Clustering of muscle acetylcholine receptors requires motoneurons in live embryos, but not in cell culture (original) (raw)
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Development of Acetylcholine Receptor Clusters on Cultured Muscle Cells
Proceedings of the National Academy of Sciences of the United States of America, 1973
Clusters of acetylcholine receptors were formed in the absence of neurons during the development of cultured chick-embryo skeletal muscle cells. The average concentration of receptors in clusters was estimated to be 9000 per /m2. At other regions of the cell, receptor concentration was 900 per 1tm2. The possibility that receptor clusters may participate in synapse formation is suggested.
Journal of Cell Biology, 1984
We have examined the effects of microtubule-and microfilament-disrupting drugs on the stability, formation, and removal of acetylcholine (ACh) receptors and ACh receptor clusters on the surface of aneurally cultured chick embryonic myotubes . (a) In muscle cell cultures, cytochalasin D (0.2 )ug/ml) or B (2 .0 Ag/ml) causes the dispersal of 50-60% of the existing clusters over a 24-h period (visualized with rhodamine-conjugated a-bungarotoxin) ; Colcemid (0.5 Ag/ml) has no affect on these clusters . The total number of cell surface ACh receptors does not decline during this period (measured by [ 1251]a-bungarotoxin binding) in the presence of either drug. (b) When cells are treated with biotinylated a-bungarotoxin and fluorescent avidin, ACh receptors are cross-linked and rapidly internalized (Axelrod, D., 1980, Proc . Natl . Acad. Sci. USA., 77 : 4823-4827). Within 6 h, I have found that 0-15% of the existing large clusters remain . Cytochalasin D or B had no effect on this removal of clusters ; however, Colcemid completely prevented the removal of clusters from the cell surface . (c) Addition of chick brain extract to chick myotubes causes an increase in the synthesis and clustering of ACh receptors (Jessell et al., 1979, Proc. Natl . Acad . Sci. USA . 76: 5397-5401) . Cytochalasin D caused a slight increase in the number of receptors synthesized in the presence of brain extract whereas Colcemid had no effect on the synthesis and insertion of new receptors into the plasma membrane induced by the brain extract . However, both drugs prevented the increase in the number of receptor clusters . These results are consistent with the hypothesis that receptor clusters are stabilized by actin-containing filaments, but that the movement of receptors in the plane of the membrane requires Colcemid-sensitive microtubules .
On the developmental regulation of acetylcholine receptor mobility in the embryonic muscle membrane
Experimental Cell Research, 1980
We have investigated the possible mechanisms underlying a developmental decrease in acetylcholine (ACh) receptor mobility in the membrane of cultured, spherical, mononucleate Xenopus embryonic muscle cells (myoballs) utilizing the method of in situ electrophoresis. We observed that between 1 and 4 days in culture, a substantial redistribution of ACh receptors can be induced by the externally applied electric field which resulted in highly asymmetrical ACh sensitivities at the cathode-and anode-facing poles of the cell. Between 5 and 8 days in culture, the extent of ACh receptor redistribution induced by the field declined to a lower level. Pretreatment with cytoskeletal disrupting agents or with a disulfide bond reducing agent before in situ electrophoresis had no effect on 2-day-old cultures but enhanced receptor mobility in 6-day-old cultures. Pretreatment with Ca*+-Mg*+-free saline (CMF), which releases cell coat material in other systems, substantially increased receptor mobility when tested on days 2, 6, and 8. On day 6, pretreatment with CMF containing cytochalasin B (CB) and colchicine produced an even greater increase in receptor mobility as compared to treatment with CB and colchicine alone. Our findings suggest that the developmental decrease in ACh receptor mobility is accounted for by at least two different mechanisms: (1) An early-developing, CMF-sensitive restriction possibly mediated by the cell coat;
Molecular Brain Research, 2001
Muscle specific kinase (MuSK) mediates agrin-induced acetylcholine receptor (AChR) aggregation on muscle membrane at the neuromuscular junction (NMJ). To examine whether MuSK enhances NMJ formation during embryonic development in vivo, the level of expression of MuSK was manipulated in Xenopus embryos and the functional consequence at the NMJ was assessed. We found that overexpression of MuSK enhanced the formation of NMJ by increasing the aggregation of AChRs at innervated regions in developing embryos. The area of AChR aggregation increased by |2-fold in MuSK injected embryos during the critical stages of NMJ formation. Interestingly, overexpression of MuSK in Xenopus embryos was found to induce the level of AChR transcript. Deletion of the Kringle domain in the MuSK construct did not attenuate the observed induction of AChR transcription and aggregation. Taken together, our findings provide the first demonstration that increased level of MuSK expression in vivo significantly elevate the aggregation and transcription of AChR at the NMJ in developing Xenopus embryos.
Acetylcholine Receptor Formation in Mouse–Chick Chimera
Experimental Cell Research, 1997
chick embryo, including axon outgrowth, synapse for-This study investigated possible interactions be-mation, and synapse elimination. The first motoneuron tween motoneurons and somitic-derived muscle cells axons leave the ventral part of the neural tube at day in the formation of neuromuscular synapses in the 3 of incubation (E3) . At the same time, somites myotome. The peculiarities of the neuromuscular synundergo morphogenesis to form sclerotome, myotome, aptic pattern in chick and mouse embryos provided a and dermatome which give rise respectively to cartimodel for studying the achievement of synaptogenesis lage, muscle, and dermis [reviewed in 3, 4]. The somitic between chick motoneurons and mouse muscle cells. cells then begin their migration from the dermomyo-In chick embryo, initial AChR clustering occurs well tome to the wing anlagen . Inside the limb bud, they before innervation of the myotome, whereas in mouse migrate actively toward the distal regions to form preembryo nerve axons invade the myotome extensively muscle masses . From E6, large nerve trunks apbefore the appearance of AChR clusters. Our approach proach the developing muscles in the limb buds, and was to replace somites from a chick host embryo with the first acetylcholine receptor (AChR) clusters are inthose derived from mouse donor embryos. We show duced to form on nearby myotubes . Between E6 and that muscle cells from mouse myotome can differenti-E10, motor nerves invade and ramify profusely in the ate in the chick embryo environment and form neurodeveloping limbs , and synapse formation is indimuscular contacts with chick motor axons. Host axons cated by both anatomical [7] and functional [9] criteria. invaded in ovo differentiating mouse myotome at a Spontaneous motility begins, and motoneurons actitime when they had not yet reached the host myotome. vate their muscles in characteristic burst patterns [9]. This particular ingrowth of motor nerves was attribut-In avian embryos, synaptogenesis leads to the forable to the mouse transplant since use of a quail somite mation of several junctions along each muscle fiber did not produce the same effect as the mouse somite, [10]. Each synaptic site is then polyinnervated by axwhich suggests that developing mouse muscles speons arising from several motoneurons [11]. During decifically modify the time course of chick axogenesis.