Ionic properties of the acetylcholine receptor in cultured rat myotubes

The acetylcholine reversal potential (Er) of cultured rat myotubes is -3mV. When activated, the receptor is permeable to K+ and Na+, but not to Cl- ions. Measurement of Er in Tris+-substituted, Na-free medium also indicated a permeability to Tris+ ions. Unlike adult frog muscle the magnitude of Er was insensitive to change in external Ca++ (up to 30 mM) or to changes in external pH (between 6.4 and 8.9). The equivalent circuit equation describing the electrical circuit composed of two parallel ionic batteries (EK and ENa) and their respective conductances (gK and gNa), which has been generally useful in describing the Er of adult rat and frog muscle, could also be applied to rat myotubes when Er was measured over a wide range of external Na+ concentrations. The equivalent circuit equation could not be applied to myotubes bathed in media of different external K+ concentrations. In this case, the Er was more closely described by the Goldman constant field equation. Under certain circumstances, it is known that the receptor in adult rat and frog muscle can be induced to reversibly shift from behavior described by the equivalent circuit equation to that described by the Goldman equation. Attempts to similarly manipulate the responses of cultured rat myotubes were unsussessful. These trials included a reduction in temperature (15 degress C), partial alpha-bungarotoxin blodkade, and activation of responses with the cholinergic agonist, decamethonium.     

Organization of acetylcholine receptor clusters in cultured rat myotubes is calcium dependent

The effect of extracellular Ca2+ concentration and myasthenic globulin on the distribution and appearance of acetylcholine receptor (AChR) clusters on rat myotubes was studied with tetramethyl-rhodamine-labeled alpha BTX. Low Ca2+ medium (2.5 X 10(-5) M) caused a time-dependent loss of AChR clusters, and a concomitant increase in small punctate areas of fluorescence. High Ca2+ concentrations (1.5 X 10(-2) M) increased the size of AChR clusters without altering AChR synthesis. These changes were not observed with other divalent ions. In the presence of myasthenic globulin, the rate of AChR turnover increases, and AChR clusters are rapidly dispersed. High Ca2+ concentration partially protects the AChR clusters from dispersal and decreases the rate of receptor turnover.     

Isolation of acetylcholine receptor clusters in substrate-associated material from cultured rat myotubes using saponin

After exposure of rat myotube cultures to saponin, less than 1% of the cellular protein was found to remain associated with the tissue culture substrate. This substrate-associated material contained approximately 10% of the acetylcholine receptors (AChRs) and greater than 80% of the large, ventral AChR clusters present in the original culture. The domain structure evident in intact cells was maintained in AChR clusters after isolation using saponin. However, vinculin, present at the clusters of intact cells, was absent from isolated clusters. Dodecyl sulfate PAGE showed that substrate-associated material enriched in AChR clusters contained a distinctive set of polypeptides, the major ones electrophoresing with apparent molecular weights of 43,000 and 49,000. Saponin extraction of cultures of established cell lines also yielded substrate-associated material with characteristics particular to the cell type.     

Neuregulin inhibits acetylcholine receptor aggregation in myotubes

The high local concentration of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction results from their aggregation by the agrin/MuSK signaling pathway and their synthetic up-regulation by the neuregulin/ErbB pathway. Here, we show a novel role for the neuregulin/ErbB pathway, the inhibition of AChR aggregation on the muscle surface. Treatment of C2C12 myotubes with the neuregulin epidermal growth factor domain decreased the number of both spontaneous and agrin-induced AChR clusters, in part by increasing the rate of cluster disassembly. Upon cluster disassembly, AChRs were internalized into caveolae (as identified by caveolin-3). Time-lapse microscopy revealed that individual AChR clusters fragmented into puncta, and application of neuregulin accelerated the rate at which AChR clusters decreased in area without affecting the density of AChRs remaining in individual clusters (as measured by the fluorescence intensity/unit area). We propose that this novel action of neuregulin regulates synaptic competition at the developing neuromuscular junction.     

Some properties of acetylcholine receptors in human cultured myotubes

The distribution and single channel properties of acetylcholine (ACh) receptors in human myotubes grown in tissue culture have been examined. Radioautography of myotubes labelled with [125I]alpha-bungarotoxin showed that ACh receptors are distributed uniformly over the myotube surface at a density of 3.9 +/- 0.5 receptors per square micrometre. Accumulations of ACh receptors (hot spots) were found rarely. The conductance and kinetics of ACh-activated channels were investigated with the patch-clamp technique. Cell-attached membrane patches were used in all experiments. A single channel conductance in the range 40-45 pS was calculated. No sublevels of conductance (substates) of the activated channel were observed. The distribution of channel open-times varied with ACh concentration. With 100 nM ACh, the distribution was best fitted by the sum of two exponentials, whereas with 1 microM ACh a single exponential could be fitted. The mean channel open-time at the myotube resting potential (ca. -70 mV, 22 degrees C) was 8.2 ms. The distribution of channel closed-times was complex at all concentrations of ACh studied (100 nM to 10 microM). With desensitizing doses of ACh (10 microM), channel openings occurred in obvious bursts; each burst usually appeared as part of a 'cluster' of bursts. Both burst duration and mean interval between bursts increased with membrane hyperpolarization. Individual channel open-times and burst durations showed similar voltage dependence (e-fold increase per 80 mV hyperpolarization), whereas both the channel closed-times within a burst and the number of openings per burst were independent of membrane potential.     

The lipid bilayer of acetylcholine receptor clusters of cultured rat myotubes is organized into morphologically distinct domains

We have studied the composition and organization of the lipid bilayer at the large, substrate-associated clusters of acetylcholine receptors (AChR) that form in cultured rat myotubes. These clusters have a characteristic morphology consisting of alternating linear domains of AChR-rich and AChR-poor membrane, the latter involved in attaching the myotube to the substrate. We partially purified AChR clusters by extracting cultured rat myotubes with the cholesterol-specific detergent, saponin. The lipid bilayer of the cluster preparation was analyzed biochemically and the substructure of the bilayers was studied morphologically using the fluorescent probes, dansyl polymyxin B, and 3,3'-di(C12H25 and C18H37) indocarbocyanine iodide (C12- and C18-diI). Our results demonstrate that preparations of AChR clusters have a lipid composition biochemically similar to that of the surrounding plasma membrane. Morphologically, however, the lipid bilayer appears to be arranged into domains that resemble the interdigitating pattern seen for the AChR. This distinctive lipid organization is not due to the use of saponin to purify clusters, as we obtained similar results with clusters isolated by physically shearing myotube cultures. The domain-like organization of the bilayer at clusters is disrupted by treatments that disperse AChR clusters in intact myotubes or that remove peripheral membrane proteins from isolated clusters. This suggests that such proteins may contribute to the organization of the bilayer. Two additional factors may also contribute to the organization of the bilayer: physical constraints imposed by sites of substrate attachment and, to a lesser extent, "boundary" lipid associated with AChR.     

Rotational diffusion of acetylcholine receptors on cultured rat myotubes

The rotational mobility of acetylcholine receptors (AChR) in the plasma membrane of living rat myotubes in culture is measured in this study by polarized fluorescence recovery after photobleaching (PFRAP). These AChR are known to exist in two distinct classes, evident by labeling with rhodamine alpha-bungarotoxin; clustered AChR that are aggregated in a pattern of highly concentrated speckles and streaks, with each cluster occupying an area of approximately 1,000 microns 2; and nonclustered AChR that appear as diffuse labeling. PFRAP results reported here show that: (a) most clustered AChR (approximately 86%) are rotationally immobile within a time scale of at least several seconds; and (b) most nonclustered AChR (approximately 76%) are rotationally mobile with characteristic times ranging from less than 50 ms to 0.1 s. External cross-linking with the tetravalent lectin concanavalin A immobilizes many nonclustered AChR. PFRAP experiments in the presence of carbachol or cytochalasin D show that the restraints to rotational motion in clusters are remarkably immune to treatments that disperse clusters or disrupt cytoplasmic actin. The experiments also demonstrate the feasibility of using PFRAP to measure rotational diffusion on selected microscopic areas of living nondeoxygenated cells labeled with standard fluorescence probes over a very wide range of time scales, and they also indicate what technical improvements would make PFRAP even more practicable.     

Cytoplasmic components of acetylcholine receptor clusters of cultured rat myotubes: the 58-kD protein

A 58-kD protein, identified in extracts of postsynaptic membrane from Torpedo electric organ, is enriched at sites where acetylcholine receptors (AChR) are concentrated in vertebrate muscle (Froehner, S. C., A. A. Murnane, M. Tobler, H. B. Peng, and R. Sealock. 1987. J. Cell Biol. 104:1633-1646). We have studied the 58-kD protein in AChR clusters isolated from cultured rat myotubes. Using immunofluorescence microscopy we show that the 58-kD protein is highly enriched at AChR clusters, but is also present in regions of the myotube membrane lacking AChR. Within clusters, the 58-kD protein codistributes with AChR, and is absent from adjacent membrane domains involved in myotube-substrate contact. Semiquantitative fluorescence measurements suggest that molecules of the 58-kD protein and AChR are present in approximately equal numbers. Differential extraction of peripheral membrane proteins from isolated AChR clusters suggests that the 58-kD protein is more tightly bound to cluster membrane than is actin or spectrin, but less tightly bound than the receptor-associated 43-kD protein. When AChR clusters are disrupted either in intact cells or after isolation, the 58-kD protein still codistributes with AChR. Clusters visualized by electron microscopy after immunogold labeling and quick-freeze, deep-etch replication show that, within AChR clusters, the 58-kD protein is sharply confined to AChR-rich domains, where it is present in a network of filaments lying on the cytoplasmic surface of the membrane. Additional actin filaments overlie, and are attached to, this network. Our results suggest that within AChR domains of clusters, the 58-kD protein lies between AChR and the receptor-associated 43-kD protein, and the membrane-skeletal proteins, beta-spectrin, and actin.     

The relationship of the postsynaptic 43K protein to acetylcholine receptors in receptor clusters isolated from cultured rat myotubes

We have examined the relationship of acetylcholine receptors (AChR) to the Mr 43,000 receptor-associated protein (43K) in the AChR clusters of cultured rat myotubes. Indirect immunofluorescence revealed that the 43K protein was concentrated at the AChR domains of the receptor clusters in intact rat myotubes, in myotube fragments, and in clusters that had been purified approximately 100-fold by extraction with saponin. The association of the 43K protein with clustered AChR was not affected by buffers of high or low ionic strength, by alkaline pHs up to 10, or by chymotrypsin at 10 micrograms/ml. However, the 43K protein was removed from clusters with lithium diiodosalicylate or at alkaline pH (greater than 10). Upon extraction of 43K, several changes were observed in the AChR population. Receptors redistributed in the plane of the muscle membrane in alkali-extracted samples. The number of binding sites accessible to an anti-AChR monoclonal antibody directed against cytoplasmic epitopes (88B) doubled. Receptors became more susceptible to digestion by chymotrypsin, which destroyed the binding sites for the 88B antibody only after 43K was extracted. These results suggest that in isolated AChR clusters the 43K protein covers part of the cytoplasmic domain of AChR and may contribute to the unique distribution of this membrane protein.     

Calmodulin and acetylcholine receptor clustering in embryonic chick myotubes

We have used the calmodulin antagonists, trifluoperazine (TFP) and calmidazolium, to study the potential role of this protein in the movement of acetylcholine receptors (AChRs) to and from the myotube membrane, as well as in the formation of clusters of AChRs within the plasma membrane. Neither calmidazolium (up to 10(-6) M) nor TFP (10(-5) M) inhibited receptor degradation or the incorporation of new receptors (12 to 24 h). In addition, neither drug blocked the increased synthesis of receptors induced by chick brain extract, nor significantly affected AChR clusters already in the plane of the membrane at the time of drug addition. However, both drugs blocked new receptor clusters (induced by a basement membrane extract from Torpedo electric organ) from forming. These results indicate that receptors can move to and from the cell membrane in a calmodulin-independent fashion, but movement in the plane of the membrane to form a cluster requires the participation of calmodulin.     

Disruption and reformation of the acetylcholine receptor clusters of cultured rat myotubes occur in two distinct stages

We have examined the redistribution of acetylcholine receptor (AChR) intramembrane particles (IMPs) when AChR clusters of cultured rat myotubes are experimentally disrupted and allowed to reform. In control myotubes, the AChR IMPs are evenly distributed within the AChR domains of cluster membrane. Shortly after addition of azide to disrupt clusters, IMPs become unevenly scattered, with some microaggregation. After longer treatment, IMPs are depleted from AChR domains with no further change in IMP distribution. Contact domains of clusters are relatively poor in IMPs both before and after cluster dispersal. Upon visualization with fluorescent alpha-bungarotoxin, some AChR in azide-treated samples appear as small, bright spots. These spots do not correspond to microaggregates seen in freeze-fracture replicas, and probably represent receptors that have been internalized. The internalization rate is insufficient to account completely for the loss of IMPs from clusters, however. During reformation of AChR clusters upon removal of azide, IMP concentration in receptor domains increases. At early stages of reformation, IMPs appear in small groups containing compact microaggregates. At later times, AChR domains enlarge and IMPs within them assume the evenly spaced distribution characteristic of control clusters. These observations suggest that the disruption of clusters is accompanied by mobilization of AChR from a fixed array, allowing AChR IMPs to diffuse away from the clusters, to form microaggregates, and to become internalized. Cluster reformation appears to be the reverse of this process. Our results are thus consistent with a two-step model for AChR clustering, in which the concentration of IMPs into a small membrane region precedes their rearrangement into evenly spaced sites.     

Agonists cause endocytosis of nicotinic acetylcholine receptors on cultured myotubes

Regulated trafficking of neurotransmitter receptors in excitable cells may play an important role in synaptic plasticity. In addition, agonist‐induced endocytosis of nicotinic acetylcholine receptors (nAChRs) in particular might be involved in nicotine tolerance and addiction. The existing evidence concerning regulated internalization of cell‐surface nAChRs is indirect and equivocal, however. In the present study, radioligand binding and fluorescence microscopy were used to show that agonists cause substantial endocytosis of nAChRs on cultured myotubes. Exposure to carbachol or nicotine caused a decrease in the intensity of fluorescent labeling of clusters of cell‐surface nAChRs that was blocked by low temperature. Overall, myotubes exposed to carbachol or nicotine bound 50–70% less [¹²⁵I]‐α‐bungarotoxin on the cell surface than untreated cells. The effect of carbachol was significant within 5 min, increased progressively for at least 4 h, and had a sensitivity of 100 nM or less. Exposure to carbachol caused the appearance or dramatic expansion of an intracellular pool of nAChRs, which were localized to discrete, largely perinuclear structures. A pulse‐chase labeling protocol allowed the selective labeling and localization of nAChRs that had been internalized from the cell surface. In untreated cells, very little internalization of nAChRs occurred over a period of 3 h, indicating that constitutive endocytosis of receptors over this period was minimal. Exposure to carbachol, however, caused a dramatic increase in the endocytosis of nAChRs. These results provide direct evidence that agonists, including the tobacco alkaloid nicotine, can cause substantial endocytosis of cell‐surface nAChRs. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 212–223, 2001     

Muscle-derived agrin in cultured myotubes: expression in the basal lamina and at induced acetylcholine receptor clusters.

The synaptic basal lamina (SBL) directs key aspects of the differentiation of regenerating neuromuscular junctions. A range of experiments indicate that agrin or a closely related molecule is stably associated with the SBL and participates in inducing the formation of the postsynaptic apparatus after damage to adult muscle. The selective concentration of agrin-related molecules in the SBL suggests that agrin is secreted locally by cellular components of the nerve-muscle synapse. In vivo studies on aneural embryonic muscle indicate that the muscle cell is one source of the agrin-like molecules in the SBL. Here we have used cultured chick muscle cells to study the expression of agrin-related molecules in the absence of innervation. Immunofluorescence and immunoelectron microscopy show that myogenic cells in culture express agrin-related molecules on their surfaces, and that at least a subset of these molecules are associated with the basal lamina. Moreover, in short term cultures agrin-like molecules accumulate on the surfaces of myogenic cells grown in unsupplemented basal media. We quantified the expression of agrin-like molecules on the cell surface using a solid-phase radioimmune assay. The expression of these molecules is relatively low during the first 6 days of culture and increases fourfold during the second week. The stimulation of the expression of agrin-related molecules in these long-term cultures requires the presence of chick embryo extract or fetal calf serum. We also characterized the expression of muscle-derived agrin-like molecules at clusters of AChR. These agrin-related molecules are not consistently colocalized at spontaneous AChR aggregates; however, they are selectively concentrated at greater than or equal to 90% of the AChR clusters that are induced by Torpedo agrin. These data, together with previous results from in vivo developmental experiments indicate that the agrin-like molecules in the synaptic basal lamina are derived at least in part from the muscle cell. In addition, the expression of agrin-like molecules can be regulated by soluble factors present in CEE and FBS. Finally, the selective localization of these molecules at induced AChR clusters, taken together with their localization in the basal lamina, suggests that agrin-like molecules secreted by the muscle cell play an important role in the formation and/or the stabilization of the postsynaptic apparatus.     

Agents that activate protein kinase C reduce acetylcholine sensitivity in cultured myotubes

We have examined acetylcholine (ACh)-elicited potentials or currents in current- or voltage-clamped cultured myotubes exposed to 12-O- tetradecanoyl-phorbol-13-acetate (TPA), a potent tumor promoter that activates protein kinase C. Although this agent had little action on either membrane resting potential or electrical resistance, a reversible decrease in ACh sensitivity was induced on 3-4-d-old chick myotubes. Depression of transmitter action by TPA was extended to 7-8-d mouse myotubes only when they were treated with phosphatidylserine. Glyceryl dioleate had effects on myotubes similar to those of TPA but with a reduced efficacy. We conclude that the activation of protein kinase C might be involved with the capacity of ACh receptors to respond to transmitter stimulation.     

Dispersal and reformation of acetylcholine receptor clusters of cultured rat myotubes treated with inhibitors of energy metabolism

The effects of energy metabolism inhibitors on the distribution of acetylcholine receptors (AChRs) in the surface membranes of non-innervated, cultured rat myotubes were studied by visualizing the AChRs with monotetramethylrhodamine-alpha-bungarotoxin. Incubation of myotubes with inhibitors of energy metabolism causes a large decrease in the fraction of myotubes displaying clusters of AChR. This decrease is reversible, and is dependent on temperature, the concentration of inhibitor, and the duration of treatment. Cluster dispersal is probably not the result of secondary effects on Ca++ or cyclic nucleotide metabolism, membrane potential, cytoskeletal elements, or protein synthesis. Sequential observations of identified cells treated with sodium azide showed that clusters appear to disperse by movements of receptors within the sarcolemma without accompanying changes in cell shape. AChR clusters dispersed by pretreating cells with sodium azide rapidly reform upon removal of the inhibitor. Reclustering involves the formation of small aggregates of AChR, which act as foci for further aggregation and which appear to be precursors of large AChR clusters. Small AChR aggregates also appear to be precursors of clusters which form on myotubes never exposed to azide. Reclustering after azide treatment does not necessarily occur at the same sites occupied by clusters before dispersal, nor does it employ only receptors which had previously been in clusters. Cluster reformation can be blocked by cycloheximide, colchicine, and drugs which alter the intracellular cation composition.     

Agonists cause endocytosis of nicotinic acetylcholine receptors on cultured myotubes.

Regulated trafficking of neurotransmitter receptors in excitable cells may play an important role in synaptic plasticity. In addition, agonist-induced endocytosis of nicotinic acetylcholine receptors (nAChRs) in particular might be involved in nicotine tolerance and addiction. The existing evidence concerning regulated internalization of cell-surface nAChRs is indirect and equivocal, however. In the present study, radioligand binding and fluorescence microscopy were used to show that agonists cause substantial endocytosis of nAChRs on cultured myotubes. Exposure to carbachol or nicotine caused a decrease in the intensity of fluorescent labeling of clusters of cell-surface nAChRs that was blocked by low temperature. Overall, myotubes exposed to carbachol or nicotine bound 50-70% less [(125)I]-alpha-bungarotoxin on the cell surface than untreated cells. The effect of carbachol was significant within 5 min, increased progressively for at least 4 h, and had a sensitivity of 100 nM or less. Exposure to carbachol caused the appearance or dramatic expansion of an intracellular pool of nAChRs, which were localized to discrete, largely perinuclear structures. A pulse-chase labeling protocol allowed the selective labeling and localization of nAChRs that had been internalized from the cell surface. In untreated cells, very little internalization of nAChRs occurred over a period of 3 h, indicating that constitutive endocytosis of receptors over this period was minimal. Exposure to carbachol, however, caused a dramatic increase in the endocytosis of nAChRs. These results provide direct evidence that agonists, including the tobacco alkaloid nicotine, can cause substantial endocytosis of cell-surface nAChRs.     

Specificity of neuronal factors which aggregate acetylcholine receptors on cultured myotubes

Neuronal factors from conditioned medium of neuroblastoma X glioma hybrid cells or isolated from embryonic pig brain aggregate acetylcholine receptors (AChR) on cultured chicken and rat myotubes. A membrane surface protein labelled with a fluorescent monospecific antibody was not aggregated with the same treatment. Antibodies against AChR block the action of the aggregating factors but do not produce large aggregates themselves. These findings indicate that the factors specifically react with the AChR on developing myotubes.     

Reorganization and stabilization of acetylcholine receptor aggregates on rat myotubes

Aggregation of acetylcholine receptors (AChRs) is an important early feature of the postsynaptic development of the vertebrae neuromuscular junction. At later stages of differentiation, aggregates are remodeled and stabilized. Aggregation of AChRs can be induced on rat myotubes in culture within 4 hr by treatment with embryonic pig brain extract (EBX). In this study, further sequential changes in the distribution of AChRs were followed by video-intensified fluorescence microscopy. These studies have revealed that groups of AChR aggregates that have formed after 4 hr in EBX are reorganized during the exposure to EBX for 20 additional hr to form a smaller number of larger, oval-shaped aggregates. We have named these two types of aggregates "4-hr aggregates" and "24-hr aggregates". This reorganization occurs by the expansion and merging of individual aggregates within a group, and by the incorporation of newly inserted AChRs. The 24-hr aggregates are an average of 15 times greater in area than 4-hr aggregates, and contain regions with an apparent AChR site density (fluorescence intensity) that is more than twice that of 4-hr aggregates. Electron microscopy of mapped 24-hr aggregates revealed that folded plasma membrane is associated with these regions, probably accounting for the elevated fluorescence. The 24-hr aggregates are more stable than 4-hr aggregates, as determined by their significantly slower disassembly after removal of EBX, elevation of temperature (38 degrees C), reduction of extracellular calcium levels (0.1 mM), or the addition of sodium azide (7 mM). This was determined by following disassembly both statistically (using fixed cultures) and by direct observations of living myotubes. These findings were confirmed by measuring the sequential changes in relative AChR site density over time in individual living myotubes. Thus, 24-hr aggregates form by the reorganization of 4-hr aggregates; exhibit a more regular, compact shape; and are more stable than 4-hr aggregates. These changes in AChR organization and aggregate stability resemble the changes occurring after the initial formation of junctional AChR aggregates during embryonic development, demonstrating additional similarities between this model system and the developing neuromuscular junction.     

Early stages in the formation and stabilization of acetylcholine receptor aggregates on cultured myotubes: sensitivity to temperature and azide

We have studied the effects of temperature and sodium azide on the formation and stability of embryonic brain extract (EBX)2-induced acetylcholine receptor (AChR) aggregates on myotubes. Sequential changes in AChR distribution were studied on living myotubes in culture by video-intensified fluorescence microscopy. Aggregate formation was temperature dependent, increasing sharply from 24-36 degrees, maximal at 36-37 degrees, and virtually blocked at 38-40 degrees. Whereas aggregate size increased rapidly with time (up to 4 hr) at 36 degrees, at 18-24 degrees small (less than or equal to 1 micron) "microaggregates" formed and accumulated for up to 10 hr. Aggregates formed within 1.5 hr at the sites of microaggregates (formed after 4 hr at 23 degrees) if the temperature was raised to 36 degrees. However, if EBX was removed, the microaggregates on 50% of myotubes disassembled within 1.5 hr. The formation of microaggregates at 23 degrees and aggregates at 36 degrees was reversibly inhibited by sodium azide. These results show that clusters of microaggregates are the precursors of aggregates, and suggest that microaggregate clouds represent a discrete, labile, ATP-dependent stage in aggregate formation. Aggregates that had formed after 4 hr in the presence of EBX disassembled slowly (within 12-14 hr) following removal of EBX at 36 degrees, and even more slowly at 23-30 degrees. However, a temperature shift to 38 degrees, or the addition of azide, resulted in a rapid but reversible disassembly of aggregates (within 4 hr). Thus, newly formed aggregates appear to be relatively stable structures, while microaggregate clouds are labile, tending to disassemble or evolve into aggregates.