Three-dimensional in vivo analysis of Dictyostelium mounds reveals directional sorting of prestalk cells and defines a role for the myosin II regulatory light chain in prestalk cell sorting and tip protrusion

During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressing cells) moved independently and directionally to for m a cluster. This is consistent with a chemotaxis model for cell sorting (a nd not differential adhesion) in which a long-range signal attracts many of the prestalk cells to the site of cluster formation. Surprisingly, the ecm AO prestalk cluster that we observed was initially found at a random locati on within the mound of this Ax3 strain, defining an intermediate sorting st age not widely reported in Dictyostelium. The cluster then moved en masse t o the top of the mound to produce the classic, apical pattern of ecmAO pres talk cells. Migration of the cluster was also directional, suggesting the p resence of another long-range guidance cue. Once at the mound apex, the clu ster continued moving upward leading to protrusion of the mound's tip. To i nvestigate the role of the cluster in tip protrusion, we examined ecmAO pre stalk-cell sorting in a myosin II regulatory light chain (RLC) null in whic h tips fail to form. In RLC-null mounds, ecmAO prestalk cells formed an ini tial cluster that began to move to the mound apex, but then arrested as a v ertical column that extended from the mound's apex to its base. Mixing expe riments with wild-type cells demonstrated that the RLC-null ecmAO prestalk- cell defect is cell autonomous. These observations define a specific mechan ism for myosin's function in tip formation, namely a mechanical role in the upward movement of the ecmAO prestalk cluster. The wild-type data demonstr ate that cell sorting can occur in two steps, suggesting that, in this Ax3 strain, spatially and temporally distinct cues may guide prestalk cells fir st to an initial cluster and then later to the tip.