribution of the extracellular protein, laminin, which paralleled that of the AchR, was noticed. At the subsequent stage studied (80-mm embryos), codistribution of the two proteins was noticed on the ventral face of the cell. Intracellular pools of AchR and 43-kD protein were followed at the EM level in 80-mm electrocytes. AchR immunoreactivity was detected within membrane compartments, which include the perinuclear cisternae of the endoplasmic reticulum and the plasma membrane. On the other hand, 43-kD immunoreactivity was not found associated with the AchR in the intracellular compartments of the cell, but codistributed with the AchR at the level of the plasma membrane. The data reported in this study suggest that AchR clustering in vivo is not initially determined by the association of the AchR with the 43-kD protein, but rather relies on AchR interaction with extracellular components, for instance from the basement membrane, laid down in the tissue before the entry of the electromotor nerve endings.

scholarly journals Agrin regulates CLASP2-mediated capture of microtubules at the neuromuscular junction synaptic membrane

2012 ◽  
Vol 198 (3) ◽  
pp. 421-437 ◽  
Author(s):  
Nadine Schmidt ◽  
Sreya Basu ◽  
Stefan Sladecek ◽  
Sabrina Gatti ◽  
Jeffrey van Haren ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptor ◽  
Three Dimensional ◽  
Postsynaptic Membrane ◽  
Dimensional Structure ◽  
Muscle Membrane ◽  
Synaptic Membrane ◽  
Dense Network ◽  
Phosphatidylinositol 3 Kinase ◽  
Neuronal Regulation

Agrin is the major factor mediating the neuronal regulation of postsynaptic structures at the vertebrate neuromuscular junction, but the details of how it orchestrates this unique three-dimensional structure remain unknown. Here, we show that agrin induces the formation of the dense network of microtubules in the subsynaptic cytoplasm and that this, in turn, regulates acetylcholine receptor insertion into the postsynaptic membrane. Agrin acted in part by locally activating phosphatidylinositol 3-kinase and inactivating GSK3β, which led to the local capturing of dynamic microtubules at agrin-induced acetylcholine receptor (AChR) clusters, mediated to a large extent by the microtubule plus-end tracking proteins CLASP2 and CLIP-170. Indeed, in the absence of CLASP2, microtubule plus ends at the subsynaptic muscle membrane, the density of synaptic AChRs, the size of AChR clusters, and the numbers of subsynaptic muscle nuclei with their selective gene expression programs were all reduced. Thus, the cascade linking agrin to CLASP2-mediated microtubule capturing at the synaptic membrane is essential for the maintenance of a normal neuromuscular phenotype.

scholarly journals 300-kD subsynaptic protein copurifies with acetylcholine receptor-rich membranes and is concentrated at neuromuscular synapses

1987 ◽  
Vol 104 (4) ◽  
pp. 939-946 ◽  
Author(s):  
ML Woodruff ◽  
J Theriot ◽  
SJ Burden
Keyword(s):  
Acetylcholine Receptor ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Synaptic Membrane ◽  
Binding Assays ◽  
Western Blots ◽  
Neuromuscular Synapses ◽  
Peripheral Membrane Proteins ◽  
Torpedo Electric Organ ◽  
Skeletal Muscle Antibodies

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four different subunits of the acetylcholine receptor and in two peripheral membrane proteins at 43 and 300 kD. We produced monoclonal antibodies against the 300-kD protein and have used these antibodies to determine the location of the protein, both in the electric organ and in skeletal muscle. Antibodies to the 300-kD protein were characterized by Western blots, binding assays to isolated membranes, and immunofluorescence on tissue. In Torpedo electric organ, antibodies to the 300-kD protein stain only the innervated face of the electrocytes. The 300-kD protein is on the intracellular surface of the postsynaptic membrane, since antibodies to the 300-kD protein bind more efficiently to saponin-permeabilized, right side out membranes than to intact membranes. Some antibodies against the Torpedo 300-kD protein cross-react with amphibian and mammalian neuromuscular synapses, and the cross-reacting protein is also highly concentrated on the intracellular surface of the post-synaptic membrane.

scholarly journals Lipid Raft-Based Membrane Compartmentation of a Plant Transport Protein Expressed in Saccharomyces cerevisiae

2006 ◽  
Vol 5 (6) ◽  
pp. 945-953 ◽  
Author(s):  
Guido Grossmann ◽  
Miroslava Opekarova ◽  
Linda Novakova ◽  
Jürgen Stolz ◽  
Widmar Tanner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Fusion Protein ◽  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Gfp Fusion Protein ◽  
Ergosterol Synthesis ◽  
Membrane Compartments ◽  
Green Fluorescent

ABSTRACT The hexose-proton symporter HUP1 shows a spotty distribution in the plasma membrane of the green alga Chlorella kessleri. Chlorella cannot be transformed so far. To study the membrane localization of the HUP1 protein in detail, the symporter was fused to green fluorescent protein (GFP) and heterologously expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In these organisms, the HUP1 protein has previously been shown to be fully active. The GFP fusion protein was exclusively targeted to the plasma membranes of both types of fungal cells. In S. cerevisiae, it was distributed nonhomogenously and concentrated in spots resembling the patchy appearance observed previously for endogenous H+ symporters. It is documented that the Chlorella protein colocalizes with yeast proteins that are concentrated in 300-nm raft-based membrane compartments. On the other hand, it is completely excluded from the raft compartment housing the yeast H+/ATPase. As judged by their solubilities in Triton X-100, the HUP1 protein extracted from Chlorella and the GFP fusion protein extracted from S. cerevisiae are detergent-resistant raft proteins. S. cerevisiae mutants lacking the typical raft lipids ergosterol and sphingolipids showed a homogenous distribution of HUP1-GFP within the plasma membrane. In an ergosterol synthesis (erg6) mutant, the rate of glucose uptake was reduced to less than one-third that of corresponding wild-type cells. In S. pombe, the sterol-rich plasma membrane domains can be stained in vivo with filipin. Chlorella HUP1-GFP accumulated exactly in these domains. Altogether, it is demonstrated here that a plant membrane protein has the property of being concentrated in specific raft-based membrane compartments and that the information for its raft association is retained between even distantly related organisms.

scholarly journals EVIDENCE FOR RECYCLING OF SYNAPTIC VESICLE MEMBRANE DURING TRANSMITTER RELEASE AT THE FROG NEUROMUSCULAR JUNCTION

1973 ◽  
Vol 57 (2) ◽  
pp. 315-344 ◽  
Author(s):  
J. E. Heuser ◽  
T. S. Reese
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Motor Nerve ◽  
Coated Vesicles ◽  
Synaptic Membrane ◽  
Nerve Terminals ◽  
Frog Neuromuscular Junction ◽  
Vesicle Membrane ◽  
Intracellular Compartments

When the nerves of isolated frog sartorius muscles were stimulated at 10 Hz, synaptic vesicles in the motor nerve terminals became transiently depleted. This depletion apparently resulted from a redistribution rather than disappearance of synaptic vesicle membrane, since the total amount of membrane comprising these nerve terminals remained constant during stimulation. At 1 min of stimulation, the 30% depletion in synaptic vesicle membrane was nearly balanced by an increase in plasma membrane, suggesting that vesicle membrane rapidly moved to the surface as it might if vesicles released their content of transmitter by exocytosis. After 15 min of stimulation, the 60% depletion of synaptic vesicle membrane was largely balanced by the appearance of numerous irregular membrane-walled cisternae inside the terminals, suggesting that vesicle membrane was retrieved from the surface as cisternae. When muscles were rested after 15 min of stimulation, cisternae disappeared and synaptic vesicles reappeared, suggesting that cisternae divided to form new synaptic vesicles so that the original vesicle membrane was now recycled into new synaptic vesicles. When muscles were soaked in horseradish peroxidase (HRP), this tracerfirst entered the cisternae which formed during stimulation and then entered a large proportion of the synaptic vesicles which reappeared during rest, strengthening the idea that synaptic vesicle membrane added to the surface was retrieved as cisternae which subsequently divided to form new vesicles. When muscles containing HRP in synaptic vesicles were washed to remove extracellular HRP and restimulated, HRP disappeared from vesicles without appearing in the new cisternae formed during the second stimulation, confirming that a one-way recycling of synaptic membrane, from the surface through cisternae to new vesicles, was occurring. Coated vesicles apparently represented the actual mechanism for retrieval of synaptic vesicle membrane from the plasma membrane, because during nerve stimulation they proliferated at regions of the nerve terminals covered by Schwann processes, took up peroxidase, and appeared in various stages of coalescence with cisternae. In contrast, synaptic vesicles did not appear to return directly from the surface to form cisternae, and cisternae themselves never appeared directly connected to the surface. Thus, during stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vesicles by way of intermediate cisternae.

Targeting of acetylcholine receptor and 43 kDa rapsyn to the postsynaptic membrane in Torpedo marmorata electrocyte

1998 ◽  
Vol 92 (3-4) ◽  
pp. 177-181 ◽  
Author(s):  
Fabrizia Bignami ◽  
Gilles Camus ◽  
Sophie Marchand ◽  
Lise Bailly ◽  
Françoise Stetzkowski-Marden ◽  
...  
Keyword(s):  
Acetylcholine Receptor ◽  
Postsynaptic Membrane ◽  
Torpedo Marmorata

Biochemistry of the renal V-ATPase.

1992 ◽  
Vol 172 (1) ◽  
pp. 219-229 ◽  
Author(s):  
S L Gluck ◽  
R D Nelson ◽  
B S Lee ◽  
Z Q Wang ◽  
X L Guo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Function ◽  
Proton Pumps ◽  
Intracellular Membrane ◽  
Acid Base Balance ◽  
Intracellular Compartments ◽  
Selective Membrane ◽  
Base Balance ◽  
Membrane Compartments ◽  
High Concentrations

In most eukaryotic cells, vacuolar H(+)-ATPases (V-ATPases) are present primarily or exclusively in intracellular membrane compartments, functioning in the acidification of the endocytic and secretory vacuolar apparatus necessary for constitutive cell function. V-ATPases also participate in renal hydrogen ion secretion in both the proximal and distal nephron, residing at high concentrations on the plasma membrane, where they are regulated physiologically to maintain the acid-base balance of the organism. Recent experiments have begun to reveal how the kidney controls transcellular proton transport while still maintaining acidification of intracellular compartments. Control may occur by recruitment of proton pumps to or away from the plasma membrane. The proton-transporting plasma membrane of intercalated cells is a specialized apparatus that translocates the enzyme between an intracellular membrane pool and the plasma membrane in response to physiological stimuli. Regulation may also occur by changes in the kinetics of the V-ATPase. V-ATPases are a family of structurally similar enzymes which differ in the composition of specific subunits. Cytosolic regulatory enzymes present in renal cells may preferentially affect V-ATPases in selective membrane compartments.

scholarly journals Tubular crystals of acetylcholine receptor.

1984 ◽  
Vol 99 (4) ◽  
pp. 1202-1211 ◽  
Author(s):  
A Brisson ◽  
P N Unwin
Keyword(s):  
Acetylcholine Receptor ◽  
Cross Sections ◽  
Asymmetric Distribution ◽  
Torpedo Marmorata ◽  
Oriented Molecules ◽  
Cell Dimensions ◽  
Average Unit ◽  
Plane Group ◽  
Unit Cell Dimensions ◽  
Tubular Crystals

Well-ordered tubular crystals of acetylcholine receptor were obtained from suspensions of Torpedo marmorata receptor-rich vesicles. They are composed of pairs of oppositely oriented molecules arranged on the surface lattice with the symmetry of the plane group p2 (average unit cell dimensions: a = 90 A, b = 162 A, gamma = 117 degrees). The receptor in this lattice has an asymmetric distribution of mass around its perimeter, yet a regular pentagonal shape; thus its five transmembrane subunits appear to have different lengths, but approximately equal cross sections. The tubes grow by lateral aggregation on the vesicle surface of ribbons of the paired molecules. Both ribbons and tubes were sensitive to dispersal by the disulphide reductant, dithiothreitol. This observation and other evidence suggest that the basic pairing interaction in the tubes may be that of the physiological dimer, involving contact between delta-subunits.

Direct involvement of a lamin-B-related (54 kDa) protein in the association of intermediate filaments with the postsynaptic membrane of the Torpedo marmorata electrocyte

1995 ◽  
Vol 108 (1) ◽  
pp. 153-160
Author(s):  
A. Cartaud ◽  
B.J. Jasmin ◽  
J.P. Changeux ◽  
J. Cartaud
Keyword(s):  
Acetylcholine Receptor ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Early Stage ◽  
Postsynaptic Membrane ◽  
Muscle Fibres ◽  
Motor Innervation ◽  
Torpedo Marmorata ◽  
Lamin B ◽  
Stage Of Development

Mechanisms by which motor innervation induces postsynaptic membrane differentiation and functional compartmentalization of the subneural sarcoplasm in skeletal muscle fibres are still poorly understood. However, transmembrane control of cytoskeletal activities by the nerve terminal may be considered. Here, we examine several properties of a 54 kDa protein, previously identified in the postsynaptic membrane of the Torpedo marmorata electrocyte with anti-lamin B antibodies, in order to study its role in the assembly of the subneural intermediate filament meshwork. Using a ligand blot assay, we show that this protein binds desmin, a type III intermediate filaments protein, at micromolar concentrations. Moreover, purified acetylcholine receptor-rich membrane fragments are able to generate arrays of desmin filaments in vitro. Immunofluorescence experiments indicate that the 54 kDa protein becomes associated with the acetylcholine receptor-rich membrane at an early stage of development of the electrocyte, and that a polarized desmin network develops concomitantly from the postsynaptic membrane. Taken together, these data show that, like karyoskeletal lamin B, the 54 kDa protein is involved in the organization of the subneural intermediate filament meshwork. Control of the assembly of the subneural cytoskeleton by components of the postsynaptic membrane may thus be a prerequisite for the functional compartmentalization of the muscle fibre triggered by motor innervation.

scholarly journals CLASP2-dependent microtubule capture at the neuromuscular junction membrane requires LL5β and actin for focal delivery of acetylcholine receptor vesicles

2015 ◽  
Vol 26 (5) ◽  
pp. 938-951 ◽  
Author(s):  
Sreya Basu ◽  
Stefan Sladecek ◽  
Isabel Martinez de la Peña y Valenzuela ◽  
Mohammed Akaaboune ◽  
Ihor Smal ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Actin Cytoskeleton ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Postsynaptic Membrane ◽  
Interaction Partner ◽  
Synaptic Membrane ◽  
Microtubule Capture

A novel mechanism is described for the agrin-mediated focal delivery of acetylcholine receptors (AChRs) to the postsynaptic membrane of the neuromuscular junction. Microtubule capture mediated by CLASP2 and its interaction partner, LL5β, and an intact subsynaptic actin cytoskeleton are both required for focal AChR transport to the synaptic membrane.

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