DULEH, SN; POPE, RK; Indiana University South Bend; Indiana University South Bend: Active Rac1B in Dictyostelium Discoideum Co-localizes with F-actin at Cell:Cell Contacts

Rac, a small G-protein in the Ras superfamily, is involved in the accumulation and organization of actin filaments. Actin filaments and associated proteins are responsible for cellular movement, cytokinesis, and phagocytosis. To determine the localization and function of active and inactive Rac1B in Dictyostelium discoideum, cell lines that express constitutively active Rac1B, constitutively inactive Rac1B, and wild-type Rac1B were established. These cell lines were constructed with Dictyostelium ORF+ cells using an inducible plasmid, pDXD-3C, containing myc-tagged Rac1B sequences. Immunofluorescence using anti-myc antibodies shows constitutively active Rac1B is localized in areas of filopodia and cell-to-cell contacts, whereas constitutively inactive Rac1B is homogenously distributed throughout the cell. Phalloidin staining demonstrates that F-actin co-localizes in regions containing active Rac1B. Cell lines expressing either mutant form of Rac1B exhibited defects in cytokinesis, phagocytosis, and multicellular development. To identify proteins that interact with Rac1B, immunoprecipitations are being performed with anti-myc antibodies to isolate the different myc-tagged forms of Rac1B and their binding partners. Understanding the function of Rac1B will lead to a better understanding of filamentous actin dynamics, and thus cell motility.