Resumo

Objetive: The small GTPase Rnd1 and STI1 are both involved with neurite outgrowth and cytoskeleton plasticity. Based on literature results we decided to investigate if Rnd1 and STI1 could specifically interact with each other and if this interaction could implicate in biological relevance. Methods: Polyclonal antibodies were generated by immunizing mice (anti-Rnd1) and rabbits (anti-STI1) with recombinant 6His-Rnd1 and 6His-STI1 respectively. These antibodies were used to investigate subcellular localization of Rnd1 and STI1 on primary hippocampal E18 neurons and for biochemichal assays. Cell lysates from HEK 293T cells co-transfected with pEGFP-STI1 and pEGFP vectors and mouse whole brain lysate were used for pull-down assay with GST-Rnd1 and GST proteins bound to glutathione-Sepharose beads. Mouse whole brain lysate was also used to co-immunoprecipitate endogenous Rnd1 with anti-STI1 antibody. Bound proteins from pull-down and co-immunoprecipitation were analysed by Westren blotting using different antibodies: anti-STI1, anti-GFP, anti-Rnd1 or anti-GST. PC-12 and N2a cells were transfected with GFP-tagged Rnd1 and STI1 proteins and used for neuritogenesis assays. COS-7 cells were transfected with the expression vectors for myr-myc-PlexinA1 (PlexA1), GFP, EGFP-Rnd1 (Rnd1), EGFP-STI1 and used for cell contraction assay. In order to investigate if STI1 and Rnd1 could co-localize in lipid rafts, indicating a putative domain for their interaction, these microdomains were purified by Nycodenz linear gradient flotation assay from brains of wild-type and PrPc knock-out mice and from surface biotinylated PC-12 cells. Thirteen Fractions were collected from the top to botton of the tube, resolved by SDS-PAGE and analyzed by Western blotting using anti-flotillin-1, anti-caveolin-1 and anti-PrPc antibodies as lipid rafts markers. Results: Pull-down and co-immunoprecipitation assays suggested that Rnd1 and STI1 can interact in vitro. Results showed also that co-expression of GFP-Rnd1 and GFP-STI1 can revert the formation of neuritic processes induced by GFP-Rnd1. Furthermore, GFP-STI1 decreases COS-7 cell contraction mediated by GFP-Rnd1 and PlexA1. Flotation assay showed that both STI1 and Rnd1 are present in lipid rafts of mice brain but also on detergent soluble membrane fractions. PC-12 biotinylated rafts indicated that STI1 could be present in the cytoplasmic side of rafts. Conclusion: These results suggest that these two proteins may interact each other and could be involved in cytoskeleton plasticity and neurite process formation. Supported by: CNPq, CAPES/REUNI, SETI-UGF.