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Article |
Address correspondence to Joshua R. Sanes, Dept. of Anatomy and Neurobiology, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: (314) 362-2507. Fax: (314) 747-1150. email: sanesj{at}pcg.wustl.edu
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Abstract |
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Key Words: acetylcholine receptor; laminin; muscle; neuromuscular junction; synapse formation
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Introduction |
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It has long been known that myotubes cultured in the absence of neurons form small, specialized domains, sometimes called "hot spots" or "plaques," in which AChRs cluster at high density (Vogel et al., 1972; Fischbach and Cohen, 1973; Anderson and Cohen, 1977; Bloch, 1979). This result, coupled with recent genetic studies in vivo, led to the idea that muscle cells can initiate postsynaptic differentiation independent of the nerve (Sanes and Lichtman, 2001; Arber et al., 2002). In contrast, the topologically complex pretzel-like arrays typical of the mature synapse have never been observed to form in the absence of innervation either in vitro or in vivo. Additionally, neurites contacting myotubes in vitro can induce AChR clusters with shapes mirroring those of the neuritemyotube contact (Anderson and Cohen, 1977; Frank and Fischbach, 1979), and neonatal denervation in vivo arrests the morphological maturation of AChR aggregates (Slater 1982b; Moss and Schuetze, 1987). Based on these observations, it is generally assumed that the branching pattern of the motor axon dictates the topology of the mature postsynaptic apparatus.
Here, we present data that call this assumption into question. While studying AChR clustering in aneural, cultured myotubes, we found conditions that promoted formation of complex, branched receptor aggregates resembling those found at adult NMJs. Further analysis revealed many parallels between these aggregates and the adult postsynaptic apparatus, including association with a similar set of molecular specializations, dependence on rapsyn and the muscle-specific kinase (MuSK) for their formation, and maturation through a similar series of transitional forms. We then exploited the experimental accessibility of the preparation to document the steps and patterns of AChR addition and removal that transform the plaque into a pretzel. Finally, we performed experiments in vivo to show that patterns of AChR addition discovered in vitro actually occur during synaptogenesis. Together, these results reveal nerve-independent mechanisms that can specify many features of the mature postsynaptic apparatus, and raise the possibility that the muscle plays an instructive role in shaping the nerve terminal arbor as the synapse matures.
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Results |
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MuSK- and rapsyn-dependent formation of aneural aggregates
Clustering of AChRs at the NMJ requires MuSK; no AChR clusters form in muscles of MuSK-/- or rapsyn-/- mice (DeChiara et al., 1996; Sanes and Lichtman, 2001). On the other hand, some agents, including soluble laminin, can stimulate MuSK-independent clustering of AChRs in cultured myotubes (Sugiyama et al., 1997; Gautam et al., 1999). At least in this respect, soluble laminin clusters AChRs by a mechanism of unknown physiological significance. To ask whether laminin-coated substrata promoted formation of aggregates by the physiologically relevant MuSK-dependent mechanism, we used a myogenic cell line derived from MuSK-/- mice. These muscle cells form small AChR clusters when treated with soluble laminin (Sugiyama et al., 1997), but did not form AChR clusters when cultured on laminin-coated substrates (Fig. 2 A, red cells). Transfection of the cells with GFP-tagged MuSK, however, restored their ability to form complex AChR aggregates (Fig. 2, A and B, green cell). Thus, unlike bath-applied laminin but like the mechanisms responsible for receptor clustering at the NMJ, substrate-bound laminin requires MuSK to form aneural AChR aggregates. We also tested myotubes from rapsyn-/- mice, because rapsyn, like MuSK, is required for all AChR clustering in vivo. Rapsyn-/- myotubes cultured on laminin-coated substrates formed no receptor clusters, although control cells did (Fig. 2, C and D).
AChR clusters are dramatically perturbed in mice lacking agrin, which activates MuSK (Gautam et al., 1996; Burgess et al., 1999). Agrin is expressed by myotubes as well as by motorneurons, but only the latter synthesize the alternatively spliced z-plus agrin isoform which is 1,000-fold more effective than z-minus agrin in promoting AChR clustering (Ferns et al., 1992; Gesemann et al., 1995). Nonetheless, z-minus agrin does have detectable clustering activity in some assays (Ferns et al., 1992). Therefore, we considered the possibility that matrix-coated substrates might act by concentrating muscle-derived z-minus agrin, or even by inducing expression of z-plus agrin. To test this hypothesis, we assayed primary myotubes cultured from mice lacking all forms of agrin (Lin et al., 2001). When such cells were grown on laminin-coated substrates, AChR clusters formed that were identical to those observed on myotubes prepared from control littermates (Fig. 2, E and F). This result is consistent with the finding that MuSK can be activated in the absence of agrin (Zhou et al., 1999; Lin et al., 2001). Likewise, adding soluble agrin to myotubes cultured on gelatin increased the number of plaques but never led to formation of more complex aggregates (unpublished data).
We also asked if formation of aneural pretzels was a specific response to laminin. Neither gelatin nor polyornithine induced aneural pretzel formation (Fig. 1 A and Fig. 2 G). However, branched structures were observed when myotubes were cultured on a substrate coated with fibronectin (Fig. 2 H). These clusters were smaller than those formed on laminin, and had more jagged edges, but their presence demonstrated that the ability to induce branched aggregates is not unique to laminin. Instead, it may be that strong adhesion promotes topological maturation (see Discussion). Consistent with this possibility, myotubes adhered more strongly to laminin or fibronectin than to gelatin or polyornithine (as determined by susceptibility to mechanical detachment; not depicted), and myotubes detached from laminin-coated substrates left behind patterned AChR aggregates, presumably embedded in adherent membrane fragments (Fig. 2 I).
Molecular features shared by aneural aggregates and NMJs
To compare the molecular architecture of aneural aggregates and NMJs, we used a panel of probes to matrix, membrane, and cytoplasmic proteins known to be concentrated at synaptic sites in vivo (Sanes and Lichtman, 1999, 2001). The distribution of rapsyn, as revealed by immunostaining, precisely matched that of AChRs in aggregates, as it does in vivo (Fig. 3 A; Sanes and Lichtman, 1999, 2001). Similarly, MuSK was enriched in AChR-rich areas, as monitored by the localization of GFP-MuSK in transfected myotubes (Fig. 2 B), although some GFP-MuSK was present in AChR-poor areas, perhaps as a consequence of overexpression. Likewise, phosphotyrosine staining was increased at areas rich in AChRs (Fig. 3 B; Qu et al., 1990). Thus, signaling components involved in NMJ differentiation are associated with aneural aggregates.
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Association of AChR aggregates with specialized nuclei
As NMJs mature in vivo, myonuclei cluster beneath the postsynaptic apparatus. Staining with a nuclear dye revealed clusters of nuclei associated with AChR aggregates in vitro (Fig. 4 A). On average, nuclear density was approximately threefold higher beneath aggregates than elsewhere in the same cells (Fig. 4 B). This result shows that axons are dispensable for local accumulation of myonuclei.
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Differences between aneural aggregates and NMJs
Some components of the mature postsynaptic apparatus were not detected at aneural aggregates. We used fluorescently coupled fasiculin-2 to probe for the presence of acetylcholinesterase, a muscle-derived enzyme localized to the NMJ, but found none at aneural aggregates (unpublished data). Its absence may reflect the fact that acetylcholinesterase accumulation is activity dependent (Rubin et al., 1980). In addition, laminin 1, which is present throughout the muscle fiber basal lamina in vivo, was concentrated at aneural aggregates, and laminin
4, which is concentrated at the mature NMJ, was not detected in vitro (Fig. 3 E and not depicted). There were also two topological features of the mature NMJ that were not evident in the aggregates: the shallow gutters in which AChR-rich branches lie and the deep folds that indent the postsynaptic membrane. The presence of folds has been documented at AChR-rich plaques in aneural myotubes (Sanes et al., 1984) and we cannot exclude the possibility that some are present at branched aggregates, but confocal and interference reflection microscopy failed to detect the striations indicative of multiple, aligned folds (unpublished data; see Marques et al., 2000 for evidence that folds can be visualized by light microscopy). Finally, there is only a single NMJ per adult muscle fiber, but some cultured myotubes bore multiple branched aggregates.
Time-lapse imaging of topological transformations
AChR aggregates at neonatal NMJs are plaque shaped. Later, ring-shaped, C-shaped, and branched forms become successively more prevalent (Slater, 1982a; Marques et al., 2000; Lanuza et al., 2002; Fig. 5, AD). A similar array of forms was present in vitro (Fig. 5, EH). More complex forms were preferentially associated with larger myotubes (Fig. 5 I), suggesting that they appeared sequentially as myotubes matured. To test this idea, we assessed the prevalence of simple and complex aggregates in cultures 36 d after inducing fusion. Aggregates resembling more mature NMJs became more common over time while clusters with simpler morphology became less prevalent (Fig. 5 J). The pace of maturation was much faster, however, in vitro than in vivo, perhaps reflecting the more rapid growth of myotubes in vitro than in vivo.
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Patterns of AChR addition to growing aggregates
The transitions documented in Figs. 5 and 6 must result from some combination of movements, additions, and losses of AChRs. To assess the patterns in which these occur, we performed two additional experiments. In the first, we asked where AChRs were added as the aggregates grew. We saturated surface AChRs with Alexa 594coupled Btx, washed away unbound label, and incubated the cells for 12 h. After this period, cells were incubated with Alexa 488Btx to label receptors inserted into the membrane during the preceding 12 h. After an additional 12 h, we stained with a third spectrally distinct conjugate, Alexa 647Btx.
This experiment revealed three features of AChR addition (Fig. 7, AD). First, each population had a distinct distribution, indicating that once AChRs were added to the cluster, they mixed with other AChR populations to only a limited extent. Second, the separate populations were systematically arranged in order of age, with younger receptors preferentially (although not exclusively) concentrated at the periphery of aggregates. Third, in aggregates that were C-shaped or branched, new AChRs were most abundant at the outer or convex margin. These results suggest complex patterning of the machinery responsible for AChR addition, loss, or mobility.
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We also observed that many of the AChRs present on the surface at t = 0 were subsequently found in intracellular particles, likely to be endocytic vesicles (Fig. 7, F and I; Fig. 6, B, C, and F; see Akaaboune et al., 1999 for discussion of similar observations in vivo). These vesicles were more abundant in areas near aggregates than in aggregate-free areas, suggesting that they were derived from the aggregate itself. This result raises the possibility that holes form at least in part by coupled processes of endocytic removal of AChR-rich membrane and its replacement with AChR-poor membrane.
Patterns of AChR addition in vivo
To learn whether patterns of receptor addition observed in vitro resemble those at normally developing NMJs, we devised the following experiment: mouse pups expressing YFP in all motor axons (Feng et al., 2000) were injected above the sternomastoid muscle with Alexa 647Btx at postnatal day five. After injection, pups were returned to their mothers for 1 d, after which the sternomastoid muscle was removed and labeled with Alexa 594Btx. This protocol revealed a pattern similar to that observed in vitro: AChRs carrying the second tag were concentrated around the periphery of the receptor cluster (Fig. 8). In addition, in aggregates that were open at one side, new AChRs were preferentially added at the outer or convex margin, just as observed in vitro (Fig. 8 A, arrowheads compare with Fig. 7 D). There was more labeling of central areas by the final color in vivo than in vitro (Fig. 7, AD, compare with Fig. 8) but this reflects, at least in part, differences in protocol: AChRs were not saturated by the first color in vivo (to avoid lethality from systemic Btx), nor were the first and final colors separated by an intermediate label. Thus, as seen in vitro, AChRs have limited mobility and are added in an annular, asymmetric fashion to growing end plates.
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Discussion |
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Why were complex aggregates not described previously? Numerous groups (including our own) have analyzed clustering of AChRs in cultured myotubes. However, in most cases myotubes were cultured on uncoated, gelatin-coated, or polycation-coated substrata, which do not promote pretzel formation. In some studies, C2 cells (Kostrominova and Tanzer, 1995; Yao et al., 1996; Adams et al., 1999), other myogenic lines (Ocalan et al., 1988), or primary myoblasts (Foster et al., 1987) were grown on laminin-coated substrates. However, in these cases postsynaptic differentiation was not assessed.
How does the substrate promote the formation of an AChR-rich domain? One possibility is that tight adhesion is critical. First, complex aggregates form exclusively at the substrate interface, and when the interface is larger, aggregates are more complex. Second, when myotubes are mechanically detached from the substrate, they leave behind postsynaptic "ghosts" enriched in AChRs, indicating a direct, tight interaction with the substrate. Third, the effect is not laminin specific: aggregates also form on fibronectin. Because fibronectin engages a largely nonoverlapping set of cell surface receptors (4ß1 and
5ß1 integrins versus the major laminin receptors
7ß1 integrin and dystroglycan; Plow et al., 2000), the effect is likely not dependent on any specific receptor, although it might require integrin signaling generally. Finally, the AChR clustering effects of bath-applied laminin, which are receptor mediated, do not require MuSK (Sugiyama et al., 1997; Burkin et al., 1998; Marangi et al., 2002), whereas pretzel formation is MuSK dependent. Together, these data suggest that substrate-bound laminin does not act through established laminin-dependent AChR clustering pathways or a unique matrix receptor, but rather supplies a sufficiently adhesive substrate to trigger muscle-intrinsic machinery.
How might adhesion initiate postsynaptic differentiation? One possibility is that the primary effect of the adhesive substrate is to cluster and thereby activate MuSK. Multimerization activates many receptor tyrosine kinases, and for MuSK in particular, clustering with antibodies (Xie et al., 1997) or by coexpression with rapsyn (Gillespie et al., 1996) promotes agrin-independent activation. Alternatively, the interaction may be autocrine; laminin may provide a scaffold upon which the myotube deposits components normally concentrated in the synaptic cleft (Fig. 3), some of which might activate MuSK. Indeed, although it was originally thought that agrin alone activates MuSK, agrin-independent but MuSK-dependent postsynaptic differentiation has now been documented in vivo (Lin et al., 2001; Sander et al., 2001; Yang et al., 2001).
Mechanisms of postsynaptic maturation
A key question raised by this study is how a uniform coat of laminin leads to formation of an elaborately patterned array. Why does the branched array resemble a pretzel rather than, for example, a snowflake? Because the sequence of steps by which patterns form in aneural myotubes is similar to that observed in vivo (Figs. 58), similar mechanisms may be involved, in which case analysis in the accessible culture system might help us to understand synaptic maturation. Our observations have revealed a set of cellular phenomena that can guide future studies of molecular mechanisms that regulate this maturation.
First, branches form from plaques in a multistep process that involves the appearance of AChR-poor perforations, their asymmetric expansion, and fusion at discrete sites along shared borders. Although individually simple and few in number, combination and iteration of these steps provides a way to understand how the highly complex topology of the adult NMJ is generated. Moreover, small differences among aggregates in the number, spacing, growth, and fusion pattern of perforations can explain the great topological variation among NMJs.
Second, the formation of AChR-poor perforations within a plaque implies the existence of a mechanism for local disappearance of AChRs without a large-scale change in the AChR density nearby. Local endocytosis is a candidate mechanism for this local removal. Endocytosis has been shown to participate in the turnover of unclustered AChRs and the dispersal of AChR plaques on cultured myotubes (Pumplin and Bloch, 1987; St. John and Gordon, 2001). Our observation that AChR-containing intracellular vesicles are conspicuous as plaques perforate is consistent with the possibility that locally controlled endocytosis may act to sculpt aggregates. Current studies are aimed at documenting the topology of endocytosis during aggregate remodeling.
Third, the AChR dynamics we observed provide a way to understand how AChR-poor perforations elaborate: new AChRs are added to aggregates circumferentially, and there is limited mixing of receptor populations added at different times. As a consequence of the limited intermixing, areas that become AChR poor remain AChR poor. As a consequence of peripheral addition, lost AChRs are replaced in a manner that leads to overall aggregate growth while preserving the central AChR-poor perforations. AChRs exhibit at least some similar behaviors in vivo: Akaaboune et al. (2002) showed that mobility of AChRs at adult NMJs is limited; we were able to show constrained mobility and circumferential addition of AChRs at developing NMJs (Fig. 8); and a study by Weinberg et al. (1981) argued for a similar pattern during growth of synapses in reinnervated adult muscle based on autoradiographic techniques.
What is the source of the patterning information? An attractive possibility is that patterns result from a self-assembly process in which cytoskeletal rearrangements drive membrane remodeling. One possible parallel is the podosome belt, a geometrically complex adhesive structure formed in another large, multinucleated cell, the osteoclast (Marchisio et al., 1984). Recent studies of these structures have provided evidence that their growth and maturation involves actin polymerization and depolymerization organized by integrins; ring formation; centrifugal expansion driven by asymmetric addition of new podosome subunits; and microtubule-based stabilization at the cell periphery (Destaing et al., 2003; Evans et al., 2003; for review see Linder and Aepfelbacher, 2003). Similarly, actin-driven movements and actin-organizing molecules (e.g., rho GTPases) have been implicated in AChR clustering (Bloch and Pumplin, 1988; Dai et al., 2000; Weston et al., 2003), although their roles in generating complex topology have not heretofore been considered. Unfortunately, although actin (stained with phalloidin) is concentrated in regions of complex AChR aggregates, attempts to demonstrate that other components of focal adhesion plaques, podosomes, or endocytic compartments (e.g., paxillin, integrin subunits, or rab5a) colocalize with the aggregates have so far been unsuccessful (unpublished data). Therefore, we do not yet have satisfying insights into the machinery that assembles these structures.
The patterns of receptor addition we observed may be common to several types of junctions. A recent study of gap junctions demonstrated that they grow by addition of subunits (connexins) in an "inside-out" pattern parallel to that documented here for AChRs (Gaietta et al., 2002). Similar processes may occur at neuronneuron synapses, but their small size has so far made it impossible to learn where receptors are added or removed as these synapses grow and remodel (Passafaro et al., 2001; Rosenberg et al., 2001). In contrast, the large size and accessibility of the aggregates we describe allow these issues to be addressed.
A postsynaptic influence on presynaptic topology?
The observation that aneural myotubes form AChR aggregates with similar morphology to those at NMJs raises the possibility that postsynaptic topology may influence presynaptic branch patterns rather than (or in addition to) the other way around. The obvious difficulty with this view is that it appears inconsistent with the sequence of events that occurs during normal development, in which the axon forms a rudimentary terminal arbor before the postsynaptic apparatus becomes branched (Sanes and Lichtman, 1999). In fact, however, many previous observations are consistent with a postsynaptic influence on presynaptic morphology. First, during normal development, some AChR-rich plaques form on portions of the myotube surface that axons have not yet contacted (Dahm and Landmesser, 1991), and even when embryonic muscles are rendered aneural in vivo, AChR plaques form in the central region that axons would have contacted (Lin et al., 2001; Yang et al., 2001; Arber et al., 2002). Thus, growing axons may sometimes contact preformed AChR clusters, rather than inducing new ones. Second, early topological transitions, including the appearance of receptor-poor perforations in the plaque, often precede local axonal remodeling (Balice-Gordon and Lichtman, 1993). This sequence has led to the hypothesis that loss of postsynaptic specializations from a restricted region leads to withdrawal of an axonal branch from that region. Third, likewise, regions of developing NMJs that are vacated by retracting axons are often reoccupied by other axon branches, a process dubbed "synaptic takeover" (Walsh and Lichtman, 2003). Fourth, when motor axons contact MuSK-/- or rapsyn-/- myotubes, in which postsynaptic differentiation is blocked, they fail to form arbors (Nguyen et al., 2000). Fifth, although neonatal denervation largely arrests postsynaptic maturation, some AChR-poor holes do appear and grow even after denervation (Slater, 1982b; unpublished data). Sixth, growth of muscle fibers during late stages of maturation or in response to androgens results in symmetrical enlargement of the postsynaptic apparatus, which is matched by presynaptic growth, maintaining precise alignment of pre- and postsynaptic structures (Balice-Gordon et al., 1990). Finally, during reinnervation, the pattern of nerve terminal branches is determined by postsynaptic morphology (Sanes et al., 1978; Rich and Lichtman, 1989). Thus, there is reason to believe that muscle-intrinsic patterning of the postsynaptic array could determine the pattern of axon terminal branches during development.
Finally, we emphasize that the ability of the myotube to generate an NMJ-like pattern does not preclude a critical role for the nerve. Postsynaptic structures may not only exert retrograde influences on nerve terminal branches but also receive anterograde influences from the nerve. A precedent is provided by studies in which focal inactivation of AChRs in small regions of adult junctions, interpreted as mimicking focal loss of neurotransmission, resulted in focal loss of AChRs, followed by withdrawal of overlying axonal branches (Balice-Gordon and Lichtman, 1994). During development, for example, areas of decreased synaptic efficacy could lead to local loss of AChRs and associated matrix molecules, which would act back to cause withdrawal of presynaptic branches. Similarly, the open side of the AChR cluster might form randomly in isolation, but locally restricted activity due to myelination of the preterminal axon may bias the polarization of the cluster in vivo. This could account for the observation that the nerve nearly always enters the NMJ from the open side (Marques et al., 2000).
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Materials and methods |
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Cells were trypsinized and replated onto 8-well Permanox chamber slides (Nalge International) for histological studies or onto 60-mm Permanox dishes for time-lapse imaging. Before plating, dishes were coated with 5 µg/ml polyornithine (Sigma-Aldrich) in distilled water for 30 min and air dried. For laminin or fibronectin coating, a 10 µg/ml solution of EHS laminin (Invitrogen) or a 20 µg/ml solution of human fibronectin (Sigma-Aldrich) in L-15 medium supplemented with 0.2% NaHCO3 was incubated over polyornithine-coated dishes overnight at 37°C, and aspirated immediately before plating cells. Myoblasts were grown to confluency and switched to DME plus 2% horse serum with penicillin and streptomycin to induce fusion. Cells were incubated at 37°C, 5% CO2 for 37 d after fusion. For MuSK rescue experiments, MuSK-/- myoblasts were transfected with either GFP-MuSK or GFP alone using FuGENE 6 transfection reagent (Roche) at the time of plating (Zhou et al., 1999).
Analysis of cultured myotubes
Live myotubes were incubated with 1 µg/ml of fluorescently coupled Btx (Molecular Probes) for 30 min to label AChRs. The cells were washed with PBS, fixed in 4% PFA in PBS, and washed again in PBS before mounting in glycerol plus 1 mg/ml paraphenylenediamine for immediate visualization or scavenged with 50 mM lysine in PBS plus 0.1% (vol/vol) Triton X-100 for immunostaining. Nonspecific staining was blocked with a solution of 2% BSA and 2% goat serum in PBS plus 0.1% Triton X-100 before overnight incubation with rabbit anti-GFP (CHEMICON International, Inc.), rabbit anti-rapsyn (Gautam et al., 1995), mouse antiphosphotyrosine (clone PY20; BD Transduction Labs), rabbit antiß2 laminin (a gift from R. Timpl, Max Planck Institut of Biochemistry, Martinsried, Germany), rabbit anti5 laminin (Miner et al., 1997), rat anti
1 laminin (clone 1914; CHEMICON International, Inc.), or rabbit antisyne-1 (Apel et al., 2000). In the case of
1 staining, cells were cultured on a substrate of 10 µg/ml of rat laminin-1 (Telios Pharmaceuticals,) with which the antibody does not cross react. Primary antibodies were detected with Cy3- or Alexa 488coupled goat secondary antibodies (Molecular Probes). In some cases cells were incubated with DAPI (Molecular Probes) for 5 min to visualize nuclei.
To distinguish newly inserted AChRs from older receptors in culture, we stained myotubes with Alexa 594Btx for 30 min, washed the cells with differentiation media, and returned them to the incubator for 1224 h. Newly inserted AChRs were then stained with Alexa 488Btx and the cells were collected. For some experiments we used Alexa 647Btx as a third label after an additional 12 h of incubation.
Epifluorescence images of fixed cells were collected on an Axioplan2 microscope (Carl Zeiss MicroImaging, Inc.) fitted with a MagnaFire CCD camera (Optronics). Multiple time point observations of individual AChR aggregates labeled with Btx were collected on a microscope (model BX50; Olympus) equipped with a cooled CCD camera (model MicroMax; Princeton Instruments Inc.) and a 100X dipping cone water immersion lens (NA 1.0). To reduce phototoxicity, the myotubes were kept in phenol red-free medium buffered with Hepes for the duration of the imaging sessions. Between time points the cells were returned to the incubator in their differentiation medium.
Confocal images were obtained on a confocal laser scanning microscope (model FV500; Olympus) equipped with Krypton/Argon/HeNe lasers using a 60X oil objective (NA 1.4). Images were analyzed with Metamorph software (Universal Imaging Corp.) and edited with Adobe Photoshop version 6. All confocal images are z stacks flattened in Metamorph. Nuclear density in postsynaptic regions was determined by counting nuclei enclosed within an elliptical border fitted to each AChR cluster. Extrasynaptic nuclear density was measured similarly for regions lacking AChR clusters.
Analysis of the NMJ
Muscles were dissected from transgenic mice in which all motor axons are marked with YFP (line YFP-16; Feng et al., 2000). Muscles were fixed overnight in 4% PFA, washed, and stained with 1 µg/ml Alexa 594Btx overnight to label AChRs. To distinguish newly inserted AChRs from older receptors in vivo, postnatal day five YFP-16 mouse pups were injected near the sternomastoid muscle with 100 µl of a 1 µg/ml solution of Alexa 647Btx in sterile saline. Pups were returned to their mothers for 1 d before they were killed and the sternomastoid fixed and stained with Alexa 594Btx. Images were obtained as described previously (Analysis of cultured myotubes).
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Acknowledgments |
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Submitted: 22 January 2004
Accepted: 18 February 2004
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References |
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