Ultrastructure of acetylcholine receptor aggregates parallels mechanisms of aggregation.

BACKGROUND: Acetylcholine receptors become aggregated at the developing neuromuscular synapse shortly after contact by a motorneuron in one of the earliest manifestations of synaptic development. While a major physiological signal for receptor aggregation (agrin) is known, the mechanism(s) by which muscle cells respond to this and other stimuli have yet to be worked out in detail. The question of mechanism is addressed in the present study via a quantitative examination of ultrastructural receptor arrangement within aggregates. RESULTS: In receptor rich cell membranes resulting from stimulation by agrin or laminin, or in control membrane showing spontaneous receptor aggregation, receptors were found to be closer to neighboring receptors than would be expected at random. This indicates that aggregation proceeds heterogeneously: nanoaggregates, too small for detection in the light microscope, underlie developing microaggregates of receptors in all three cases. In contrast, the structural arrangement of receptors within nanoaggregates was found to depend on the aggregation stimulus. In laminin induced nanoaggregates receptors were found to be arranged in an unstructured manner, in contrast to the hexagonal array of about 10 nm spacing found for agrin induced nanoaggregates. Spontaneous aggregates displayed an intermediate amount of order, and this was found to be due to two distinct population of nanoaggregates. CONCLUSIONS: The observations support earlier studies indicating that mechanisms by which agrin and laminin-1 induced receptor aggregates form are distinct and, for the first time, relate mechanisms underlying spontaneous aggregate formation to aggregate structure.

A quantitative evaluation of SAGE.

Serial Analysis of Gene Expression (SAGE) is an innovative technique that offers the potential of cataloging both the identity and relative frequencies of mRNA transcripts in a given poly(A(+)) RNA preparation. Although it is a very effective approach for determining the expression of mRNA populations, there are significant biases in the observed results that are inherent in the experimental process. These are caused by sampling error, sequencing error, nonuniqueness, and nonrandomness of tag sequences. The quantitative information desired from SAGE experiments consists of estimates of the number of genes and the frequency distribution of transcript copy numbers. Of additional concern is the extent to which a given tag sequence can be assumed to be unique to its gene. The present study takes these mathematical biases into account and presents a basis for maximum likelihood estimation of gene number and transcript copy frequencies given a set of experimental results. These estimates of the true state of genomic expression are markedly different from those based directly on the observations from the underlying experiments. It also is shown that while in many cases it is probable that a given tag sequence is unique within the genome, in larger genomes this cannot be safely assumed.

Fetal membrane distention: determination of the intrauterine surface area and distention of the fetal membranes preterm and at term.

OBJECTIVE: This study was undertaken to obtain an accurate measurement of the intrauterine surface area and the degree of distention of the apposed fetal membranes preterm and at term. STUDY DESIGN: Serial longitudinal images of the uterus in 23 women between 25 and 41 weeks' gestation were obtained by ultrasonography. A Mathematica (Wolfram Research, Inc, Champaign, Ill) program assembled a 3-dimensional image and calculated the intrauterine surface area for each patient. The surface areas of the placental amnion and membranes were measured in vitro after delivery. From these measurements the degree of distention of each fetal membrane in vivo was calculated. RESULTS: The mean calculated intrauterine surface areas were as follows: 1037 +/- 70 cm(2) (25-29 weeks' gestation, n = 4), 1376 +/- 121 cm(2) (30-34 weeks' gestation, n = 4), and 1876 +/- 307 cm(2) (37-41 weeks' gestation, n = 15, P =.0021 by Wilcoxon rank sum test). The surface areas of the expelled membranes at 25 to 29, 30 to 34, and 37 to 41 weeks' gestation were 737 +/- 61 cm(2), 855 +/- 77 cm(2), and 1115 +/- 149 cm(2), respectively. The ratios of intrauterine surface area to the area of the expelled membrane and hence a measure of the degree of distention in vivo were 1.4 +/- 0.05 at 25 to 29 weeks' gestation (n = 4), 1.6 +/- 0.2 at 30 to 34 weeks' gestation (n = 4), and 1.7 +/- 0.3 at term (n = 15). CONCLUSION: The intrauterine surface area in vivo increases during gestation. The surface area of the fetal membranes as measured in vitro increases to a lesser extent. The fetal membranes are therefore distended in vivo.

Structural organization of developing acetylcholine receptor aggregates.

We report the first quantitative ultrastructural analysis of newly formed acetylcholine receptor aggregates. Aggregates were induced in Xenopus muscle cell cultures with agrin, labeled with gold particles, and detected using high resolution scanning electron microscopy. Aggregates are readily discernible at the ultrastructural level within 2 h of stimulation by agrin. The size and density profiles of the developing aggregates show that receptors reach maximal density very quickly in small "nano-aggregates" and that the aggregation process is not limited by the diffusion rate of the receptor. Quantitative analysis of label locations indicates that the receptor distribution within aggregates is nonrandom. Instead, the newly aggregated receptors appear to be bound to a localized scaffold conforming to a hexagonal (close-packed) geometry with a spacing of approximately 9.9 nm.

Common molecular mechanisms in field- and agrin-induced acetylcholine receptor clustering.

1. The aggregation of acetylcholine receptors at the developing neuromuscular junction is critical to the development and function of this synapse. In vitro studies have shown that receptor aggregation can be induced by the finding of agrin to the muscle cell surface and by the electric field-induced concentration of a (nonreceptor) molecule at the cathodal cell pole. 2. We report here on the interaction between agrin binding and electric fields with respect to the distribution of receptors and agrin binding sites. 3. (a) Pretreatment of cells with agrin completely blocks the development of field-induced receptor clusters. (b) Field-induced aggregation of receptors precedes the field-induced aggregation of agrin binding sites by approximately 30 min. (c) Electric fields prevent agrin-induced receptor clustering despite the presence of agrin binding sites and freely diffusing receptors. 4. These results indicate that another membrane component-but not the agrin binding site and not the receptor-is required for agrin-induced receptor clustering. They also suggest that electric fields and agrin cause receptor clustering via common molecular mechanisms.

Density and diffusion limited aggregation in membranes.

Aggregation of membrane molecules is a crucial phenomenon in developing organisms, a classic example being the aggregation of post-synaptic receptors during synaptogenesis. Our understanding of the molecular events involved is improving, but most models of the aggregation or concentration process do not address binding events on the molecular level. An exception is the study of diffusion limited aggregation, in which the aggregation process is simulated on a molecular level. In this analysis, however, important physical parameters such as molecular size, diffusion constant and initial density are not addressed. Thus no predictions about the rate at which such aggregates will form is possible. In the present work the model of diffusion limited aggregation is extended to incorporate these parameters and make the corresponding predictions.

Synapse elimination, the size principle, and Hebbian synapses.

Synapse elimination at the vertebrate neuromuscular junction reduces a polyinnervated population of muscle fibers to a monoinnervated state. The function of this developmental phenomenon (if any) is unproven. A theoretical analysis of Hebbian (correlation) rules connecting presynaptic and postsynaptic activity and synaptic strength at the neuromuscular junction is presented. The following points are demonstrated: (1) Correlational competition leads to the reduction of polyinnervation to a stable monoinnervated state; (2) the competition gives rise to the size principle over a wide range of the plausible parameter space; (3) over a significant subrange, the competition selectively eliminates topographically incorrect synapses; and (4) in cases in which topographic projection errors overwhelm the system, both error correction and the development of the size principle are disrupted. Correlational competition may explain contradictory experimental results concerning the effects of stimulating or silencing subpopulations of motor neurons. It may also explain an otherwise puzzling instance of a breakdown in the size principle seen in humans undergoing neural regeneration. Taken together, these findings suggest a novel hypothesis for the function of synapse elimination at the neuromuscular junction: the establishment of the size principle.

Acetylcholine receptor clustering is triggered by a change in the density of a nonreceptor molecule.

Acetylcholine receptors become clustered at the neuromuscular junction during synaptogenesis, at least in part via lateral migration of diffusely expressed receptors. We have shown previously that electric fields initiate a specific receptor clustering event which is dependent on lateral migration in aneural muscle cell cultures (Stollberg, J., and S. E. Fraser. 1988. J. Cell Biol. 107:1397-1408). Subsequent work with this model system ruled out the possibility that the clustering event was triggered by increasing the receptor density beyond a critical threshold (Stollberg, J., and S. E. Fraser. 1990. J. Neurosci. 10:247-255). This leaves two possibilities: the clustering event could be triggered by the field-induced change in the density of some other molecule, or by a membrane voltage-sensitive mechanism (e.g., a voltage-gated calcium signal). Electromigration is a slow, linear process, while voltage-sensitive mechanisms respond in a rapid, nonlinear fashion. Because of this the two possibilities make different predictions about receptor clustering behavior in response to pulsed or alternating electric fields. In the present work we have studied subcellular calcium distributions, as well as receptor clustering, in response to such fields. Subcellular calcium distributions were quantified and found to be consistent with the predicted nonlinear response. Receptor clustering, however, behaves in accordance with the predictions of a linear response, consistent with the electromigration hypothesis. The experiments demonstrate that a local increase in calcium, or, more generally, a voltage-sensitive mechanism, is not sufficient and probably not necessary to trigger receptor clustering. Experiments with slowly alternating electric fields confirm the view that the clustering of acetylcholine receptors is initiated by a local change in the density of some non-receptor molecule.

Local accumulation of acetylcholine receptors is neither necessary nor sufficient to induce cluster formation.

Acetylcholine receptors (AChRs) accumulate at developing neuromuscular junctions in part via lateral migration of diffusely expressed receptors. Using a model system--cultured Xenopus muscle cells exposed to electric fields--we have shown that AChRs, concentrated at the cathode-facing cell pole, continue to aggregate there after the field is terminated (Stollberg and Fraser, 1988). These observations are consistent with the possibility that the field-induced increase in receptor concentration triggers the aggregation event. Only 2 other molecular events could initiate the electric field-induced receptor aggregation: (1) a local increase in the density of some other molecules, or (2) a voltage-sensitive mechanism. Treatment of muscle cell cultures with neuraminidase changes the cell surface charge and has been reported to reverse the direction of electromigration for AChRs and concanavalin A binding sites (Orida and Poo, 1978). Using digitally analyzed fluorescence videomicroscopy, we find that AChRs in neuraminidase-treated cultures accumulate at both cell poles in an electric field. After termination of the field, the AChR continues to aggregate at the cathode-facing pole, as in cells not treated with neuraminidase. However, receptor density decreases at the anode-facing pole, indicating that elevated AChR density does not initiate receptor aggregation. Cells pretreated with neuraminidase and trypsin (which blocks receptor aggregation) display reversed receptor distributions compared to untreated controls, indicating that electromigration has indeed been reversed. The rate at which neuraminidase- and trypsin-treated cells approach steady-state distributions indicates a receptor diffusion constant of approximately 1.2 x 10(-9) cm2/sec, consistent with a diffusion trap mechanism of receptor aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine receptors and concanavalin A-binding sites on cultured Xenopus muscle cells: electrophoresis, diffusion, and aggregation.

Using digitally analyzed fluorescence videomicroscopy, we have examined the behavior of acetylcholine receptors and concanavalin A binding sites in response to externally applied electric fields. The distributions of these molecules on cultured Xenopus myoballs were used to test a simple model which assumes that electrophoresis and diffusion are the only important processes involved. The model describes the distribution of concanavalin A sites quite well over a fourfold range of electric field strengths; the results suggest an average diffusion constant of approximately 2.3 X 10(-9) cm2/s. At higher electric field strengths, the asymmetry seen is substantially less than that predicted by the model. Acetylcholine receptors subjected to electric fields show distributions substantially different from those predicted on the basis of simple electrophoresis and diffusion, and evidence a marked tendency to aggregate. Our results suggest that this aggregation is due to lateral migration of surface acetylcholine receptors, and is dependent on surface interactions, rather than the rearrangement of microfilaments or microtubules. The data are consistent with a diffusion-trap mechanism of receptor aggregation, and suggest that the event triggering receptor localization is a local increase in the concentration of acetylcholine receptors, or the electrophoretic concentration of some other molecular species. These observations suggest that, whatever mechanism(s) trigger initial clustering events in vivo, the accumulation of acetylcholine receptors can be substantially enhanced by passive, diffusion-mediated aggregation.

Neuronal acetylcholine receptors: fate of surface and internal pools in cell culture.

Chick ciliary ganglion neurons have nicotinic ACh receptors that mediate synaptic input to the cells. Ultrastructural studies with a monoclonal antibody that recognizes the neuronal ACh receptor have previously shown that, in addition to a predominantly synaptic location for the receptors on the neuron surface in vivo, substantial amounts of intracellular receptor are present as well. Here we report that intracellular receptor and smaller receptor-related components make up at least two-thirds of the total antibody binding sites associated with the ciliary ganglion neurons in cell culture. The intracellular sites for the most part represent integral membrane components that bind to concanavalin A when solubilized, indicating that the components are glycosylated. Sucrose gradient analysis shows that the intracellular material includes a 10 S component, likely to represent assembled receptor, along with species sedimenting in the 5-9 S range. Blocking the surface sites with unlabeled antibody and measuring the appearance of the new receptor on the cell surface with radiolabeled antibody indicates that the receptors are inserted into the plasma membrane at a rate equivalent to about 4% of the total surface receptor per hour. The transit time for newly synthesized receptor to reach the cell surface appears to be 2-3 hr. These observations suggest that about 5% of the intracellular receptors are transported to the cell surface in culture. The half-life of ACh receptors in the plasma membrane was estimated by 2 different approaches to be about 22 hr. Surface and internal sites respond in a qualitatively similar way to external agents that specifically modulate cholinergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional blockade of neuronal acetylcholine receptors by antisera to a putative receptor from brain.

Antisera to a putative acetylcholine receptor purified from chick brain specifically inhibit the acetylcholine response of chick ciliary ganglion neurons in cell culture. The putative brain receptor and a similar membrane component previously identified on ciliary ganglion neurons appear to be functional nicotinic acetylcholine receptors in the nervous system and are clearly distinct from membrane components in the tissues that bind alpha-bungarotoxin.

Characterization of a component in chick ciliary ganglia that cross-reacts with monoclonal antibodies to muscle and electric organ acetylcholine receptor.

Chick ciliary ganglion neurons have previously been shown to contain a component that shares an antigenic determinant with the "main immunogenic region" of the alpha-subunit in nicotinic acetylcholine receptor from skeletal muscle and electric organ. Ultrastructural studies of antibody binding in the ganglion have shown that the cross-reacting antigen exposed on the surface of the neurons is located predominantly in synaptic membrane. Here we show that the neuronal antigen can be identified in detergent extracts of ciliary and sympathetic ganglia, but not in extracts of heart, liver, spinal cord, retina, or dorsal root ganglia. In the ciliary ganglion the component is present as an integral membrane constituent, and, when detergent solubilized, it sediments as a 10 S species and binds to concanavalin A. The component is distinct from the alpha-bungarotoxin-binding site on the neurons since toxin-binding sites and antibody-binding sites can be precipitated separately in ganglion extracts. The component reaches peak levels per ganglionic protein between embryonic days 8 and 12. These are some of the properties expected for the nicotinic acetylcholine receptor on ciliary ganglion neurons.