PROTEOMIC ANALYSIS OF DROSOPHILA GASTRULATION

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DESCRIPTION (appended verbatim from investigator's abstract): We are interested in understanding the mechanisms and proteins involved in cell shape maintenance and change using Drosophila mesoderm morphogenesis as a model system. The mesoderm is formed by the invagination of a large rectangular patch of cells on the ventral surface of the embryo in a process called ventral furrow formation. This invagination is brought about in 15 minutes by a series of cell shape changes that transform the columnar-shaped epithelial cells into wedge-shaped furrow cells, which eventually become migratory and form the mesoderm layer. Genetic analysis has revealed the genes involved in the signaling cascade that leads to the specification of the ventral furrow cells. The final genes in this signaling cascade are two transcriptional regulators, twist and snail. Embryos that are mutant for either gene fail to form a ventral furrow. Genetic dissection has not been able to identify any cytoskeletal-associated or structural proteins as mechanical mediators of the cell shape changes. This is probably due to the combined effects of maternal contribution, pleiotropic function and functional redundancy. We have developed a novel biochemical approach, called difference gel electrophoresis (DIGE), for identifying protein changes that occur during ventral furrow formation. This new technique is a modified two-dimensional polyacrylamide gel electrophoresis method that involves labeling of proteins prior to electrophoresis with differently-colored fluorescent dyes. Proteins from two different cell types are labeled with the different dyes, combined, and then run on the same electrophoresis gel. After electrophoresis, a pair of fluorescence images is recorded with a large format fluorescence imager. Proteins that are common to both cell types appear as two-colored spots; proteins that are unique to one cell type or the other only contain one color dye. This allows for rapid detection of protein differences, so called "difference-proteins." These difference-proteins are then identified by mass spectrometry (MS). To date, more than twenty difference-proteins have been detected. These include maternally supplied and post-translationally modified proteins. Three of these proteins have been identified by MS: two proteasome subunits and transferrin. The aims of this proposal are to use DIGE/MS to identify the remaining VF difference-proteins for cloning and antibody production and to determine the role these difference-proteins play in cell shape modulation and ventral furrow morphogenesis. For the latter, we will use time-lapse-microscopy in conjunction with gene knock-out and ectopic expression of the cloned difference-proteins.