Negative Regulation of Yeast WASp by Two SH3 Domain-Containing Proteins  Avital A. Rodal, Amity L. Manning, Bruce L. Goode, David G. Drubin  Current Biology  Volume 13, Issue 12, Pages 1000-1008 (June 2003) DOI: 10.1016/S0960-9822(03)00383-X

Figure 1 Proteins Used in This Study (A) Domain structure of the proteins used in this study. (B) Coomassie-stained 12.5% (left) and 10% (right) acrylamide gels of purified proteins. Current Biology 2003 13, 1000-1008DOI: (10.1016/S0960-9822(03)00383-X)

Figure 2 Activity of Full-Length Las17 (A and B) A total of 3 μM rabbit muscle actin (1% pyrene labeled) was polymerized in the presence of the indicated concentrations of Arp2/3 complex, Las17, or the WA fragment of Las17. (C) The concentration of barbed ends was calculated by using the formula [barbed ends] = rate of polymerization/(rate constant [actin monomers]). Arbitrary fluorescence units were converted to a concentration of filamentous actin by assuming a final actin filament concentration of 2.9 μM. The rate constant for rabbit muscle actin polymerization at pH 7.5 is 8.7 μM−1s−1[56]. Current Biology 2003 13, 1000-1008DOI: (10.1016/S0960-9822(03)00383-X)

Figure 3 Effects of Profilin on Las17 Activation of Arp2/3 Complex (A–C) (A) A total of 3.5 μM or (B and C) 3 μM rabbit muscle actin with a 1% pyrene label was polymerized in the presence of the indicated concentrations of Arp2/3 complex, Las17, the Las17 WA fragment, preformed actin filaments, or profilin. Current Biology 2003 13, 1000-1008DOI: (10.1016/S0960-9822(03)00383-X)

Figure 4 Effects of Sla1 and Bbc1 on Las17 Activity (A–C) (A) A total of 3 μM or (B and C) 3.5 μM rabbit muscle actin (1% pyrene labeled) was polymerized in the presence of the indicated concentrations of Arp2/3 complex, Las17, Bbc1, or the Sla1 fragment. (A) Sla1 inhibits Las17 at a half-maximal concentration of 200 nM Sla1. (B) Sla1 and Bbc1 have additive inhibitory effects on Las17. (C) The first and second SH3 domains of Sla1 are required for inhibition of Las17 activity on Arp2/3 complex. Arp2/3 complex was used at 15 nM, Las17 was used at 15nM, and the Sla1 SH3 fragments were used at 1 μM. Percent inhibition was normalized to the activity of wild-type Sla1SH3. The bars show the average and standard deviation from at least two independent experiments. Current Biology 2003 13, 1000-1008DOI: (10.1016/S0960-9822(03)00383-X)

Figure 5 Genetic Interactions between SLA1 and BBC1 (A) DDY2513 (sla1Δ bbc1Δ ) cells containing SLA1 plasmids were grown to saturation in selective media, plated in ten-fold serial dilutions on selective media, and grown at the indicated temperatures for 4 days. (B) Rhodamine-phalloidin staining and phase microscopy of sla1 bbc1Δ mutant cells shifted to 37°C for 2 hr. (C) Las17::GFP localization (live cells, top two panels) and Las17::GFP/actin colocalization (fixed cells, bottom four panels) in sla1 bbc1Δ cells shifted to 37°C for 2 hr. Current Biology 2003 13, 1000-1008DOI: (10.1016/S0960-9822(03)00383-X)