Myxobacteria build their species-specific fruiting bodies bycell movement and then differentiate spores in specific placeswithin that multicellular structure . New steps in the developmentalaggregation of Myxococcus xanthus were discovered through aframe-by-frame analysis of a motion picture . The formation andfate of 18 aggregates were captured in the time-lapse movie.Still photographs of 600 other aggregates were also analyzed.M . xanthus has two engines that propel the gliding of its rod-shapedcells: slime-secreting jets at the rear and retractile piliat the front . The earliest aggregates are stationary massesof cells that look like three-dimensional traffic jams . We proposea model in which both engines stall as the cells' forward progressis blocked by other cells in the traffic jam . We also proposethat these blockades are eventually circumvented by the cell'scapacity to turn, which is facilitated by the push of slimesecretion at the rear of each cell and by the flexibility of the myxobacterial cell wall . Turning by many cells would transform a traffic jam into an elliptical mound, in which the cells are streaming in closed orbits . Pairs of adjacent mounds are observedto coalesce into single larger mounds, probably reflecting thefusion of orbits in the adjacent mounds . Although fruiting bodiesare relatively large structures that contain 10⁵ cells, no long-range interactions between cells were evident . For aggregation, M. xanthus appears to use local interactions between its cells.

 
Myxobacteria build fruiting bodies that have a wide varietyof shapes, each serving to differentiate 1 of the approximately40 different myxobacterial species [25] . Cylindrical and sphericalmasses of cells constitute the stalks, branches, and sporangioles[packages of myxospores] of these fruiting bodies . Althoughthe shapes are relatively simple, they represent inherited patternsof cell movement . Their heritability over a billion years of speciation is demonstrated by the strong correlation between the morphology of fruiting bodies and their molecular phylogenybased on comparing 16S RNA sequences in these members of thedelta subgroup of proteobacteria [36] . Fruiting body development is initiated by starvation, and the structures are built entirelyby cell movement . There is little cell division because starvationhas induced the cells to give a stringent response; Myxococcusxanthus responds to starvation by arresting ribosome biosynthesis[8], by expressing a subset of its genes [20], and by adopting appropriate new patterns of cell movement [15] . We sought todefine these movement patterns and their role in building a fruiting body.

A new understanding of the mechanics of gliding motility andof its regulation in M . xanthus provided the starting pointfor the present investigation . Myxococcus cells, which are 5to 7 µm long and 0.5 µm in diameter, glide overa surface, leaving behind a trail of slime [3, 5, 37] . Hodgkindiscovered that M . xanthus has two gliding engines encoded bynonoverlapping sets of genes [9] . One engine is now consideredto be an array of jets that are located at both ends of thecell and that secrete slime from one end at a time [41] . Theother engine is a polar cluster of retractile type IV pili locatedat one end of the cell [16, 38, 21] . Using both engines, M.xanthus glides in one direction for a while and then reverses.A reversal is not accomplished by making a U-turn but by exchanginghead for tail, as shown in a movie by L . Jelsbak attached toreference 15 . Studies of frz mutants have revealed a cytoplasmic chemosensory pathway that can alter the probability of reversing direction [2] . Kinetic measurements of swarm expansion showthat the two engines most often cooperate in moving the cell [17] . Cooperation strongly suggests that the two engines areswitched from pole to pole by a tightly regulated and concertedmechanism . Individually, slime secretion [known as A motility]and pilus retraction [known as S motility] endow the cells withdifferent swarm patterns, which combine in A⁺S⁺ cells [9] . Bothengines are needed for development because A^-S^- mutants cannotbuild fruiting bodies [9] . Nevertheless, most A^-S⁺ and A⁺S^- strains are able to build fruiting bodies, although they buildmore slowly than A⁺S⁺ [Y . Cheng and D . Kaiser, unpublished observations].Although either engine is sufficient, this finding suggeststhat the coordination mechanism, which was intact in all ofthe A^-S⁺ and A⁺S^- strains tested, plays a role in development.

Wireman and Dworkin showed that M . xanthus and the closely related M . fulvus construct spherical fruiting bodies raised on a short stalk [recent isolates from soil] or raised mounds [domesticated laboratory strains] and then sporulate [40] . Reichenbach etal . made a remarkable series of time lapse movies that comparethe fruiting body development of different species [26-29]. These films show that Stigmatella and Chondromyces spp., whosefruiting bodies have multiple sporangioles raised on a thick stalk, begin just like M . xanthus by raising a mound . Peripheral cells of M . xanthus, located outside but immediately adjacent to the mound, do not differentiate into spores [23] . As a consequenceonly mound cells sporulate [14, 30] . The developmental dataraise the question of how M . xanthus regulates its movementso as to build a mound and then to sporulate within it . We reporthere two new movement patterns that help to construct its fruitingbodies.

 
Submerged culture on glass. The procedure is modified after that of Kuner and Kaiser [19].First, 2 ml of an exponential culture of DK1622 in Casitone-Trismedium [CTT], at a density of 2.5 Klett units, was added toa 2.5-cm diameter Corning Bionique culture dish with a metalbaseplate, a Teflon core, and a Teflon gasket, with a microscopecoverslip forming the bottom of the dish . After the culturehad grown for 20 h at 31°C without agitation, the CTT fluidwas removed by aspiration; the culture was next washed oncewith 3 ml of H₂O, which was replaced with 10 mM morpholinepropanesulfonicacid-1 mM CaCl₂, and then the culture was returned to 31°Cto starve and to complete fruiting body development . A diagramof the setup is Fig . 3A . At the times indicated, the cultureswere mounted on a mechanical stage of a Leitz Labovert invertedmicroscope to preset X and Y coordinates, with the dish coverslipdirectly above the objective lens at room temperature . The dishwas photographed and then returned to the incubator . Specimenswere photographed by using a phase contrast stop in the condenserto provide oblique illumination.

 
SAC. Two sterile 0.5-mm-thick silicone rubber gaskets [Grace Biolabs,Bend, Oreg.] were placed on top of a flame-sterilized glass microscope slide, creating a small well whose bottom half was filled with 1.5% agar in clone-fruiting [CF] medium . After theagar hardened, the M . xanthus culture was spotted onto it and allowed to dry for 5 min, and then the well created by the second gasket was filled with CF liquid medium . The final layers ofa submerged agar culture [SAC] apparatus consisted of the coverslip,a gasket containing CF liquid, a gasket containing agar, anda slide . The M . xanthus population is between the agar and theCF liquid . A diagram of the setup is shown in Fig . 3B, and additionaldetails are given in Fig . 1 of reference 39].

 
SAC cultures were maintained at 25°C with a heated stage[Brook, Lake Villa, Ill.] for the acquisition of time-lapseimages . Video microscopy was performed on a Nikon Eclipse E800microscope [Nikon, Melville, N.Y.] by using long working distanceobjectives . Digital images were acquired from an analog videosource by using a Scion LG-3 video capture card and Scion Imagesoftware [Scion, Frederick, Md.] . Phase-contrast images weregenerated with a charge-coupled device camera [Optronics Engineering,Goleta, Calif.] . Images were saved at regular intervals [either20 or 60 s per frame], and background noise was reduced by averagingthe video rate images over a period of 2 s . Images were savedas sequentially numbered TIFF files and assembled into time-lapsemovies by using Quicktime [Apple Computer, Cupertino, Calif.].

 
Morphological stages in aggregation. As Myxococcus cells settle from suspension onto the glass floorof a submerged culture dish and grow beneath the CTT liquidmedium to confluence, they also move to form plate-like multicellulardomains [19] . These domains range from 0.2 to 0.4 mm in diameter,and in each the rod-shaped cells lay side by side with theirlong axes roughly parallel, as can be seen in the scanning electronmicrograph of Fig. 1, frame 1 . Where two adjacent domains intersect, the difference in their cell orientation prevents a smooth abutment, and a slightly elevated ridge is formed . An intersection offour domains and their ridges is evident in the upper rightquadrant of frame 1 . A light microscope image taken with a x6.3 objective lens under oblique illumination [Fig . 1, frame 2]of a submerged culture 1 h after the replacement of CTT [growth] medium with morpholinepropanesulfonic acid-Ca starvation buffer to start development emphasizes the ridges because they scattermore light than the plate-like domains . With the help of a mechanical stage equipped with a vernier scale, the specimen was repositioned with a [measured] accuracy of ±5 µm, the averagelength of one cell . In this way, the very same microscopic fieldwas photographed at 1, 5, 8, 11, and 24 h to show how the cellarrangement changes as fruiting bodies develop . To obtain asignificant sample of the culture, eight different microscopefields were photographed at these time points . A representativefield is illustrated in Fig. 1, frames 2, 3, 4, 5, and 6, whichshow successive time points.

The other seven fields showed the same type of changes overtime as those in Fig . 1, and a description of the features general to all eight cultures follows . At 5 and 8 h, the original network of ridges has generally faded; then, especially clearly at 8h, a few of the original elements became brighter [Fig . 1, frames 3 and 4] . Higher-magnification views showed no pale cells, cell fragments, or other signs of cell lysis at 8 h [see, for example, Fig . 2] . For that reason, the fading and brightening implies that cells are collecting at certain points . The bright elements of the network at 8 h appeared smaller and higher in cell density than the ridges at 1 h, implying that the bright elements are condensations . These condensates at 8 h are also asymmetric,with tails that lead in several directions away from their centers.Some tails are still evident at 11 h; one is indicated withan arrow in Fig . 1, frame 5 . One such condensate is, shown in the Fig . 2, 8-h photo . That scanning electron photomicrograph shows an elongated asymmetric heap of cells whose long axisis 80 µm and short axis 15 µm, which can be seento contain many hundreds of cells [19] . Viewed by phase-contrast light microscopy, one 8-h elongated heap and parts of two othersare evident in the middle of Fig . 2, frame 1, and at the edges of that field.

 
Since precisely the same field was photographed from 1 to 24h, it was possible to superimpose the 8-h photos onto the corresponding1-h photos of Fig . 1 and of the other culture fields [not in the figure] . Superimposition revealed that all of these elongated heaps had been constructed on a ridge of the 1-h network . Moreover, the tails of these 8-h aggregates superimposed on other ridges that had branched from the first in the 1-h network . By 11 h,some of these asymmetric aggregates enlarge and round up, whileothers fade [compare Fig . 1, frames 4 and 5] . It is as if the rounded aggregates have drawn in cells from the space between aggregates because the tails [arrow in frame 5] have vanishedas the aggregate rounds up [frame 6], and fewer cells are evidentbetween aggregates [Fig . 1 and 2] . By 24 h, there are very fewcells outside the rounded aggregates, as shown by the paucityof points that scatter light in the field outside them [Fig. 1 and 2].

As mentioned, superimposition of the 24-h photos onto the earlier photos showed that all of the nascent fruiting body aggregatesarose from asymmetric aggregates at 8 h, which in turn arosefrom elements of the 1-h network of ridges . However, only certainelements of the original network nucleated a fruiting body.Considering the 1-h ridge pattern as an irregular network thatcovers the surface [diagrammed in Fig . 3C], three classes ofgeometric objects were distinguished: a face, i.e., a singledomain surrounded by edges, an edge where two domains overlap,and vertices where three domains intersect . Point-to-point superimpositionwas made on 43 nascent fruiting bodies, and 36 of these tracingscould be completed without ambiguity . [There was ambiguity whena ridge was so broad that the adjacent facets were difficultto discern.] According to the 36 unambiguous traces, none ofthe fruiting bodies arose from a face, 34 arose from an edge,and 2 may have arisen from an edge or possibly a vertex [becausethe ridges at the vertex were broad] . Thus, 36 fruiting bodiescould be traced back to a ridge in the initial domain pattern.In every case, the long axis of the asymmetric aggregates at 8 h corresponded to the orientation of the ridge in the domain pattern . The field in Fig . 1, frame 2, initially had 133 ridgesthat produced 13 fruiting bodies . Thus, a small fraction of ridges give rise to a fruiting body.

Kuner and Kaiser [19] showed that fruiting bodies in submergedculture, such as those in Fig . 1, frame 6, and older containviable, heat-resistant spores . Sporulation in submerged culturehas been amply confirmed in other studies [33, 34] . Sporulationis the last step in fruiting body development, and it takesplace even when fruiting bodies are submerged . The same fieldsviewed in Fig . 1 were also photographed at x16 in phase contrast to view individual cells . Figure 2, frame 1, shows that mostof the cells between the 8-h asymmetric aggregates and the 11-h symmetric aggregates have disappeared by 24 h, when sporulation has commenced . Julien et al . showed that peripheral cells arenot expressing C-signal-dependent genes and, for that reason,are unable to sporulate [14] . Cells located between different aggregates at 11 and 24 h are likely to be the peripheral rods described by O'Connor and Zusman [22] . The 24-h image in Fig. 2 [frame 3] and those photographed at x16 at five other sites[not shown] suggest that peripheral rods in submerged culturewould eventually lyse.

To check whether fruiting bodies are clustered in submerged culture, their spatial distribution was analyzed . Thirty-four photographs, like Fig . 1, frame 6, and containing a total of 653 fruiting bodies, were randomly sampled by using a squareof fixed size . The size of the sampling square was such thatabout one fruiting body would be obtained per sample . As itturned out, the average number of fruiting bodies per samplesquare ranged from 0.28 to 2.1 over all 34 photos . For eachphotograph, the number of sample squares that captured no fruitingbodies, those with 1, 2, or 3 fruiting bodies,the total number of fruiting bodies captured in the samples,and the total number of samples were recorded . If, on the onehand, the spatial distribution were very orderly, the numbers of fruiting bodies per sample are expected to be mostly 1's,a few 2's, a few 0's, and probably no 3's . On the other hand,if fruiting bodies were independently distributed, then thenumbers of samples with 0, 1, 2, or 3 fruitingbodies would have Poisson statistics . The proper Poisson distributionwould be set by the average number of fruiting bodies per unitarea for each photograph . Table 1 shows ² tests of the hypothesisthat the data are described by the Poisson distribution appropriatefor each of the 34 photographs [1] . The level of significanceis too high for all plates to reject the hypothesis that fruitingbodies are distributed independently . Complete independencemight not be expected if nascent aggregates that form closeto one another often fuse, as is observed in the agar culturesdescribed below . Such fusion would create a deficiency of sampleswith two or more fruiting bodies and a corresponding excess of samples with none . Keeping the possibility of fusions in mind, the distribution of fruiting bodies is consistent with independent sites of aggregation on the 0-h network of ridges.

 
Based on fruiting body locations, at least two steps can bediscerned at which the sites of mature fruiting bodies are determined.The first is the selection of a ridge to become an asymmetricaggregate . Of the 133 edges in Fig . 1, frame 2, 22 bear an aggregate at 8 h . The second step is the selection of an asymmetric aggregate to enlarge into a symmetric fruiting body; of the 22 fruiting bodies marked at 8 h, 13 remained at 24 h . The number of different microscopic cell arrangements in a culture of 10⁸ cells wouldbe very large, as the field shown in Fig . 2, frame 1, makesplain . In Fig . 2 and the other photos [not shown], no repeatingstructures, apart from the aggregates themselves, were apparent.Rather, the independence of fruiting body sites is consistentwith determination by the unique microarrangements of cells,any one of which has a very low probablility of nucleating anaggregate.

Morphogenesis in SAC. Fruiting body development in SAC has been photographed in timelapse by Welch, originally for the purpose of tracking cellsin traveling waves [39] . Beyond the waves, Welch's photographsdocumented the patterns of cell movement during aggregationin detail . A frame-by-frame analysis of the movie revealed theearly formation of stationary aggregates, similar in their temporaryimmobility [for many hours] to the asymmetric aggregates describedabove in Kuner submerged culture . The Welch movies also showinteractions between traveling waves and the stationary aggregates.Finally, the Welch photos show how stationary aggregates subsequentlymove . Welch's submerged cultures differ in three ways from thosephotographed for Fig . 1 and 2 [see diagram in Fig . 3] . First, the cells are moving and aggregating on a thin layer of agarin CF medium rather than on the glass surface of a microscopecoverslip in buffer in A . Second, all of the fruiting bodiesform along the perimeter of the initial inoculum of cells inSAC [Fig . 3B] because there is a physical barrier, probablysurface tension, to outward movement of cells at the edge [39]. In Fig . 3A, fruiting bodies formed at spatially random locationsall over the glass substrate . Finally, traveling waves are strongin the setup of Fig . 3B, whereas they are not observed in culturesof the Fig . 3A type . This indicates that movement is generallymore organized in the Fig. 3B condition . Despite these differences,the cultures depicted in Fig . 3A and B show the same two stages: fixed aggregate formation that is followed by selective enlargement of certain aggregates . With a photograph taken every 1.5 min, the Fig . 3B cultures offer greater time resolution and more structural detail than those of Fig . 3A, in which photographswere taken at 3- to 5-h intervals.

A time-lapse movie of M . xanthus fruiting body development can be downloaded from http://cmgm.stanford.edu/devbio/kaiserlab/.

Starting from a uniform cell density across the culture at 0min [Fig . 4], cells swarm outward from the center of the culture, first accumulating to form a band of elevated cell density around the original edge of the culture . Cells within that band tend to be oriented parallel to the edge, as described previously[39] . Initially, the band appears uniformly dark around itscircumference, as shown in Fig . 4 [206 min] . Then, ca . 140 min later, density variations can be perceived when two lumpy setsof cells appear to travel around the band: one set moving clockwiseand the other set moving counterclockwise . At their start, theselumps are evident in the time-lapse video, but they are notresolved in a single still photograph . Shortly, however [Fig.4, 435 min], dark, arc-shaped zones are evident at the outeredge of the band in the still photographs, each arising fromthe outer edge of a lump . Two such arcs are indicated by whitearrows in the 435-min frame, and others are evident but arenot labeled . Even though the arcs appear to have arisen frommoving lumps, these arcs are stationary; they can be recognizedlater at the same position through at least 812 min . At 435min the inner edge of the band directly opposite an arc wasa moving lump of optical density . After 435 min, the stationaryarcs widen across the band toward its inner edge [Fig. 4, 678min] . As the arc spreads across the band, the arc becomes anelongated teardrop, whose point aims toward the center of theculture spot [located vertically above the top of Fig. 4 [678min]].

 
Recall that the cells within the peripheral band have theirlong axes oriented tangent to the culture edge [39] . By 678 min, the moving lumps of these cells have now sharpened into tangentially directed moving ridges, the crests of travelingwaves, as a consequence of the focusing action described byIgoshin et al . [10] . By 678 min, the lumps have become a clockwiseand a counterclockwise set of traveling waves, moving aroundthe band [Fig . 4] [39] . The ridges of these waves make acuteangles with the edge, and it becomes apparent that the tear-shapedfixed aggregates are regularly spaced at wavelength intervalsin Fig. 4 [678 min] . At that time, 18 aggregates could be distinguished,reading from left to right, and these were named A1 to A18.

The moving waves appear to wash over these 18 aggregates, whereas the latter remain fixed at their position . At this time theopacity of a wave crest is comparable to the opacity of an aggregate.As a consequence, the total opacity at the position of an aggregate fluctuates periodically as the waves move over it . For example, aggregate A12 was clocked over several cycles, and it has aperiod of six to seven movie frames of 1.5 min each . If thestart of a cycle is taken when a wave crest coincides with thecenter of an aggregate, then the space between adjacent fixedaggregates at the start has a low optical density . The densityof the aggregate plus the wave crest extends across the widthof the band [Fig . 4, 807 min] . Shortly thereafter [812 min],the wave crest is 180° out of phase with the aggregates,and the space between aggregates is darkened by a relativelybroad and diffuse distribution of cells in the wave . It canbe seen at 812 min that many cells remain stationary becausenone of the aggregates move; the waves move . In Fig. 4, 812min, the waves are very plain between aggregates A12, A13, A14,A15, and A16 . As the waves travel, they progress to the nextset of registrations with aggregates at 816 min in Fig . 4 . Cyclingis readily observed if the movie is stepped from frame B17951to frame B17957, one frame per step.

After many waves have washed, so to speak, over the aggregates, they can be seen to have grown larger . By comparing each aggregatein Fig . 4 at 816 min with its initial arc at 435 min, one can appreciate the growth . In the interval from 435 to 816 min,the center of each aggregate remains at the same position alongthe edge, showing no detectable lateral movement . This symmetricalgrowth of the aggregates suggests that some wave cells havebeen added to the aggregates . It also suggests that the newcells have not inserted into the asymmetric core of the aggregatebut rather encircle the core . Many cells along the crest ofa wave might cross an aggregate at about the same time, offeringmany opportunities to be deposited . A reverse process is alsoobserved . Some aggregates dissipate, losing their density aswaves wash over them; it is as if cells can leave an aggregateto rejoin the wave system . A3, A4, A9, A10, A11, A15, A16, A17,and A18 disappear with time; none of them are opaque at 1,419 min in Fig . 4.

Returning to 678 min, a few clumps of cells can be seen as dots beyond the edge of the original culture . [They appear belowthat edge in Fig . 4.] Before their appearance, no cells had been observed to migrate away from the edge into the area wherethe dots are found . The dots were not apparent at 0 min; theybecame visible at 206 min . Individual cells are too small tobe seen at the magnification used for the movie, but after 206min there will have been time for cells to aggregate into thesenow-visible clumps . Because these cells are few, they were nottracked . Suffice it to say that their capacity to enlarge byfusing with each other indicates that these are clumps of motilecells . At least three of them close to the perimeter of theculture spot were observed to fuse with an aggregate by meansof thin, slime-like ribbons at 816 min.

The period of stationary aggregate dissipation and growth comesto an end when some aggregates are seen to move . In every case,adjacent aggregates fused with one another, joining at the pair'scenter of mass . In Fig . 4, 1,419 min, A12 and A13 are seen to be moving and fusing . Then, at 1,924 min, A7 and A8 fuse . Later, fused aggregates are observed to fuse with another . Again, itis always adjacent aggregates that fuse . Eventually, only threelarge aggregates remain in this sector of the culture to becomefruiting bodies in Fig . 4, 3,866 min; originally there had been 18 stationary aggregates . Secondary fusion shows that one fusion does not exhaust the capacity of a motile aggregate to fuse, and these properties suggest a mechanism of fusion describedin the Discussion.

Just as small aggregates fuse with each other, A5 fuses withthe wave system . This later fusion of an aggregate with thewaves differs from the loss of cells from an early nonmotileaggregate back to the waves described above in that A5 is motile.First, A5 bends to the right, taking up an orientation thatconfronts the waves, then the interior of A5 flows rapidly towarda wave . This flow suggests that most cells within a motile aggregateare streaming [15] . Initially, the flow leaves behind a thin,U-shaped, empty shell [Fig. 4, 2,475 min] . Finally, and moreslowly, the shell also dissipates, giving up almost all itsdensity to the wave system [Fig . 4, 2,682 min] . In addition,the left part of the fused A7 and A8 at 3,866 min becomes anothershell, apparently by fusion with waves.

Moving waves are still evident at 2,682 min . However, their optical density has fallen because the waves may have suffereda net loss of cells to the growing, motile aggregates . The wavesmay also have gained cells from the center of the culture spotdue to outward swarming . Nevertheless, the wavelength changedlittle from 678 to 2,682 min . Since the wavelength is expectedto depend on the cell density in the wave crests [10], the crestdensity appears to have remained high . This could be explainedby the focusing effects . In any case by 3,866 min, the wavesare no longer visible . All visible cells are concentrated inthe three large aggregates evident in Fig . 4 . All three pulsefor a short time at approximately the frequency of the travelingwaves . Although pulsing is not evident in a still image, itis quite marked in movie frames B19820 to B19980 [http://cmgm.stanford.edu/devbio/kaiserlab/].

Cell movement in the vicinity of enlarging aggregates. In mixtures of a few fluorescent [i.e., green fluorescent protein-labeled] M . xanthus cells and many nonfluorescent cells, individual fluorescentcells can be tracked even in regions of high total cell density.Using a ratio of 1 fluorescent/300 nonfluorescent cells, L. Jelsbak has made several time-lapse movies of cells moving in the vicinity of fruiting body aggregates that are enlargingon the surface of a slab of agarose [L . Jelsbak, unpublishedresults] . Jelsbak photographed at 15 h of development beforesporulation had begun, and he has made these images availableto us for cell tracking . Table 2 shows data from four of Jelsbak's movies with a total of 13 aggregates, all nonfluorescent, and68 fluorescent cells . During the 15-min movies, some of the68 tracked cells appeared for all or part of their trajectoriesto coincide with an aggregate, as if they were moving from theadjacent less-organized mass of cells onto the top surface ofan aggregate . Since the photographs were taken from the topof the culture downward, these fluorescent cells must be incontact with the top surface of the aggregate . Other fluorescentcells in the same microscopic field never coincided with anaggregate during their 15-min period of photography and presumablyare near the top of the surrounding mat of cells at lower densitybecause they are less well organized . Table 2 shows the numberof fluorescent cells coincident with the dense image of an aggregateat some point in their trajectory, the number never coincident,and the fraction of the microscopic field's area that is occupiedby its aggregates . For the spatial distribution of fluorescentcells, the data of Table 2 shows that, although aggregates accountedfor only 15% of the average area of the frames, 60% of the fluorescent cells were coincident with an aggregate . Since the coincidentcells are bright and moving, they are not within the aggregate,adhering to it, or trying to penetrate it . The bias favoringcoincidence suggests that cells approaching an aggregate fromthe neighborhood may turn to glide on the aggregate's surfaceand may be trapped there for a while . These cells may be contributingto the enlargement of an early aggregate, as reported abovefor Kuner submerged cultures and for SAC.

 
The data on cell movement and on changes in the shape and sizeof aggregates reported here give evidence of two new steps in aggregation . The first step is the formation of stationary clusters of cells; they were observed in both Kuner submerged cultureand in SAC . In SAC, 18 such clusters were observed to form simultaneously, spaced one ripple wavelength apart along the culture edge [Fig. 4] . Every pair of countermigrating wave crests initiated an aggregate . The location and regular spacing of those aggregates suggests the following mechanism . At the time, the edge is aband of relatively high cell density; the traveling waves areevident as "lumps" of cell density . Although obscure in stillimages, the moving lumps can be perceived at movie speed [http://cmgm.stanford.edu/devbio/kaiserlab/]. A regular spacing of one wavelength suggests that the aggregates form at intersections of a rightward-moving wave, a left-moving wave, and the high-density edge of the peripheral band, as diagrammed in Fig . 5 . There would be more cells at such intersections thananywhere else . Cells in a wave are moving when they arrive at the intersection, and band cells may also be moving though with less regularity . Both A and S motilities are known to be requiredfor traveling waves [32] . Moving cells might stop at the triple intersections, because, like so many motor cars arriving on roads from several opposite directions, the cells find themselves stuck in a traffic jam that inhibits each other's movement.Worse than cars on flat roads, cells could enter their jam fromoutside the plane, and the cell jams would be three-dimensional.Mechanically, both engines would be expected to stall if a cell'sforward motion were blocked by other cells . Wolgemuth et al.have detailed that argument for the force generated by the A-engine[41], and S motility could also be blocked by a barricade . Liketraffic jams, the aggregates remain stationary for more than6 h in SAC.

 
Aggregates also formed at sites where cells had piled up inKuner submerged cultures . At these sites, two plate-like domainsof cells, each with a different cell orientation and thus outof register with each other and overlapping, are located . Kuneraggregates are asymmetric and elongated along the edge wheretwo domains overlap [Fig . 1 and 2] . However, only a fraction of the edges nucleate an aggregate because each has a unique microarrangement of cells according to the history of how that arrangement came about . As M . xanthus glides, the cell leaves a trail of slime, and that trail guides other cells that happento cross it [5, 15, 41] . The trails are not observable in confluentcultures such as are shown in Fig . 1 and 2, but they do constrain the future movements of each cell and thus its potential for aggregation . The scanning electron micrograph image of one such aggregate has the appearance of a flattened traffic jam [seeFig. 2, 8 h] . No traveling waves were observed in these cultures, and the aggregates remained stationary from 8 h to 24 h . However, during these 16 h the aggregates grew in size and gained circular symmetry . It is as if their cells were orbiting around . Indeed, cells were observed to turn and to glide on the surface of an aggregate [Table 2], and cells at the base of such aggregates have been tracked in circular orbits, as shown by Sager and Kaiser [see Fig . 6 in reference 31] . Cell vortex patterns havebeen observed in scanning electron micrographs of nascent fruitingbodies [24].

 
After they enlarged for about 6 h, the stationary aggregatesin SAC began to fuse with each other . A model, based on theobservations described above, for the conversion of a stationarytraffic jam into a motile aggregate is presented in Fig . 6.The gradual loss of density in the waves and concurrent gainin density of the aggregates from 435 min to 3,866 min [themovie or Fig. 4] suggest that there is net transfer of cellsfrom wave crests to enlarging aggregates . We suggest that thetransferred cells circulate in orbits around a core consistingof a stationary traffic jam, enlarging and rounding the aggregates.That circulation is represented by thin arcs within the aggregatesin Fig. 6 . The ability of motile aggregates to fuse with each other clearly shows that their constituent cells are moving. The organization of that movement is suggested by other experiments in which equal numbers of cells were found by tracking to be circulating clockwise and counterclockwise in the nascent fruiting bodies [31] . No reversals were observed in experiments of trackswithin an aggregate; however, reversals were observed in cells outside the aggregate [31].

M . xanthus cells are flexible because their peptidoglycan is organized as articulating plates [4] . The long and flexiblemyxobacterial cells would be expected to bend and turn at a barrier if they were being pushed by slime secretion from their rear [41] . Moreover, the movies of Reichenbach and Kuhlwein show cells turning after collision with another cell [18] . Finally,the tracking data of Table 2 suggest that, when cells encountera dense aggregate, they turn and move along its surface . Asa result of their capacity to turn, stalled cells within thetraffic jam core of an enlarging aggregate would begin to navigatearound the cells within the jam that block their forward progress,either by turning as just described or by reversing at each blockade . These movements that would break the jam are represented in Fig . 6 by the lighter gray of the core in an enlarging aggregatethan in the traffic jam . According to the model, an aggregatewould become motile and capable of fusion only after all ofthe cells of the traffic jam had begun to circulate, as in the motile aggregate represented in Fig . 6 . The central hole shownin the motile aggregate was observed by Sager as a region of low cell density [31] . Two such motile aggregates would be expectedto have the ability to fuse with one another, creating one largercirculation that retained the capacity to fuse again . Indeed,serial fusion is observed in SAC between 816 min and 3,866 min[see movie and Fig . 4].

Several consecutive fusions result in an aggregate of fruiting body size [as evident in the movie from 1,419 to 3,866 min andFig. 4] . Other experiments have shown that many cells circulating within an aggregate the size of a fruiting body transmit C signal to each other as they stream at high cell density [11, 12, 15].Repeated C signaling between cells within a nascent fruitingbody aggregate would be expected to raise the level of C signalon cells, via the act positive feedback loop, up to the thresholdfor sporulation [6, 7] . Finally, myxospore differentiation wouldbe induced [15, 35].

The model of Fig . 6 combines the structural data from two typesof submerged culture . Cell movement is more highly organized in SAC [Fig . 3B] than in Kuner cultures [Fig. 3A]: Kuner fruitingbodies arise randomly on the lawn of cells [Table 1], whereasin SAC aggregates are initiated at one-wavelength intervalsaround the edge of the lawn or patch of cells [Fig . 5] . Despitea difference in initial organization, the aggregates at 11 hin Kuner submerged culture [Fig . 1 and 2] have the same rounded shape and about the same size as the mounds in SAC, suggesting that cells in the Kuner fruiting bodies are circulating in thesame way as those in SAC . Since traffic jams and circulatingcells are observed in both types of submerged cultures, we suggestthat these two structures are likely to be used in the formationof fruiting bodies under other conditions, including withina patch of cells that is open to the air [40] . Because the sitesof fruiting body formation in open agar plate cultures are unpredictable, we have been unable to trace enlarging aggregates backward in time to their initiating aggregate, as we have done in SAC [Fig. 4] . The experiments in Kuner submerged culture and SAC predict that the initial aggregates will be a kind of traffic jam, and Jelsbak and Søgaard-Andersen recently reported smalljam-like clusters of fluorescent cells on agar plate cultures[see Fig. 2 [6 h] in reference 13].

 
This study was supported by Public Health Service grant GM23441to D.K . and by postdoctoral fellowship GM20356 to R.W . [bothfrom the National Institute of General Medical Sciences].

Brooke Danaher expertly tracked cells in the Welch and Jelsbak movies . We thank L . Jelsbak for access to four of his movies.O . Igoshin, G . Oster, and M . Alber contributed helpful suggestions.

 
* Corresponding author . Mailing address: Departments of Biochemistry and Developmental Biology, Stanford University, Stanford, CA 94305 . Phone: [650] 723-6165 . Fax: [650] 725-7739 . E-mail: kaiser@cmgm.stanford.edu.

Present address: Biology Department, Syracuse University, Syracuse, NY 13244.

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