Analysis of racE in cytokinesis:
RacE was identified as a novel small GTP-binding protein essential for cytokinesis in Dictyostelium. We would now like to determine how racE functions in cytokinesis. One approach is to identify proteins that interact specifically with racE and whose interactions are important in cytokinesis. Towards that goal we have generated a series of chimeric proteins containing varying proportions of racE and racC (a closely related small GTP-binding protein also from Dictyostelium). These chimeric proteins have been expressed, behind both GFP and FLAG epitopes, in wild-type and racE mutant cells. We can now take advantage of the GFP and FLAG epitopes to immunoprecipitate the chimeric proteins from appropriate cell lysates and look for proteins that co-precipitate. These proteins will be isolated and analyzed for their involvement in cytokinesis.
Dimerization of racE:
While conducting the above experiments we observed a band on our gels that corresponded to twice the molecular weight of racE. Furthermore, this band cross-reacted with anti-racE antibodies in western blot analysis. To confirm that racE actually does have the potential to self-dimerize we expressed both GFP- and FLAG-tagged racE in the same cell line. GFP-racE was then immunoprecipitated from cell lysates prepared from these cells. The immunoprecipitates were analyzed by western blotting using both anti-GFP and anti-FLAG antibodies. Interestingly, FLAG-racE was found in immunoprecipitates of GFP-racE, confirming that racE is capable of forming self-dimers. Additional studies suggest that this dimerization is mediated through the C-terminal end of the protein. Current studies, involving site-directed mutagenesis and deletion analysis, are aimed towards identifying which residues are important in racE dimerization.
Analysis of pats1, a novel gene required for cytokinesis:
We have recently identified the pats1 gene as a large open reading frame which is required for proper cytokinesis to take place in Dictyostelium cells in suspension culture. This open reading frame was identified through REMI screening for cytokinesis-specific genes. We have identified several interesting protein domains in the translated product of the pats1 open reading frame. These include a region homologous to myotubularin, a mammalian protein phosphatase, a region corresponding to the GTP-binding pocket of small GTP-binding proteins, and a protein kinase domain. In addition, we have also identified 13 leucine-rich repeats and 8 WD-40 repeats. The combination of these domains and repeats suggests that the pats1 protein may be a multi-functional protein important in cytokinesis. We are now intensively studying this gene/protein on several fronts. We have begun to over-express several of the domains, independently, in both wild-type and pats mutant cells. Interestingly, over-expression of the protein kinase domain results in a cytokinesis defect in attached cells. We are now in the process of cloning other domains, independently and as larger parts of the pats1 protein, to examine the role of these other domains. We are also interested in generating a full-length clone of the pats1 gene. With this we plan to begin mutational and deletion analyses, focusing on the identified domains and specific amino acid residues within those domains. We are also interested in pursuing structural analyses of the pats1 protein. To identify interacting proteins we plan to conduct co-immunoprecipitation experiments. We will take advantage of epitope tagging antibodies generated against several synthetic peptides from different pats1 protein domains. These epitopes and antibodies may also be useful in determining the intracellular location of the pats1 protein (particularly in dividing cells).
Expression of yeast and mammalian centrosomal proteins in Dictyostelium:
Recently, it has been shown that centrosomes, the organelle responsible for the organization of microtubules, may also play an important role in the temporal regulation of cytokinesis, as well as in the placement of the actin/myosin contractile ring. In collaboration with Stephen Doxsey, we are in the process of expressing several centrosome-associated proteins (from yeast and mammalian sources) in Dictyostelium and testing these cells for defects in cytokinesis. Observation of such a phenotype would open up Dictyostelium for further studies in the role of the centrosome in cytokinesis.
Isolation of additional cytokinesis-defective cell lines:
As part of the Cell Biology course (Bio 137), for the last two years we have conducted a semester-long REMI mutagenesis screen for cell lines unable to complete cytokinesis when grown under suspension conditions. In both years we have isolated several such cell lines and are currently attempting to identify the gene disruptions in these cell lines that result in their cytokinesis-defective phenotype. Through the identification and characterization of additional genes required for cytokinesis we hope to shed some insight into the nature of how this important event is regulated in cells.