The effect of rat spinal cord explants and cell-free nerve extract on acetyicholine receptor site density and distribution was studied using 125I-and rhodamine-labeled abungarotoxin on Le, a cloned rat muscle cell line. Control Lt myotubes have a low and uniform distribution of acetylcholine receptors (20 i 3 sites per pm3 in the present study) The addition of spinal cord explants caused an increase in average receptor site density of abut 6 times on myotubes within 2 mm of the explant, while a smaller increase of 3 times was observed at distances greater than 5 mm. The formation of high-density patches of receptors was also stimulated. These observations suggested that a diffusible substance originating from the explant was responsible for these changes. Cell-free homogenates of the central nervous system were prepared and found to produce the same effects. The effect of the homogenate was not strongly dependent on the age of the fetus from which the tissue was isolated, and fetal liver had little or no effect. The active component~s) appears to be a protein(s) with a molecular weight of abut 100,000. Because the nerve homogenates make the L6 cells resemble primary muscle cultures, we suggest that a common factor is responsible for regulating the acetylcholine receptor in the two types of muscle culture. The normally acetylcholine receptor-poor L4 cells may provide a more sensitive assay for these factors than do primary muscle cultures.Many studies on neurotrophic phenomena have dealt with the control of synthesis and distribution of the acetylcholine receptors (AcChR) in embryonic and adult innervated and denervated muscle. One favored system for these studies has been muscle cells grown in culture [see recent reviews by Nelson (1) and Gutman (2)].In primary muscle cultures the AcChR tends to form in high density patches on myotubes. Several authors have suggested that these patches play a role in the subsequent development of the cells (3,4).The L6 line of cloned muscle cells (5) resembles primary muscle cultures in its developmental sequence and ability to form synaptic connections (6). Furthermore, the conductance change per AcChR in response to AcCh is the same in L6 as in primary rat myotubes (7). However, these cells have a uniform distribution and considerably lower site density of AcChR than the myotubes in primary cultures (7-9). These characteristics make L6 a desirable system for studying the possible control of AcChR distribution in skeletal muscle prior to innervation.The present study deals with the effects of isolated explants of nervous system tissue and of cell-free nerve extracts on AcChR in L6 cells. We have found that both are capable of inducing an increase in average AcChR site density and the formation of high-density patches.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 2035 METHODSThe acetylcholine rec...

The product of an integrated transgene provides a convenient and cell-specific reporter of intracellular protein catabolism in 103 muscle cells of the nematode Caenorhabditis elegans. The transgene is an in-frame fusion of a 5'-region of the C. elegans unc-54 (muscle myosin heavy-chain) gene to the lacZ gene of Escherichia coli [Fire and Waterston (1989): EMBO J 8:3419-3428], encoding a 146-kDa fusion polypeptide that forms active beta-galactosidase tetramers. The protein is stable in vivo in well-fed animals, but upon removal of the food source it is inactivated exponentially (t1/2 = 17 h) following an initial lag of 8 h. The same rate constant (but no lag) is observed in animals starved in the presence of cycloheximide, implying that inactivation is catalyzed by pre-existing proteases. Both the 146-kDa fusion polypeptide (t1/2 = 13 h) and a major 116-kDa intermediate (t1/2 = 7 h) undergo exponential physical degradation after a lag of 8 h. Degradation is thus paradoxically faster than inactivation, and a number of characteristic immunoreactive degradation intermediates, some less than one-third the size of the parent polypeptide, are found in affinity-purified (active) protein. Some of these intermediates are conjugated to ubiquitin. We infer that the initial proteolytic cleavages occur in the cytosol, possibly by a ubiquitin-mediated proteolytic pathway and do not necessarily inactivate the fusion protein tetramer.

No abstract

The product of an integrated transgene provides a convenient and cell-specific reporter of intracellular protein catabolism in 103 muscle cells of the nematode Caenorhabditis elegans. The transgene is an in-frame fusion of a 5'-region of the C. elegans unc-54 (muscle myosin heavy-chain) gene to the lacZ gene of Escherichia coli [Fire and Waterston (1989): EMBO J 8:3419-3428], encoding a 146-kDa fusion polypeptide that forms active beta-galactosidase tetramers. The protein is stable in vivo in well-fed animals, but upon removal of the food source it is inactivated exponentially (t1/2 = 17 h) following an initial lag of 8 h. The same rate constant (but no lag) is observed in animals starved in the presence of cycloheximide, implying that inactivation is catalyzed by pre-existing proteases. Both the 146-kDa fusion polypeptide (t1/2 = 13 h) and a major 116-kDa intermediate (t1/2 = 7 h) undergo exponential physical degradation after a lag of 8 h. Degradation is thus paradoxically faster than inactivation, and a number of characteristic immunoreactive degradation intermediates, some less than one-third the size of the parent polypeptide, are found in affinity-purified (active) protein. Some of these intermediates are conjugated to ubiquitin. We infer that the initial proteolytic cleavages occur in the cytosol, possibly by a ubiquitin-mediated proteolytic pathway and do not necessarily inactivate the fusion protein tetramer.

Abstract. In situ hybridization of chick cultured muscle cells using exonic DNA probes for both AChR or-subunit and the catalytic subunit of AChE, revealed major differences in the distribution of label both over nuclei and in their surrounding cytoplasm, although some overlap in these distributions exists. For the AChR a-subunit there is a highly skewed distribution of labeled nuclei, with 35% of the nuclei being relatively inactive (<0.25 times the mean label) and ,o10% being very heavily labeled (>2.5 times the mean label). In contrast the nuclei labeled with the exonic probe for the AChE transcripts had a more Gaussian distribution, yet with some slight skewness in the direction of a few heavily labeled nuclei. There was also a difference in the cytoplasmic distribution of the label. The AChR a-subunit mRNA was mainly within 4 #m of labeled nuclei while the AChE mRNA was more widely distributed throughout the cytoplasm, possibly within a 10 #m rim around labeled nuclei. An intronic probe for the AChE gave the identical distribution of nuclear label to that of the exonic probe (but without any cytoplasmic label). In addition, calibration of the technique indicated that per myotube the AChE transcript is about sixfold more abundant than the AChR c~-subunit transcript.