No abstract

Starvation induces free‐living Dictyostelium discoideum amoebae to form slugs that typically contain 100,000 cells. Only recently have sufficient clues become available to suggest how coordinated cell actions might result in slug movement. We propose a “squeeze‐pull” model that involves circumferential cells squeezing forward a cellular core, followed by pulling up of the rear. This model takes into account the different classes of cells in the slug; it is proposed that prestalk cells are engines and prespore cells are the cargo.

Abstract. While the role of myosin II in muscle contraction has been well characterized, less is known about the role of myosin II in non-muscle cells. Recent molecular genetic experiments on Dictyostelium discoideum show that myosin II is necessary for cytokinesis and multicellular development. Here we use immunofluorescence microscopy with monoclonal and polyclonal antimyosin antibodies to visualize myosin II in cells of the multicellular D. discoideum slug.M YOSIN II is found in all myocytes and in most eukaryote cells (Kom and Hammer, 1988;Warrick and Spudich, 1987) where it is implicated in cytokinesis (Fujiwara and Pollard, 1976;De Lozanne and Spudich, 1987;Knecht and Loomis, 1987; KitanishiYumura and Fukui, 1989), the control of cell shape (Wessels et al ., 1988), the motility of cells (Yumura et al., 1984;Rubino et al., 1984;Spudich and Spudich, 1982 ;Honer, 1988), the maintenance of cell polarity (Fukui et al., 1990), and the capping of membrane receptor proteins (Pasternak et al ., 1989). In multicellular organisms, myosin II may also play an important role in cell-cell interactions . Studies suggest that actomyosin bands are responsible for folding sheets of epithelia during gastrulation (Odell et al., 1981;Lee et al., 1983) and that myosin II is involved in both mouse morula compaction (Sobel, 1983 ; and resistance to stress in the embryonic chick area opaca (MonnetTschudi and Kucera, 1988) . Other studies involving gene disruption indicate that myosin II is necessary for normal multicellular development in Diciyostelium discoideum (see below) .D. discoideum is a simple, mobile eukaryote that can exist as an amoeba or as a multicellular aggregate (Bonner, 1967;Raper, 1984). D. discoideum thus lends itself to the study of both single cells and three-dimensional tissues. The cytoskeletal proteins ofindividual amoebae have been widely studied (Spudich and Spudich, 1982;Rubino et al., 1984;Fukui et al., 1987;Condeelis et al., 1987;Knecht and Loomis, 1987;De Lozanne and Spudich, 1987;Gerisch et al., 1989), however, those in multicellular aggregates have not .Mutant D. discoideum cells that lack myosin II survive and undergo (albeit slow) amoeboid movement; such cells differentiate into the two types which normally constitute a slug (prestalk and prespore cells) and theyaggregate to form loose mounds ofcells (Knecht and Loomis, 1987 A subpopulation of peripheral and anterior cells label brightly with antimyosin II antibodies, and many of these cells display a polarized intracellular distribution of myosin II. Other cells in the slug label less brightly and their cytoplasm displays a more homogeneous distribution of myosin II. These results provide insight into cell motility within a three-dimensional tissue and they are discussed in relation to the possible roles of myosin II in multicellular development . Wessels et al ., 1988 ;Peters et al ., 1988;Manstein et al., 1989) . Atthis point, development is blocked : aggregates of myosin-deficient mutants fail to become mobile-they do not form slugs with norm...

Cellulose is one of the commonest structural biopolymers. How cellulose is organized in extracellular matrices is a mystery. Here we investigate a model system, the extracellular matrix (ECM) of Dictyostelium discoideum which is composed of proteins and cellulose. A group of glycoproteins, the sheathins, which colocalize with cellulose in the ECM of D. discoideum are characterized. Sheathins are dimeric or trimeric forms of molecular mass 53-68 kDa, where the monomers are 12-35 kDa. The sheathin subunits are similar but not identical proteins. The sheathin family comprises sheathin 68, (68-kDa trimer); sheathin 62, (62-kDa dimer); sheathin 55, (55-kDa dimer), and sheathin 53, (53-kDa dimer). The subunits which assemble into the four sheathins represent at least three gene products: ShC, ShD, and ShE which are linked by disulphide bonds. Protein sequence analysis shows two of the sheathin genes encode products ShC and ShD with very similar amino terminal sequences. This group of D. discoideum ECM glycoproteins has homology with two other much larger ECM proteins of D. discoideum, ST430 and ST310, which are located in a more dispersed fashion in the ECM. Sheathins are tightly but non-covalently associated with the ECM, and this association requires strong denaturing conditions for disruption, e.g., SDS or 8 M urea. Sheathins form a component of the "cell prints" which are believed to have a role in cell-ECM interactions and slug cell migration.

Time-lapse video light microscopy was used to study the emergence and maturation of the migratory slug from a D. discoideum aggregate. The anterior part, the tip of this simple multicellular organism, establishes migration prior to the definition of the rear, and hence the length of the slug. It was found that newly formed slugs of wild-type strain WS380B can reach lengths greater than 1 cm, yet mature slugs of this strain are rarely longer than 2-3 mumm. Often the tip extended out of the aggregation mound upon an arching pillar of cells. After the tip first touched the substratum, it commenced migration with a rapid succession of movement steps. Here we show that at the initiation of migration, a differential rate of cell movement along the developing slug axis results in a series of complicated changes, before the stable and mature shape of the slug is formed. Our results lead to new conclusions about D. discoideum slug formation and shape maintenance. Evidence is presented for regulation of slug length.

Time-lapse video recordings of migrating multicellular slugs of Dictyostelium discoideum were subjected to image analysis. A transient ‘collar-like’ structure was identified at the anterior end of the slug. This collar remains stationary in the wild- type strain WS380B; it is observed shortly after the advancing tip contacts the substratum. Stationary collars formed approximately every 12min; they were matched with patterns revealed on the underside of slime trails with FITC-coupled monoclonal antibody MUD50. It is proposed that stationary collars are involved with the forward movement of the slug. The mutant strain HU2421 lacks the MUD50-epitope and forms collars which do not remain stationary but move backwards along the slug to collect at a ‘waist’ region. The slipping-collars observed in the mutant correlated with very slow migration rates. We propose that HU2421 moves slowly because it lacks traction.