Figure 3.

Basal Rho activity is required for the maintenance of Tiam1/Rac–induced cell–cell contacts in NIH3T3 fibroblasts. (A) Serum is required for Tiam1-induced cell–cell contacts. Phase-contrast pictures of NIH3T3 fibroblasts expressing C1199 Tiam1 showing cells grown in the presence of serum, after 24 h serum starvation, and 5 h after addition of serum to 24 h serum-starved cells. Bar, 10 μM. (B) Rho activity is required for Tiam1-induced cell–cell contacts. C1199Tiam1-expressing NIH3T3 fibroblasts were stained for Tiam1 (DH antibody, green) and F-actin (red) after 24 h of serum starvation, 24 h of serum starvation followed by addition of 5 μM LPA (8 h), and treatment for 12 h with 30 μg/ml C3 transferase toxin (in the presence of serum). Bar, 25 μM. (C) Cell lysates (from 5 × 10⁶ cells) of control and C1199Tiam1-expressing NIH3T3 fibroblasts were assayed for Rho and Rac activities. Cells were grown in serum (lane 1) or 24 h serum-starved (lanes 2–7) before stimulation with LPA (5 μM, 15 min) as indicated. Lysates were incubated with GST-C21 (lanes 1–5) or GST-PAK-CD (lanes 6 and 7), and bound GTPases were analyzed with antibodies against RhoA or Rac1.