Figure 5. Memo is required for... | Rockefeller University Press

Figure 5.

$Figure 5. Memo is required for localization of RhoA and mDia1 to membrane ruffles and lamellipodia. (A and B) SKBR3 cells expressing EGFP-RhoA or EGFP-mDia1 (A) or EGFP-Cdc42 or EGFP-Rac1 (B) constructs and control or Memo siRNA, as indicated, were treated with 5 nM HRG for 10 min. EGFP-RhoA and EGFP-mDia1 were recruited to cell membrane and ruffles (arrowheads) in control but not in Memo-depleted cells, whereas localization of EGFP-Cdc42 and EGFP-Rac1 was independent of Memo. (C) Cell body (CB) and lamellipodia (Lp) of control and Memo siRNA–expressing cells were fractionated. Equal amounts of proteins were analyzed by Western blotting to reveal the relative amounts of RhoA in the respective fractions. RhoA is decreased in the lamellipodia-enriched fraction but is unchanged in the cell body of Memo knockdown cells. (D) Bacterially produced Memo was incubated with GDP-loaded GST-RhoA or GTPγS-loaded GST-RhoA, GST-RhoA L63, GST-Cdc42, or GST-Rac1 bound to glutathione beads. RhoGTPase-associated Memo was visualized by Western blotting. Ponceau S protein staining (bottom) shows equivalent amounts of GST fusion proteins. (E) Cells were transfected with Memo siRNA and EGFP-RhoA or EGFP-RhoA-CCKVL. MTs were visualized by incorporation of β-tubulin–DsRed. RhoA-CCKVL localizes to membrane ruffles (arrowheads) and allows MT outgrowth in Memo-depleted cells. Bars, 10 μm.$

Memo is required for localization of RhoA and mDia1 to membrane ruffles and lamellipodia. (A and B) SKBR3 cells expressing EGFP-RhoA or EGFP-mDia1 (A) or EGFP-Cdc42 or EGFP-Rac1 (B) constructs and control or Memo siRNA, as indicated, were treated with 5 nM HRG for 10 min. EGFP-RhoA and EGFP-mDia1 were recruited to cell membrane and ruffles (arrowheads) in control but not in Memo-depleted cells, whereas localization of EGFP-Cdc42 and EGFP-Rac1 was independent of Memo. (C) Cell body (CB) and lamellipodia (Lp) of control and Memo siRNA–expressing cells were fractionated. Equal amounts of proteins were analyzed by Western blotting to reveal the relative amounts of RhoA in the respective fractions. RhoA is decreased in the lamellipodia-enriched fraction but is unchanged in the cell body of Memo knockdown cells. (D) Bacterially produced Memo was incubated with GDP-loaded GST-RhoA or GTPγS-loaded GST-RhoA, GST-RhoA L63, GST-Cdc42, or GST-Rac1 bound to glutathione beads. RhoGTPase-associated Memo was visualized by Western blotting. Ponceau S protein staining (bottom) shows equivalent amounts of GST fusion proteins. (E) Cells were transfected with Memo siRNA and EGFP-RhoA or EGFP-RhoA-CCKVL. MTs were visualized by incorporation of β-tubulin–DsRed. RhoA-CCKVL localizes to membrane ruffles (arrowheads) and allows MT outgrowth in Memo-depleted cells. Bars, 10 μm.