Title

Function and localization of rac proteins in Dictyostelium

Date of Completion

January 1999

Keywords

Biology, Cell

Degree

Ph.D.

Abstract

Rho family proteins regulate polymerization of actin cytoskeleton in a variety of cell types. Thus far, 14 rac genes in rho family have been identified in Dictyostelium. To begin the characterization of each rac protein function, racA and racD was fully cloned using inverse PCR and 3-RACE. Surprisingly, the predicted amino acid sequence of racA is three times bigger than other rac genes. Unusual Ser and Thr stretches are found at similar location of racA and racD. Moreover, both racA and racD do not carry a conserved CAAX box at the C-terminus that is critical for the lipid modification. Further characterization of these two novel rac proteins will be pursued. ^ Function of racB and racC was studied by generating mutant cell lines conditionally overexpressing wild type (WT-) and constitutively activated (CA-) version of racB and racC. Cells expressing WT-racB had a flattened shape and some of CA-racB expressing cells were also flat with ruffling edges. However, majority of CA-racB cells detached and formed numerous actin-containing protrusions. It is likely that racB induced actin polymerization to increase the number of the protrusions which resulted in detachment of cells due to the lipid tension. On the other hand, cells overexpressing WT-racC formed spherical actin containing structures called petalopods, and increased phagocytosis by three fold. CA-racC expression led to an irregular cortex shape and enlarged contractile vacuole structure. Therefore, racC seems to regulate globular polymerization of actin cortex leading to the shape changes. ^ To examine the localization of rac proteins, GFP-fusion of racC and rac1B was expressed in Dictyostelium cells. Increase in GFP-rac1B signal was found at the macropinocytic cups and phagocytic cups, which was accompanied by actin polymerization On the other hand, GFP-racC was evenly distributed in the plasma membrane and the cytoplasm without significant changes in fluorescence intensity during macropinocytosis or phagocytosis. ^ In summary, racB, racC and rac1B have a different localization and function to control actin cytoskeleton. racA and racD are distinguished from other rac proteins by their unique genetic structures. Thus, rac proteins so far tested seem to have a unique function controlling actin cytoskeleton in Dictyostelium. ^