Rac, and Myosin II to Control Cell Mechanics and Cytokinesis

Dictyostelium Strains, Cell Culture, and Plasmids Dictyostelium discoideum strains include wild type Ax3 (Rep orf+ cells), cortI, RacE, RacE (REMI insertional mutant), and myosin II heavy chain null mutant (mhcA (HS1)) [1-3] (Table SI). RacE: 14-3-3 suppression experiments were performed in both RacE mutant backgrounds with similar results. Cells were grown in Hans’ enriched HL-5 media (1.4xHL-5 plus 8% FM) plus 60 U/ml penicillin, 60 g/ml streptomycin sulfate and plus or minus selecting drugs, depending on whether the cells were transformed with an episomal plasmid (Table SI). Electroporation was performed to transform plasmids into Dictyostelium cells using a Genepulser-II electroporator. Cells transformed with pLD1A15SN-based plasmids were selected with 10-15 g/ml of G-418, and cells transformed with pDRH plasmid were selected with 30 g/ml of hygromycin. Cells were propagated at 22C on 10-cm plates. For suspension culture, cells were grown in 10-ml culture volumes in 125-ml Erlenmeyer flasks at 200 rpm. Cell densities were measured using a hemacytometer. LimEcoil-GFP was constructed in pDRH according to Schneider et al., 2003 [4]. 14-3-3GFP was constructed from an in-house GFP-tagging cassette and cloned into pLD1A15SN [3]. 14-3-3(K49E)-GFP was constructed using QuickChange II Site-Directed Mutagenesis Kit (Agilent Technologies) and the pLD1A15SN: 14-3-3-GFP as the host plasmid. The complete 143-3(K49E)-GFP cDNA sequence was confirmed by sequencing. GFP-RacE and GFP-tubulin plasmids were described previously [3, 5]. The mCherry-RacE expression plasmid was constructed by first generating an mCherry-tagging cassette in pBlueScript, similar to our GFPtagging system described previously [3, 5], and then sub-cloning the mCherry-RacE into pDRHyg [5]. The pLD1A15SN: Enl-tr plasmid was described previously [6].