Journal of Cell Science 104, 457-466 (1993) 457 Printed in Great Britain © The Company of Biologists Limited 1993

Patterns in Dictyostelium discoideum: the role of myosin II in the transition from the unicellular to the multicellular phase

Susannah Eliott1, Gregory H. Joss1, Annamma Spudich2 and Keith L. Williams1 1School of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia 2Department of Cell Biology, Stanford University School of Medicine, Stanford, California 94305, USA

SUMMARY

Dictyostelium discoideum amoebae which lack the elongation, cells within the mutant mound eventually myosin II gene are motile and aggregate to form rudi- cease translocation altogether as the terminal shape of mentary mounds, but do not undergo further morpho- the mound is reached and only intracellular particle logical development (Manstein et al., 1989). Here we use movement is observed. Scanning electron micrographs scanning electron microscopy, light microscopy, show that the surface of the wild-type mound consists immunofluorescence and computer analysis of time- of flattened cells which fit neatly together. The myosin lapse video films to study how D. discoideum myosin null null cell mound has an uneven surface, the orientation cells of strains HS2205 and HS2206 aggregate. Myosin of the cells is chaotic and no tip is formed. This is con- null cells are sufficiently coordinated in their move- sistent with the results of synergy experiments in which ments to form two-dimensional aggregation streams, myosin null cells were absent from the tips of chimeric although mutant cells within streams lack the elongated HS2205/AX2 slugs and pre-culminates. Immunofluores- shape and parallel orientation of wild-type strains. In cence microscopy using prespore and spore cell mark- the wild-type, cell movements are coordinated, cells ers reveals that a prestalk/prespore pattern forms usually joining streams that spiral inwards and upwards within the mutant mound but that terminal spore differ- as the mound extends into the standing papilla. In the entiation is incomplete. These results are discussed in aggregates of mutant strains, cell movements are relation to the role of myosin II in aggregation and mor- chaotic, only occasionally forming short-term spirals phogenesis. that rotate at less than half the speed of wild-type spi- rals and frequently change direction. Unlike the situa- Key words: myosin II, morphogenesis, Dictyostelium tion in the wild-type where spirals continue with mound development, cytoskeleton

INTRODUCTION (Williams et al., 1986; De Lozanne and Spudich, 1987; Eliott et al., 1991). D. discoideum is a soil amoeba that can Animal morphogenesis involves precisely timed cell differ- exist as single cells or as a structured multicellular aggre- entiation and cell migrations. For co-ordinated cell move- gate, depending on the stage of the asexual lifecycle. Mol- ments to occur during morphogenesis, cells must be able ecular genetic techniques involving the use of homologous to respond to extracellular signals and alter their shape and recombination have made it possible to construct myosin II movements accordingly. There is increasing evidence that null cells from which the myosin heavy chain gene has been the cytoskeleton is involved in many aspects of morpho- excised (Manstein et al., 1989). Though the mutant amoe- genesis including cell migration, cytodifferentiation and bae are able to migrate on a surface and aggregate to form developmental gene expression (Ben-Ze’ev, 1991, for rudimentary mounds, further development is blocked; these review). We are interested specifically in the role of myosin cells are unable to form the organized multicellular ‘slug’ II in animal morphogenesis. Actin and myosin II form a or mature fruiting body (Knecht and Loomis, 1987; De contractile unit that is thought to be involved in the fold- Lozanne and Spudich, 1987; Wessels et al., 1988; Peters et ing of epithelial cell sheets during gastrulation in chick (Lee al., 1988). This implies an important role for myosin II in et al., 1983) and Drososphila embryos (Young et al., 1991; tissue formation. The timing of developmental gene see also Odell et al., 1981) and the compaction of the mouse expression in the mutant is apparently normal (Knecht and morula (Sobel, 1983, 1984). Loomis, 1988), indicating that the block in morphogenesis We use the cellular slime mould, Dictyostelium dis - is due to a spatial or mechanical abnormality. coideum, to study the cytoskeleton and cell-cell interactions Using immunofluorescence techniques, it has been shown during development of a simple three-dimensional tissue that the myosin II protein is distributed unevenly in cells 458 S. Eliott and others of D. discoideum wild-type slugs, being concentrated in the 1987, 1988; De Lozanne and Spudich, 1987). HS2205 mounds cortex of peripheral and anterior cells, and evenly through- were fixed after all streams had either collected into the mounds out the cytoplasm in the inner posterior cells (Eliott et al., or begun to break up (between 17 and 19 h depending on initial 1991). This distribution corresponds with the prestalk-pre- cell density). For comparison, tipless aggregates of AX2 cells were spore pattern, a finding that is consistent with prestalk cells developed as described above and fixed after 7 h development. providing the major motive force for slug movement All mounds were vapour fixed in 1% (v/v) glutaraldehyde and 2.5% (w/v) paraformaldehyde in starvation buffer (25 mM MES, (Inouye and Takeuchi, 1980; Williams et al., 1986; Voet et 2 mM MgSO4, 0.2 mM CaCl2, pH 6.8) for approximately 2 h. al., 1984; Yumura, 1992). Cells at the periphery of the slug After fixation, plastic replicas were made of the mounds using are polarised with respect to myosin II, resembling indi- Exaflex hydrophobic dental impression material (Kerr) as vidual motile amoebae. We suggest that these cells have a described by Williams and Green (1988) for plant meristems. This specialised role in the morphogenesis and migration of the technique requires minimal fixation and handling of tissue and whole organism. thus reduces artefacts due to conventional preparation for SEM. Here we study the aggregation and development of two The replicas were coated with gold and viewed under a Philips myosin null mutant strains, HS2205 and HS2206, in order 505 scanning electron microscope. to elucidate the role of myosin II in normal D. discoideum development. We show that cells lacking myosin II are able Time-lapse videomicroscopy and computer to form aggregation streams that appear similar to wildtype analysis strains on casual observation. However, cell shape is abnor- For video observation and analysis of cell movements during mal in both streams and mounds. Myosin null cells do not aggregation, cells were filmed using a low-light-sensitive camera undergo the coordinated spiralling movements observed for (Mintron, CCD, MTV-1801CB) attached to a Zeiss OPM1 micro- scope fitted with a f100 or f150 mm lens and recorded on a wild-type cells as they approach the aggregation centre. National time-lapse cassette recorder, NV-8051. Myosin null cell Although coordinated movement is absent in mutant streams and aggregates were filmed for 2 to 8 h periods within a mounds, a distinctive prespore pattern is established. time frame of 12 to 24 h after plating. AX2 and AX3 streams and Mutant mounds are unable to form tips and, when mixed aggregates were filmed for 30 min to 5 h periods within a time with wild-type cells, are absent from the tips of chimeric frame of 5 to 12 h after plating. slugs and preculminates, suggesting that myosin II may be Speed of spiral motion (min per revolution) in aggregation involved in the process of tip formation (a factor that may mounds was determined from time lapse video segments by mea- be crucial in the inability of myosin null cells alone to suring the amount of time taken for a number of cells in the outer progress morphologically beyond the mound stage). third of a rotating aggregate to move 1/4 or 1/2 way around the circumference of the mound. For computer analysis, images taken at 10 s intervals using the above microscope and camera were stored directly in a 386 com- MATERIALS AND METHODS puter using a Matrox PIP(1024) image board. Cell movements were tracked using a program which maps individually selected Strains and development of cells points through consecutive frames (Breen et al., unpublished data). For light, immunofluorescence and scanning electron microscopy This program measures flow of identifiable features rather than (SEM) and synergy experiments, the D. discoideum myosin null delineating the cells and tracking them individually. Cell-cell mutants, HS2205 and HS2206 (Manstein et al., 1989) and two boundaries and intracellular vesicles were chosen as points to be wild-type axenic strains, AX3 (Loomis, 1969) and AX2 (Watts tracked because they could be easily seen and tracked for several and Ashworth, 1970), were used. Amoebae were grown on slime minutes. mould nutrient agar (SM) with Klebsiella aerogenes. To develop aggregates, cells were collected from a lawn of K. aerogenes, Immunofluorescence washed three times in Bonner’s salts solution and 2-25 ml drops 6 6 HS2206 amoebae (prepared as described above) were allowed to at a density of between 2 ´ 10 and 4 ´ 10 cells/ml were pipet- develop for 19, 23, 30 and 65 h at 21(±1)˚C before fixing in 2.5% ted onto water agar (1% Noble agar in distilled water with 250 (w/v) paraformaldehyde in PBS (0.15 M potassium phosphate; mg/ml dihydrostreptomycin sulphate) which formed thin (i.e. 2.75 0.9% (w/v) NaCl, pH 7.2) for 15 h at room temperature. Mounds ml agar: for filming and photography) or thick (i.e. 30 ml agar: were then encased in molten water agar and cubes bearing 1 to 4 for scanning electron and immunofluorescence microscopy) layers mounds were washed in PBS overnight at 4˚C. Cubes were infil- in 9 cm petri dishes. Drops were air dried for 5-10 min and cells trated with OCT embedding compound (Miles-Tissue Tek II) for allowed to develop in an illuminated room at 21(±1)˚C and 70- 2 days at 4˚C, and 3-4 mm sections perpendicular to the agar sur- 80% relative humidity. In the case of thin water agar plates, agar face on which the mounds had developed were cut at - 22(±2)˚C within each Petri dish was kept moist with circular strips of filter and picked up onto slides treated with chrome alum-gelatine paper soaked in Bonner’s salt solution. (Krefft et al., 1984). Slides bearing frozen sections were washed in PBS for approximately 2 h and blocked with 5% (w/v) skim Light microscopy milk for 30 min. After three 5-min washes in PBS, sections were Aggregating amoebae of strains AX3 and HS2206 were devel- labelled with MUD3 supernatant (a mouse monoclonal antibody oped on thin water-agar plates as described above and pho- that recognises a spore coat protein (SP96; Voet et al., 1985) or tographed with an Olympus camera (PM10.ADS) attached to a MUD1 (a monoclonal antibody that recognises glycoprotein PsA Zeiss Universal microscope. on the surface of prespore cells; Krefft et al., 1983) for 1 h and washed in PBS. Sections were blocked a second time for 10 min, Preparation of aggregation mounds for scanning washed again in PBS (2 ´ 10 min) and labelled with FITC-con- electron microscopy jugated sheep anti-mouse IgG (Sigma), diluted 1:25 with PBS, for Myosin null cells take much longer to aggregate than the parental 45 min. After washing in PBS (3 ´ 20 min), sections were wild-type strain (see Manstein et al., 1989; Knecht and Loomis, mounted with Aquamount (Gurr). Control slides were treated as Role of myosin II in Dictyostelium development 459 described above except that they were incubated with the second tography. Cells within the AX3 mound that did not spiral antibody only. moved towards the aggregation centre in concentric circles. Whether or not spiralling commenced later in aggregation Development of synergised slugs and early in this exceptional aggregate was not followed. On the other culminates hand, only 2 of the 18 HS2206 aggregates filmed under- AX2 and HS2205 amoebae were collected from bacterial lawns went significant spiralling motion. In these two cases, spi- and washed as described above. AX2 or HS2205 cells were stained ralling was observed during early aggregation (between 12 with a fluorescent membrane dye, DiIC16 (Molecular Probes; 20 and 15 h development) of HS2206 cells but was short-term, mg/ml) in starvation buffer on a shaker for 15 min at a total cell seldom lasting more than 12 min (or one quarter of a rev- concentration of approximately 106 cells/ml. Stained and unstained cells were washed four times in starvation buffer. olution), and the cells frequently changed direction. The Unstained AX2 cells were mixed with stained HS2205 cells at a speed of spiral motion was determined for one of the mutant ratio of 8:1. Stained AX2 cells were mixed with unstained AX2 aggregates and a wild-type aggregate with the same diam- cells at a ratio of 1:1 as a control. Mixed cell suspensions were eter (200 mm). Spiralling motion in the HS2206 aggregate centrifuged (2000 revs/min) and the cell pellets vortexed and was slower (49 min per revolution) than in the AX3 aggre- pipetted onto water agar plates. Petri dishes were enclosed in gate (21 min per revolution). square black polyvinyl chloride containers in an illuminated room Spiralling motion was observed to continue in AX3 and at 21(±1)˚C and 70-80% relative humidity. Slugs developed and AX2 aggregates as the wild-type mounds underwent fur- migrated towards light entering a 3 mm hole in the side of each ther morphogenesis, extending upwards into standing fin- container. gers (also observed by Clark and Steck, 1979). Spiralling Chimeric slugs and pre-culminates were fixed in 3% was never observed to cease during the period of filming paraformaldehyde and 0.02% glutaraldehyde in starvation buffer for 2 to 6 hours, washed in PBS and whole mounts observed under of such aggregates. Any co-ordinated movements observed a fluorescence microscope. in early HS2206 aggregates, on the other hand, gave rise To check for the stability of DiIC16 over time, stained AX2 to chaotic movements that finally ceased altogether after cells were observed under a fluorescence microscope at regular approximately 19 h; at this stage only membrane ruffling intervals over a period of 30 hours. After approximately 12 hours and intracellular particle movement could be seen. As cell much of the dye had been internalised and was seen as vesicles migration stopped within a mutant mound, any incoming inside the cell, which nevertheless remained strongly fluorescent. streams still attached to the mound also ceased moving After 24 hours, however, a large proportion of spores appeared to towards the aggregation centre and began to break up and have lost the dye. To check for transfer of dye to unstained cells, form separate smaller aggregates. stained cells were mixed with unstained cells in known propor- Movement in wild-type and mutant strains was analysed tions and cells were counted at regular intervals. Only very slight staining of some unstained cells was observed after prolonged in detail using a computer tracking program and represen- periods (this could have been due to phagocytosis by unstained tative data is shown in Fig. 2. The HS2206 aggregate shown cells of fluorescent cell debri and other particles). in Fig. 2B,D was filmed 12 h after plating and was repre- sentative of the majority of mutant aggregates, which did not show any spiral motion. HS2206 cells moved chaoti- cally once within the mound and no apparent coordination RESULTS of cell movement was indicated during the period of analy- sis (Fig. 2D). Spiralling motion was clearly observed in the Mutant amoebae initially form unbroken AX3 aggregate analysed (Fig. 2A,C). aggregation streams With low power observation it could be seen that myosin- Mutant mounds have an uneven surface and do null cells were able to form long continuous aggregation not form tips streams (Fig. 1B) similar to AX3 (Fig. 1A). At high power, Once aggregation is complete, wild-type aggregates appear however, it was apparent that the mutant cells (Fig. 1D) did as smooth hemispherical mounds which then form tips and not have the elongated morphology and parallel orientation undergo further morphogenetic movements that give rise to of cells in wild-type aggregation streams (Fig. 1C). HS2206 the slug and fruiting body. Scanning electron micrographs cells in streams were more irregular in their shape and size of plastic replicas indicated that mature myosin null mutant and more flattened (Fig. 1D) when compared to AX3 cells, aggregates are irregularly shaped cellular masses (Fig. which exhibited a more three-dimensional cylindrical 3B,D) without the smooth surface or ordered alignment of appearance (Fig. 1C). During later aggregation, wild-type cells apparent in their wild-type counterparts (Fig. 3A,C). cell streams collected into the central aggregate towards This lack of organisation was apparent during the whole which they were migrating, while many HS2206 streams period of mound formation and at no stage was a tip broke up, eventually forming many smaller aggregates. The observed to form on mutant aggregates. The example illus- beginning of this breakup is apparent in Fig. 1B. trated for HS2205 had a layer of slime sheath covering the mound, indicated by cells with shrunken sheath stretched Spiralling is defective in aggregating myosin-null over them (Fig. 3D). This was confirmed by immunofluo- cells rescence on cryosections of HS2206 mounds using an anti- It has often been observed that cells in aggregation streams body (MUD62) that labels sheath material (data not shown). form spirals as they approach the aggregation centre (Clark Low-temperature scanning electron microscopy was also and Steck, 1979). Spiralling motion was observed in 19 of employed to confirm that the dental impression material the 20 AX3 and AX2 aggregates filmed by time-lapse pho- used did not significantly alter the shape of aggregates. In 460 S. Eliott and others

Fig. 1. Phase-contrast micrographs of aggregating wild-type AX3 and myosin-less HS2206 amoebae. HS2206 cells initially form continuous aggregation streams (b) that look similar to AX3 streams (a) at low magnification. At higher magnification it can be seen that HS2206 cells within streams (d) are flattened and lack the elongated shape and parallel orientation of AX3 cells (c). High-magnification pictures (c,d) were image enhanced. Bars: a and b, 1 mm; c and d, 50 mm. this technique aggregation mounds were plunged into liquid was apparent in mutant mounds (Fig. 4A,B). By 23 h nitrogen, transferred to a cryotransfer system and examined MUD1 labelling indicated the presence of prespore cells in in a Cambridge 3600 Stereo scanning electron microscope. the outer regions of mounds (Fig. 4C). At this stage MUD3 The results confirmed those observed with the replica tech- labelling was still absent. At 30 and 65 h, MUD1 labelled nique, but they are not shown here, due to the formation the entire centre of aggregates but was absent from the base of ice crystals, which partially obscured the image. and a single cell layer across the top of mutant mounds (Fig. 4E,G,I,K). MUD3 labels prespore vesicles inside pre- Prespore/prestalk pattern in mutant mounds spore cells and the surface of spores in wild-type fruiting Disruption of the myosin heavy chain gene in D. dis - bodies (Voet et al., 1985). In myosin null mutant mounds, coideum apparently does not significantly affect the however, labelling of prespore vesicles was weak after 30 expression of a range of developmentally regulated genes, h (Fig. 4F) and surface labelling of mature spores was not despite the delay in aggregation and the morphogenetic often observed, even after 65 h (Fig. 4F,H). Cells with aberrations of these cells (Knecht and Loomis, 1988). Thus MUD3 labelling of intracellular vesicles were labelled spore and stalk cells are expected to be formed eventually strongly by MUD1, indicating that most prespore cells within mutant mounds. In wild-type strains, terminal differ- failed to differentiate normally into mature spores, result- entiation of spore and stalk cells is normally complete ing in a low spore to nonspore cell ratio. When mature within 24 h from the onset of starvation (Knecht and spores were apparent, they varied considerably in their size Loomis, 1988). To determine the pattern and timing of pre- and shape (Fig. 4J,L). Control sections incubated with spore and spore cell differentiation in myosin null aggre- second antibody alone showed no labelling. gates, frozen sections of 19-, 23-, 30- and 65-h-old mounds were labelled with a prespore-specific antibody, MUD1 and Myosin null cells are absent from the tips of a spore coat-specific antibody MUD3 (identifying the SP96 synergised slugs and early culminates antigen). At least 6 mounds from each stage were viewed Multicellular development of myosin null cells can be res- and representative examples are shown in Fig. 4. After 19 cued if they are mixed with sufficient wild-type cells at the h development, negligible MUD1 and no MUD3 labelling onset of starvation (Knecht and Loomis, 1988). We syner- Role of myosin II in Dictyostelium development 461

Fig. 2. Computer analysis of cell movements in AX3 (after 6 h development) and HS2206 (after 12 h development) aggregation mounds. Visible cellular features were chosen (a and b) and tracked by computer at 10-s intervals over a 5-min (AX3) and an 8-min (HS2206) period (see Materials and methods). Round dots indicate initial points chosen and tracks indicate direction and distance traversed. In AX3 aggregates, cells moved in spirals (c) that continued as the aggregate elongated to form the ‘standing finger’. In HS2206 mounds, cell movements were slower and uncoordinated (d). Bars: a and b, 50 mm. gised AX2 cells with stained HS2205 cells in order to of slugs and early culminates was seen in all aggregates observe where the myosin null cells go within chimeric observed from three separate experiments. In control exper- slugs and pre-culminates. Since much of the fluorescent dye iments in which stained AX2 cells were mixed with appears to be lost from spores during sporulation, we did unstained AX2 cells, stained cells were distributed along not check mature fruiting bodies. the entire length of chimeric slugs (Fig. 5D). Slugs made from mixtures of HS2205 and AX2 cells were very long, with tails that extended back to the site of DISCUSSION aggregation by as much as 1 cm. A gradation of stained null cells was observed along the length of chimeric slugs, Although myosin null cells are generally inefficient in their being absent from the extreme tip and more numerous chemotactic response, they are sufficiently coordinated in towards the rear (Fig. 5A,B). The boundary between their movements to form two-dimensional streams. Essen- unstained cells in the tip and stained cells at the base of the tially all coordination ceases, however, once cells are within tip was distinct (Fig. 5B). In some cases, however, very the multicellular aggregate. This implies that other mole- few mutant cells were observed in the entire front half of cules can be involved in cell interactions in two-dimen- the slug. Many slugs were seen to leave behind trails and sions, but that myosin II becomes essential for proper cell- clumps of cells, some of which underwent varying degrees cell interactions in a three-dimensional array. The inability of further development but rarely formed slugs or fruiting of the mutant cells to undergo coordinated movement and bodies. progress beyond the mound stage may be due to the cells During culmination of synergised slugs, the stained having insufficient strength to generate sufficient force for HS2205 cells were still absent from the tip and some the coordinated movement of large groups of cells, and/or appeared to collect into a kind of girdle at the base of the could relate to confused chemotactic signalling within the tip (Fig. 5E,F). The absence of mutant cells from the tips cell mass. 462 S. Eliott and others

Fig. 3. Scanning electron micrographs of plastic replicas of representative AX2 (a and c) and HS2205 (b and d) aggregation mounds. AX2 cells form smooth hemispherical mounds within about 7-8 h of development. The cells covering the surface of the wild-type mound are flattened and fit neatly together (a,c). HS2205 mounds form after approximately 17-19 h development and are generally asymmetrical. The outer layer of cells in HS2205 mounds is not flattened and, in this example, is covered in a layer of extracellular matrix, indicated by the wrinkles in the mounds surface (arrowheads in d). Bars: a and b, 100 mm; c and d, 50 mm.

Myosin null cells form mounds that are roughly hemi- mutants that lack two F-actin cross-linking proteins, alpha spherical but lack the smooth surface and ordered appear- actinin and gelation factor, and thus have greatly reduced ance of wild-type mounds at an equivalent stage of devel- cortical viscoelasticity (Witke et al., 1992). These cells opment. The irregular surface of mutant mounds probably aggregate normally but rarely develop beyond the tipped reflects the lack of cortical tension within myosin null cells aggregate stage. as described by Pasternak et al. (1989). Myosin II is con- Individual myosin null cells have a less polarised shape centrated in the posterior and outer lateral cortex of wild- than wild-type cells, and their movements are consequently type cells at the periphery of the early aggregate, suggest- less directed (Peters et al., 1988; Wessels et al., 1988). This ing that, in normal development, these cells could exert a lack of directed movement was also observed in this study centripetal force on the aggregation centre (Yumura et al., in both aggregation streams and mounds. Another striking 1984); further morphogenetic events may rely on the feature of myosin null cells in streams was their flattened strength (i.e. cortical tension) of these outer cells. This is appearance. Normal migrating amoebae have a three- consistent with recent evidence from D. discoideum double dimensional, elongated appearance and myosin II is con- Role of myosin II in Dictyostelium development 463

Fig. 4. Im m u n o fl uorescence micrographs of frozen sections from 19 (a,b), 23 (c,d,), 30 (e,f,j,l) and 65 (g,h,i,k) h old HS2206 mounds. Mounds were labelled with MUD1 (a,c,e,g,i) or MUD3 (b,d,f,h,j) antibodies. After 19 h development, very little labelling was apparent with MUD1 (arrowheads in a) and no labelling with MUD3 (b) antibodies. In 23-h-old mounds MUD1 labelling begins to appear at the periphery of the mound (arrowheads in c); MUD3 labelling is still absent (d). After 30 h MUD1 labelling has extended into the centre of the aggregate but is absent from cells along the base (arrowheads in e) and a thinner, less visible layer along the top. MUD3 labelling is apparent at this stage (f,j), sometimes labelling mature spores (large arrowhead) but more frequently labelling prespore vesicles (small arrowheads) (j and l are higher magnification shots of another section through a 30-h mound); mature myosin null-cell spores vary in their size and shape. After 65 h, mounds have begun to dry out and have a compacted appearance (g,h,i,k; i and k are details of the section shown in g). MUD1 labels the centre of the aggregate (g) but is still absent from a layer of cells at the periphery (large arrowhead in i and k) and along the base and top of the mound (arrowheads in g and i). MUD3 strongly labels prespore vesicles after 65 h (h); mature spores were rarely seen at this stage. Bars: a-h, 100 mm; i-l, 20 mm. 464 S. Eliott and others centrated in the posterior cortex (Yumura et al., 1984). In Steck, 1979; Robertson and Grutsch, 1981) its importance wild-type aggregates cell movements are coordinated, with in development has not been thoroughly investigated. It has streams of cells invariably forming spirals as they join the been proposed that orbital motions drive morphogenetic aggregation centre. Although spiralling in D. discoideum movements and slug migration and give the multicellular aggregation is well documented (Durston, 1973; Clark and stages their characteristic circular axis (Clark and Steck,

Fig. 5. Fluorescence micrographs of chimeric slugs and early culminates. In a-c and e-f HS2205 cells stained with a fluorescent membrane dye were mixed with unstained AX2 cells; in d stained AX2 cells were mixed with unstained AX2 cells as a control. The direction of migration for slugs in a-d is right to left. Mixtures of HS2205 cells and AX2 cells form slugs with very long tails and a gradation of mutant cells is seen extending from the base of the tip to the rear (a). Myosin null cells are absent from the tips of HS2205/AX2 mixed slugs and early culminates (a-c and e-f). The border between stained and unstained cells in slugs is clearly defined (arrowhead in b). In early culminates, myosin null cells collect around the base of the tip forming a kind of girdle (small arrowheads in f); the culminate in f is at a later stage of development than that pictured in e (the large arrowhead indicates the top of the tip). In control experiments, stained AX2 cells were distributed throughout the entire length of the slug (d). Bars: a and d, 300 mm; b,c, 10 mm; e,f, 200 mm. Role of myosin II in Dictyostelium development 465

1979). Spiralling was rarely seen in myosin null cell aggre- results suggest that myosin null cells destined to form the gation and when it did occur did not last long and quickly tip are unable to sort out within the aggregate. gave way to locally chaotic cell movements. In all mutant It is interesting that, despite the apparent chaos of the mounds observed by time lapse filming, cell migrations myosin null cell aggregates, a distinctive pattern was appeared to cease after 17 to 19 h development. observed within mature mutant mounds; prespore and spore The lack of spiral motion in myosin null cell aggrega- cells localised to the centre of the aggregate while a layer tion may be due to the mutant cells being unable to respond of unlabelled cells (presumably prestalk cells) was observed appropriately to morphogenetic signals once within the along the base and a thinner layer along the top of mutant multicellular array. In aggregation streams, cells move as a mounds. Since this pattern was established well after cell monolayer or as a layer only a few cells thick; waves of migrations within the mounds appeared to cease, it is likely cAMP are unidirectional and the cells move in one direc- that the observed pattern was generated by positional cues tion towards the aggregation centre (Durston, 1973; Robert- rather than through sorting out of differentiated cells. Evi- son and Grutsch, 1981). Aggregation-competent amoebae dence for positional information in the establishment of the in which the myosin heavy chain gene has been truncated prepore/prestalk pattern has been presented previously by are essentially normal in their response to cAMP (Peters et Krefft et al. (1984) and Williams et al. (1989). al., 1988). In the three-dimensional structure, however, The inability of myosin II null cells to form tips was also most cells are surrounded on all sides by other cells and observed in synergy experiments in which null cells stained signalling must become more complex. The polarised shape with a fluorescent membrane dye were mixed with and myosin II distribution of wild-type cells may be essen- unstained wild-type cells. Null cells were absent from the tial for cells to receive and respond appropriately to extra- extreme tips of mixed slugs and early culminates, suggest- cellular signals within a three-dimensional mass. ing that wild-type cells might be able to rescue null cell Responding to signals within three dimensions may also development by providing a tip for the cellular mass. Fur- require cells to be polarised with respect to their membrane ther experiments are required, however, to establish signal receptors. Unlike wild-type cells, myosin null cells whether null cells are absent from the tips of mixed slugs are unable to cap cell surface proteins cross-linked by con- because they lack an essential feature for tip formation canavalin A (Pasternak et al., 1989; Fukui et al., 1990). (such as cell shape or proper signalling etc.) or whether Analysis of particle transport in the plasma membrane of they simply join the aggregate too late to form the tip myosin null mutant amoebae indicates that myosin II causes (myosin-deficient cells move at less than half the speed of mobile particles to be drawn into focal points (Jay and the parent strain; Wessels et al., 1988). Elson, 1992). These results imply that the actomyosin This research was supported by a Macquarie University meshwork in wild-type cells is linked to proteins in the Research grant and an ARC program grant to Keith Williams, and plasma membrane and thus could be involved in regulating a National Institute of Health grant GM40509 to Jim Spudich. We their distribution, as has been suggested for a number of thank Nick Rasmussen for help with the replica technique and mammalian cells (Gingell and Owens, 1992, for a review). Fran Thomas for help with the scanning electron microscopy. We During the formation of the tight aggregate, almost contin- also thank James Spudich, Malcolm Jones, Manuela Fuchs and uous secretion of cAMP causes a down-regulation of the Ines Carrin for helpful discussions, and Ron Oldfield for time and cAMP receptor and a concomitant redistribution of the patience with the light microscopy. receptor into patches in the plasma membrane (Wang et al., 1988). 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