3-DIMENSIONAL LOCALIZATION OF WILD-TYPE AND MYOSIN-II MUTANT-CELLS DURING MORPHOGENESIS OF DICTYOSTELIUM

Dictyostelium amoebae that lack myosin II (mhcA(-)) are unable to unde rgo morphogenesis. The cells aggregate slowly to form hemispherical mo unds, but the mounds never extend a tip upward. Expression of developm entally regulated genes appears normal in the absence of morphogenesis . When mixed with an excess of wild-type cells, some mutant cells form differentiated spores; however, rescue is extremely inefficient (Knec ht and Loomis, 1988). In order to assess how morphogenesis is normally accomplished and why mutants lacking myosin II cannot develop, a new method has been developed that allows individual amoebae to be localiz ed and tracked at high resolution within the multicellular organism du ring development. Amoebae are labeled with a fluorescent dye at the be ginning of starvation, mixed with an excess of unlabeled cells, and al lowed to develop. The three-dimensional position of labeled cells in t he multicellular organism is then determined using a laser scanning co nfocal microscope. Using this methodology, we have shown that labeled wild-type cells are randomly distributed throughout the organism and c omplete development normally. When labeled mhcA(-) mutant cells are mi xed with a 20-fold excess of wild-type cells, they are nonrandomly loc alized even at the earliest stages of development. Mutant cells in agg regation streams are found primarily at the edges of the streams and m any cells never become part of the streams or are left behind as the w ild-type cells complete aggregation. Those that are incorporated into the aggregate are found at the edge and base, the backs of slugs and t he base of the fruiting bodies. A few mutant cells can be found in the sorus, where they presumably become spores. The segregation of mhcA(- ) mutant cells to the outside of wild-type aggregation streams argues that the mutant cells are unable to penetrate a mass of adhered, wild- type cells. We hypothesize that mutant cells lacking cortical integrit y are unable to generate sufficient protrusive force to break the adhe sion of wild-type cells to each other. This would make the mutants inc apable of moving through a mass of cells (either mutant or wild type) or of changing shape when adhered to other cells. We propose that muta nts lacking myosin II are unable to accomplish morphogenesis because t hey cannot move correctly in a three-dimensional mass of adhered cells . (C) 1995 Academic Press, Inc.