![Figure 1. Defects in Cdc42 signaling result in a size-dependent uptake defect. (A) Cdc42 depletion leads to a kinetic defect in cell spreading on invasin. Control (shScr)- and Cdc42 (shCdc42)-depleted COS1 cells were plated onto invasin-coated glass coverslips, and spreading was visualized by phase-contrast time-lapse imaging. Numbers indicate time (in minutes). Bar, 10 µm. (B) Quantification of A. The area occupied by the cell was measured at each time point. Data are presented as fold increase in area, normalized to time = 0. (C) Cdc42-depleted cells internalize small particles but fail to internalize large particles efficiently. Control, Cdc42-depleted, and Rac1 (Rac1 shRNA [shRac1])-depleted COS1 cells were incubated with small (Y. pseudotuberculosis), medium (2.9-µm beads), or large (4.5-µm beads) invasin-coated particles. Uptake was then quantified microscopically. (D) FcR-mediated uptake in Cdc42-depleted cells is size dependent. Small (1.5 µm) or large (4.5 µm) IgG-coated beads were incubated with control or Cdc42-depleted COS1 cells expressing FcγRIIA, and uptake was quantified microscopically. (E) NWASP is required for the internalization of large particles but not for small particle uptake. NWASP+/+ and NWASP−/− MEFs were incubated with 4.5- and 1.5-µm particles, and uptake was quantified microscopically. (F) WAVE2 is required for internalization of small particles. WAVE2+/+ and WAVE2−/− MEFs were incubated with Y. pseudotuberculosis, and uptake was quantified microscopically. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.](https://cdn.rupress.org/rup/content_public/journal/jcb/200/1/10.1083_jcb.201204090/4/jcb_201204090_fig1.jpeg?Expires=1767784888&Signature=gL4Cuk4Zv9o4NVuGpxBmnNJSLo4cl0qwxvMWGMFYwgx9y2nK07Zf2xp4f2Yec7Y5dtFWWHmr0TVBu8U4EC~Rz1R4pm~l4KjQmE2A8wGP8za0h2Ci30bo3P-NFeLp8tqUaWhv02Q0sNR2HnxVO6alp27phN3Fmcpw6A8HnzrEQrw~q1NrLJFEA6S0AhjKxkUKVZ7l9oF~ICDkz7iZiXjyERMqbmXXoBq2GTtsXaPFt219EkM~VWwFsNI5H2DxBGZFDHAEzFFpDXl1jP-MOuPav1g1hIe45kcJeGWNFc7qSP8lVj0G7XP8KHYYFNzZk9OMLmys71TeGxpy5DglwLcX7A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Defects in Cdc42 signaling result in a size-dependent uptake defect. (A) Cdc42 depletion leads to a kinetic defect in cell spreading on invasin. Control (shScr)- and Cdc42 (shCdc42)-depleted COS1 cells were plated onto invasin-coated glass coverslips, and spreading was visualized by phase-contrast time-lapse imaging. Numbers indicate time (in minutes). Bar, 10 µm. (B) Quantification of A. The area occupied by the cell was measured at each time point. Data are presented as fold increase in area, normalized to time = 0. (C) Cdc42-depleted cells internalize small particles but fail to internalize large particles efficiently. Control, Cdc42-depleted, and Rac1 (Rac1 shRNA [shRac1])-depleted COS1 cells were incubated with small (Y. pseudotuberculosis), medium (2.9-µm beads), or large (4.5-µm beads) invasin-coated particles. Uptake was then quantified microscopically. (D) FcR-mediated uptake in Cdc42-depleted cells is size dependent. Small (1.5 µm) or large (4.5 µm) IgG-coated beads were incubated with control or Cdc42-depleted COS1 cells expressing FcγRIIA, and uptake was quantified microscopically. (E) NWASP is required for the internalization of large particles but not for small particle uptake. NWASP+/+ and NWASP−/− MEFs were incubated with 4.5- and 1.5-µm particles, and uptake was quantified microscopically. (F) WAVE2 is required for internalization of small particles. WAVE2+/+ and WAVE2−/− MEFs were incubated with Y. pseudotuberculosis, and uptake was quantified microscopically. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
