Proteomic analysis of cell shape changes during gastrulation in Drosophila

Margaret Young
Carnegie Mellon University

My research project in the Minden lab involved the study of proteins and mechanisms involved in cell shape changes during development. A Drosophila model was used to study the embryonic process of ventral furrow formation. This process involves a series of cell shape changes that cause the formation of the mesoderm layer of the embryo. Mutational analysis has identified several genes which are involved in a signaling cascade that triggers ventral furrow formation, but this approach has not identified any cytoskeletal proteins or structural proteins involved in this process. An important thing to remember here is that the embryo contains maternally contributed proteins, in addition to zygotically expressed genes, and that many proteins are regulated by post-translational modification. For this reason, a direct protein analysis may be more useful than genetic screens for identification of the protein changes that cause the furrow to form.

For this purpose, the Minden lab has developed a new approach to identify protein changes between different cells. The technique, difference gel electrophoresis (DIGE), is a modified two-dimensional polyacrylamide gel electrophoresis method. Two protein samples are labeled separately with different fluorescent dyes. The samples are then mixed and analyzed. The mixed sample is first run on an isolelectric focusing gel to separate proteins on the basis of charge; the second dimension is an SDS-PAGE to separate the proteins on the basis of size. The gel is then fluorescently imaged to determine whether there are any protein differences. If a protein is the same in both cell samples, there will be one spot with both colors. If a protein is found in only one sample, then the spot will show only one of the dyes. This method can identify differences in level of expression as well as post-translational changes, such as phosphorylation or proteolysis, which might change the protein in such a way that makes ventral furrow formation possible. Such changes cannot be identified through RNA analysis. The spots that change are called "difference-proteins". Once these proteins are detected with DIGE, they are identified by mass spectrometry (MS).

So far, over fifty difference-proteins have been found, and many of them have been identified. My project was to determine the function of four of these proteins in ventral furrow formation and cell shape change. The proteins that I studied were determined to be time-dependent in ventral furrow formation by DIGE; they were belle, eIF-4e, squid and a dynamin-like protein. I used time-lapse microscopy to monitor the effect of reducing protein abundance with RNA interference. Now that the defects have been confirmed I will examine proteome changes in response to altered amounts of the proteins-of-interest. This will allow us to assemble a molecular framework for the proteins involved in driving cell shape change during ventral furrow formation .