Three-Dimensional Localization of Wild-Type and Myosin II Mutant Cells during Morphogenesis of Dictyostelium

David A Knecht; Eric Shelden

doi:10.1006/dbio.1995.1227

Dictyostelium amoebae that lack myosin II ( mhcA -) are unable to undergo morphogenesis. The cells aggregate slowly to form hemispherical mounds, but the mounds never extend a tip upward. Expression of developmentally 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 (Knecht 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 localized and tracked at high resolution within the multicellular organism during development. Amoebae are labeled with a fluorescent dye at the beginning of starvation, mixed with an excess of unlabeled cells, and allowed to develop. The three-dimensional position of labeled cells in the multicellular organism is then determined using a laser scanning confocal microscope. Using this methodology, we have shown that labeled wild-type cells are randomly distributed throughout the organism and complete development normally. When labeled mhcA - mutant cells are mixed with a 20-fold excess of wild-type cells, they are non-randomly localized even at the earliest stages of development. Mutant cells in aggregation streams are found primarily at the edges of the streams and many cells never become part of the streams or are left behind as the wild-type cells complete aggregation. Those that are incorporated into the aggregate are found at the edge and base, the backs of slugs and the 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 integrity are unable to generate sufficient protrusive force to break the adhesion of wild-type cells to each other. This would make the mutants incapable of moving through a mass of cells (either mutant or wild type) or of changing shape when adhered to other cells. We propose that mutants lacking myosin II are unable to accomplish morphogenesis because they cannot move correctly in a three-dimensional mass of adhered cells.

Three-Dimensional Localization of Wild-Type and Myosin II Mutant Cells during Morphogenesis of Dictyostelium

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