Proc. Nate Acad. Sci. USAVol. 80, pp. 449-453, January 1983Cell Biology

Diffusely distributed acetylcholine receptors can participate incluster formation on cultured rat myotubes

(fluorescence photobleaching recovery/mobility/cell surface/diffusion/patches)

M. STYA AND D. AXELRODBiophysics Research Division, University of Michigan, Ann Arbor, Michigan 48109

Communicated by J. L. Oncley, October 25, 1982

ABSTRACT On aneurally cultured rat primary myotubes,10% of the acetylcholine receptors (AcChoR) are found to be ag-gregated and immobilized in endogenous clusters while the re-mainder are diffusely distributed and partially mobile. This paperreports that AcChoR in clusters can be gathered from AcChoR indiffuse areas during the course of normal myotube development.AcChoR were fluorescently labeled with rhodamine-conjugateda-bungarotoxin, and all existing clusters in a circumscribed regionof the culture dish were irreversibly photobleached by a slightlydefocused laser beam, the movement of which was controlled bya lens mounted on a joystick translator. This procedure leaves in-tact only the fluorescent label on the diffusely distributed Ac-ChoR. Observation of the myotubes after several hours of incu-bation revealed cluster fluorescence redevelopment. This clusterfluorescence must have consisted ofAcChoR that previously werediffusely distributed. The majority (but not all) of cluster fluo-rescence redevelopment occurred in the location of a previouslybleached cluster. About half of the redeveloped clusters have anannular shape. The major conclusions of this study are (i) diffuselydistributed AcChoR can become clustered; (ii) endogenous clustersappear to form, at least in part, by "trapping" receptors as theydiffuse in from surrounding regions; (iii) cluster formation is anongoing process in cultured rat myotubes; and (iv) colchicine (amicrotubule-disrupting agent) inhibits cluster formation.

Both lateral mobility and immobility of acetylcholine receptors(AcChoR) play a role in the establishment and maintenance ofmyoneural junctions. Anderson and Cohen (1) have shown thatthe AcChoR are gathered into clusters at the point where anerve contacts a muscle cell in an amphibian culture system;this redistribution of receptors indicates that the AcChoR arefree to move in the membrane. Most of these nerve-musclecontacts go on to become functional synapses at which theAcChoR are immobilized.On aneurally cultured rat myotubes, approximately 10% of

the AcChoR are found to be aggregated in endogenous clusters(also called "patches") while the remainder are uniformly dis-tributed over the entire membrane. Some ofthe uniformly dis-tributed AcChoR are mobile while those in clusters are virtuallyimmobile (2).The mechanism underlying the immobilization of clustered

AcChoR is not yet understood. Some workers have postulatedthe existence of an immobile intramembrane or submembranefilamentous cytoskeleton that anchors the immobile AcChoR(3). It should be noted, however, that microtubule- and micro-filament-disrupting agents have no effect on the mobility ofAcChoR, whether mobile or immobilized in clusters (4, 5).

It is also possible that the difference in mobility reflects adifference in the structure or biochemistry of the receptors.

Indeed, biochemical differences between two coexisting classesof receptors, junctional and extrajunctional AcChoR on dener-vated adult muscle fibers, have been reported (for review, seerefs. 6 and 7).

Whatever mechanism is involved in the immobilization ofclustered AcChoR, it is of interest to know whether such clus-tered receptors are uniquely capable of aggregation or, alter-natively, whether they can exchange with the diffusely distrib-uted ones in the cell membrane.We here examine the ability ofdiffusely distributed AcChoR

to become incorporated into clusters during normal cell devel-opment in culture. Using a variation of the fluorescence pho-tobleaching recovery technique, we show that (i) diffusely dis-tributed AcChoR can become clustered; (ii) endogenous clustersappear to form, at least in part, by "trapping" receptors as theydiffuse from the surrounding regions; (iii) cluster formation isan ongoing process in cultured myotubes; and (iv) the presenceof colchicine (a microtubule-disrupting agent) inhibits clusterformation.

MATERIALS AND METHODSCulture Preparation and Treatment. Primary rat myotube

cultures were prepared as described (8) and were plated indishes with glass coverslip bottoms in 2 ml of Dulbecco's mod-ified Eagle's medium/10% fetal calfserum (both from GIBCO)/0.5% gentamycin (at 1 mg/ml in stock solution; Schering, Ken-ilworth, NJ) containing tetrodotoxin (TTX; 0.6 Ag/ml; Sigma).Medium was not changed after plating. Experiments were car-ried out on myotubes in 6- to 7-day-old cultures.

Fluorescent Toxins. Toxins were fluorescently labeled asdescribed by Ravdin and Axelrod (9). Myotube cultures wereexposed to tetramethylrhodamine-labeled a-bungarotoxin (R-BTX; toxin obtained from Miami Serpentarium) in medium at37°C for 1 hr and then washed several times in phosphate-buff-ered saline (Pi/NaCl; GIBCO) containing TTX at 0.6 jig/ml.R-BTX labeling is irreversible on the time scale of our experi-ments.

Selective Photobleaching. Fluorescence of cells was viewedwith an inverted microscope (Leitz Diavert), using either aX50, n.a. = 1.00, water-immersion objective or a X40, n.a.= 0.75, phase-contrast water-immersion objective and an argonion laser set at A = 514 nm. A small (4-mm2) region was delin-eated on each culture dish by scraping off the surrounding cells.Within the circumscribed region, patches were located by ob-serving the cells under epi-illumination by a completely defo-cused laser beam (power = 0.1 W). Photobleaching of wholeclusters was carried out with the laser power unchanged but the

Abbreviations: AcChoR, acetylcholine receptor(s); BTX, a-bungaro-toxin; R-BTX, rhodamine-labeled BTX; R-CTX, rhodamine-labeled a-cobratoxin; TTX, tetrodotoxin; Pi/NaCl, phosphate-buffered saline; F-BTX, fluorescein-labeled BTX.

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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450 Cell Biology: Stya and Axelrod

FOCUSING LENS ONJOYSTICK TRANSLATOR

FIG. 1. Optical and electronic apparatus.

beam converged to a slightly out of focus spot (radius at e-2 in-tensity 5 Aum) on the cell membrane. A focusing lens mountedon a joystick translater (Fig. 1) made it possible to sweep thisspot across any region of the myotube membrane, thus per-mitting selective and irreversible bleaching of the fluorescenceof the clustered AcChoR while leaving the fluorescence of thediffusely distributed ones intact.

After all the clusters in the circumscribed region werebleached, the cells were returned to complete medium and in-cubated at 37TC for 6.5-7.5 hr. At the end of this "recoveryperiod," the cells were washed several times with Pi/NaCl/TTX and then observed with epi-illumination by a completelydefocused laser beam at the same power.

In some experiments, cells were relabeled with R-BTX afterthe post-recovery observation to label the AcChoR that hadbeen incorporated into the membrane since the time of firstlabeling.

Video Camera and Photography Mural. The circumscribedregion on one culture dish was photographed in its entirety atthree points in the experiment. Photography was done beforethe patches were bleached, at the end of the recovery period,and after the cells had been relabeled for 1 hr with R-BTX. Tolimit the length oftime the cells were exposed to laser light (i. e.,to limit the amount of nonselective bleaching of the fluores-cence label), the photography was carried out as a two-step pro-cess. First, the cells were viewed with a silicon-intensified tar-get video camera (Cohu 4400); the field of view was recordedon a video tape recorder (Panasonic NV 8030) and simulta-neously displayed on a television monitor (Panasonic WV 5300).By moving the culture dish on the stage of the microscope, theregion ofinterest could be scanned completely. Later, the videotape was played back and the television screen was photo-graphed with a 35-mm camera with Tri-X film (Kodak). Nega-tives were contact printed, and the resulting small prints wereglued together to reconstruct a mural of the entire circum-scribed region.

RESULTSR-BTX-labeled AcChoR clusters before and immediately afterphotobleaching are shown in Fig. 2. After the 6.5- to 7.5-hrrecovery period, a number of faint clusters could be seen. Be-

FIG. 2. (A) Two unbleached clusters. (B) Upper cluster has beenbleached. The laser light required to photograph the clusters the firsttime also caused some nonspecific photobleaching of the probe. Hence,the intensity of the unbleached cluster is less in B than in A. Bar =50 jAm.

Proc. Nad Acad. Sci. USA 80 (1983)

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Proc. NatL Acad. Sci. USA 80 (1983) 451

cause all of the clustered AcChoR fluorescence was previouslybleached, we conclude that this redeveloped cluster fluores-cence must have arisen from previously diffusely distributedAcChoR. Because the BTX-AcChoR complex is extremely sta-ble (10), it is unlikely that this redeveloped cluster fluorescenceis due to dissociation and rebinding of R-BTX. During 31 ex-periments, 3,063 clusters were bleached and, after recovery,622 clusters, 20% of the number originally bleached, were ob-served. The number of reformed clusters varied with each ex-periment, ranging from 4% to 46% of the number originallybleached. As shown in Fig. 3, these clusters were generallyfaint, but the internal structure of lines and speckles that is char-acteristic of endogenous clusters was unmistakably present.On the other hand, when the recovery period after photo-

bleaching was only a 0.5-1.0 hr, very few clusters were seen.During five experiments, 856 clusters were bleached and 40were observed after recovery, 5% of the number originallybleached (range: 4-7% of the number originally bleached).These clusters generally lacked the internal structure charac-teristic of endogenous clusters.When colchicine, a known microtubule disrupter, was added

to the medium during the recovery period, the number ofclus-ters observed at the end of the 6.5- to 7.5-hr recovery perioddecreased sharply and in a dose-dependent manner (Fig. 4).The number of clusters observed after the cultures were sub-sequently relabeled with R-BTX (thus revealing any clustersthat had formed entirely of AcChoR incorporated into themembrane during the recovery period) decreased even moresharply, again in a dose-dependent manner.To determine whether the clusters observed at the end of

the recovery period (both before and after relabeling) occurredat sites on the membrane where clusters had originally beenbleached, we compared photographic murals of the circum-scribed region on one culture dish at three time points duringthe experiment: before the clusters were bleached, at the end

FIG. 3. Typical redeveloped cluster fluorescence. Bar = 50 an.

en NCrl

v 40 _- h After relabeling_J\

(a 30 -

o-j

o 20. \ Before1 relabeling

1. I I I _1 2 3 4COLCHICINE (10-8M)

FIG. 4. Effect of colchicine on cluster redevelopment. The numberof redeveloped clusters, expressed as a percentage of the number ofclusters originally bleached, is plotted against the colchicine concen-tration. The total numbers of clusters bleached at each concentrationof colchicine were 10 nM, 716; 20 nM, 606; 40 nM, 576; controls, 3,063.

of the recovery period, and after relabeling. The results of thecomparison are summarized in Fig. 5. Briefly, most of the re-developed cluster fluorescence was found in regions whereclusters had been bleached, but a minority developed in pre-viously cluster-free regions.

Slightly more than half of the faint clusters were annular inshape (58%), a ring of fluorescence surrounding a dark center(Fig. 3). On relabeling, the majority of these annular clustersretained their annularity; only 18% did not. Native clusters arerarely annular; only 1 out of 41 had that shape.We performed experiments to verify that photobleaching

does not destroy the toxin-binding ability of the AcChoR norcause the toxin to dissociate from the receptors. Myotube cul-tures were exposed to rhodamine-labeled a-cobratoxin (R-CTX)in medium for 1 hr at 37TC. They were then washed severaltimes in Pi/NaCl/TTX and all clusters in a circumscribed regionwere bleached. R-CTX was dissociated from the receptors byincubating the labeled cells in medium containing curare (Bal-timore Biological Laboratory; 1.4 mg/ml) (11). Addition offlu-orescein-labeled BTX (F-BTX) to this medium relabeled all oftheAcChoR remaining intact after the photobleaching step. Outof 174 clusters bleached in this experiment, essentially all couldbe revisualized by curare treatment and F-BTX relabeling.Since all of the revisualized clusters showed the characteristicstructure ofendogenous clusters, we conclude that the bleachedlabel was replaced by unbleached label and the underlyingstructure was unaltered by photobleaching.

In a separate experiment, myotube cultures were exposedto R-BTX at 370C for 50 min. They were then washed in Pi/NaCl/TTX and a stripe was bleached across all clusters. Thecells were immediately relabeled with R-BTX (370C for 55 min),washed with Pi/NaCl/TTX, and observed. All visible clustersstill exhibited the bleached stripe, indicating that the toxin-bearing bleached fluorophore was still bound to the AcChoRin those regions. Hence, photobleaching destroys the fluoro-phore without causing toxin to dissociate from the AcChoR.

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80%

ofreformed

clusters20x%

2 2 % of bleached

clusters

6%

of

relabeled

clusters38%

FIG. 5. Location of cluster-fluorescence redevelopment as shownby photographic murals. In this experiment, 41 clusters were bleached,19 clusters were observed after recovery, and 34 clusters were seen on

relabeling. (A) Cluster fluorescence redevelops in the location of a pre-viously bleached cluster (80% of redeveloped clusters). (B) Cluster flu-orescence redevelops in a new location (20% of redeveloped clusters).(C) Cluster fluorescence does not redevelop nor appear on relabeling(22% of bleached clusters). (D) Cluster fluorescence does not redevelopbut appears on relabeling in the location of a previously bleached clus-ter (6% of relabeled clusters). (E) Cluster fluorescence in a new locationappears only on relabeling (38% of relabeled clusters). Not all myo-tubes survived; 37% of the bleached clusters were on myotubes thatvanished during the recovery period.

DISCUSSIONWe have shown that cluster fluorescence redevelops within 7.5hr after previous cluster fluorescence has been selectively andirreversibly photobleached. From this result, we conclude thatthe redeveloped cluster fluorescence must emanate from pre-

viously diffusely distributed AcChoR-i.e., that diffusely dis-tributed AcChoR can become clustered.

Numerous previous investigations have shown that cluster-ing of diffusely distributed cell surface proteins can be exog-

enously triggered (12, 13)-in particular, the redistribution ofAcChoR induced by neural contact (1, 14) or neurally derivedfactors (15, 16). This paper reports an ongoing spontaneous clus-tering of diffusely distributed AcChoR that is not contingent on

addition of exogenous agents. Furthermore, it is not yet clearwhether AcChoR clusters induced by neural contact in a Xen-opus coculture system originate from AcChoR in previouslyexisting endogenous clusters or from diffusely distributed re-

ceptors. In this study, we have unambiguously demonstratedclustering of diffusely distributed AcChoR, a finding consistentwith the hypothesis that the mechanism that mediates AcChoRaggregation does not reside in the receptors themselves.

It is possible to explain our results by postulating more com-

plex mechanisms of receptor movement-e.g., an internaliza-tion mechanism that "shuffles" AcChoR in and out of clustersvia special membrane-enclosed vesicles. Although it is difficultto rule out such a possibility, the annular shape of many of theredeveloped clusters argues against it.

Previously, it has been shown that the lifetime of the clus-

Proc. Natl. Acad. Sci. USA 80 (1983)

tered AcChoR on the membrane surface is no greater than thatof the diffusely distributed ones and is less than that ofthe clus-ter as a whole (2). Endogenous clusters are clearly not staticstructures; Bloch (5) has reported that de novo cluster formationis an ongoing process in myotubes. We report here (Fig. 5) thevarious mechanisms involved in cluster maintenance. Rede-velopment ofcluster fluorescence in the location ofa previouslybleached cluster (Fig. 5A) indicates that existing cultures cantrap nonclustered AcChoR as they diffuse in from surroundingregions. Such a passive diffusion-trap mechanism has also beensuggested for the induced localization of AcChoR at the site ofnerve-muscle contacts (17, 18). The annular shape of the faintclusters observed at the end of the recovery period in our ex-periments is consistent with this mechanism.

Weinberg et al (19) found similar annuli when developingectopic end plates were labeled with "WI-labeled BTX one ortwo days after treatment with unlabeled BTX. The unlabeledBTX blocked the old receptors and the new receptors were re-vealed to be added at the periphery of these neurally inducedend plates. However, when we performed an analogous exper-iment with R-BTX and our aneurally cultured myotubes, newreceptors were found throughout the endogenous clusters andno annuli were ever seen. This suggests that the insertion ofAcChoR occurs uniformly throughout an endogenous cluster,a suggestion supported by the finding that, when the myotubesare relabeled with R-BTX 7 hr after selective cluster photo-bleaching, about half of the annular faint clusters thicken andan additional 18% fill in entirely. These observations indicatethat the incorporation ofnew AcChoR also plays a role in clustermaintenance. The finding that cluster fluorescence may notdevelop during the recovery period but may then appear onrelabeling in the location of a previously bleached cluster (Fig.5D) indicates that on occasion cluster maintenance dependsalmost entirely on the incorporation of new AcChoR.

Cluster-fluorescence redevelopment in a region of the mem-brane where no cluster was present initially (Fig. 5B) indicatesthe formation of a new cluster, at least partially from AcChoRalready present on the membrane. Cluster-fluorescence ap-pearance in a new location only on relabeling (Fig. SE) indicatesthe formation of a new cluster, mainly from AcChoR insertedinto the membrane during the recovery period. Cluster for-mation and turnover is apparently an ongoing process in cul-tured myotubes, using AcChoR newly incorporated into themembrane, "old" diffusely distributed AcChoR, or both. Fail-ure of cluster fluorescence to develop in a location where a clus-ter has been bleached combined with failure to appear thereon relabeling (Fig. 5C) indicates a cluster that has a very slowAcChoR incorporation rate. Such a cluster may be in the processof disappearing.

It is unlikely that photobleaching destroys the underlyingstructure of a cluster because 65% of the bleached clusters re-developed cluster fluorescence either during the recovery pe-riod or on relabeling. Using R-CTX as a label that can be pho-tobleached and then removed by curare, we showed directlythat the AcChoR in a cluster retain their ability to bind R-BTXeven after photobleaching. Photobleaching does not dissociatethe toxin from AcChoR, as shown by the fact that immediaterelabeling of stripe-bleached clusters failed to obliterate thebleaching pattern. It is still possible that photobleaching some-how affects the rate of internalization of AcChoR. Although wedid not test for this effect, its presence would not alter the con-clusion that diffusely distributed AcChoR can become clus-tered.

It would be relevant to see whether the reverse process canoccur-i.e., whether clustered AcChoR can become diffuselydistributed. Such an experiment, however, would require that

452 Cell Biology: Stya and Axelrod

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Proc. Natd Acad. Sci. USA 80 (1983) 453

we photobleach all diffusely distributed AcChoR, leaving onlythe label on clustered receptors intact. Because only 10% oftheAcChoR on myotubes are aggregated into clusters (2), the flu-orescence remaining after photobleaching would be too faintto detect, especially if this fluorescence were diffusely distrib-uted.We suggest that AcChoR are immobilized by a submem-

branous anchoring network and that any AcChoR can becomeattached to an anchor. It is possible that the characteristic struc-ture of endogenous clusters, that discrete pattern of lines andspeckles, is a manifestation ofthe arrangement ofthe anchoringnetwork. Cytoplasmic vinculin appears to be associated withthis anchoring network (20). It is not yet-clear whether the vin-culin actually composes the anchoring network or merely co-distributes with it.

The ability of colchicine to inhibit reaggregation of clustersafter dispersal with sodium azide (5), as well as the spontaneousredevelopment of clusters from diffusely distributed AcChoRin unpoisoned cells (Fig. 4), suggests the involvement of mi-crotubules in cluster formation. Note, however, that existingclusters cannot be dispersed by treatment with colchicine (4,5), indicating that cluster stability is not dependent on micro-tubules. A complete picture of cluster structure remains to beelucidated.We are grateful to C. M. Stya for plating most ofthe cell cultures used

in this study. This work was supported by National Institutes of HealthGrants NS 14565 and NS 17017.

1. Anderson, M. J. & Cohen, M. W. (1977)J. PhysioL (London) 268,757-773.

2. Axelrod, D., Ravdin, P., Koppel, D. E., Schlessinger, J., Webb,W. W., Nelson, E. L. & Podleski, T. R. (1976) Proc. Nati Acad.Sci. USA 73, 4594-4598.

3. Prives, J., Christian, C., Penman, S. & Olden, K. (1980) in Tis-sue Culture in Neurobiology, eds. Giacobini, E., Vernadakis, A.& Shahar, A. (Raven, New York), pp. -35-52.

4. Axelrod, D., Ravdin, P. M. & Podleski, T. R. (1978) Biochim.Biophys. Acta 511, 23-38.

5. Bloch, R. (1979)1. Cell Biol 82, 626-643.6. Fambrough, D. M. (1979). Physiol Rev. 59, 165-227.7. Tipnis, U. R. & Malhotra, S. K. (1980) Can.J. Physiol PharnmacoL

58, 445-458.8. Axelrod, A. (1980) Proc. Natl Acad. Sci. USA 77, 4823-4827.9. Ravdin, P. & Axelrod, D. (1977) AnaL Biochem. 80, 585-592, and

correction (1977) 83, 336.10. Devreotes, P. & Fambrough, D. M. (1975)J. Cell Biol 65, 335-

368.11. Patrick, J., Heineman, S. F., Lindstrom, J., Schubert, D. &

Steinbach, J. H. (1972) Proc. Natl Acad. Sci. USA 69, 2762-2766.12. De Petris, S., Raff, M. C. & Malucci, L. (1973) Nature (London)

New Biol 244, 275-277.13. Taylor, R. B., Duffers, W. P. H., Raff, M. C. & de Petries, S.

(1971) Nature (London) New Biol 233, 225-229.14. Frank, E. & Fischbach, G. D. (1979) J. Cell Biol 83, 143-158.15. Jessell, T. M., Siegel, R. E. & Fischbach, G. D. (1979) Proc. NatL

Acad. Sci. USA 76, 5397-5401.16. Podleski, T. R., Axelrod, D., Ravdin, P., Greenberg, I., John-

son, M. M. & Salpeter, M. M. (1978) Proc. NatL Acad. Sci. USA75, 2035-2039.

17. Poo, M.-M. (1982) Nature (London) 95, 332-334.18. Edwards, C. & Frisch, H. L. (1976)J. Neurobiol 7, 377-381.19. Weinberg, C. B., Reiness, C. G. & Hall, Z. W. (1981)1. Cell

Biol 88, 215-218.20. Bloch, R. J. & Geiger, B. (1980) Cell 21, 25-35.

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