Reduced membrane perturbation at the nascent phagosome in Cdc42-depleted cells. (A–D) Characterizing phagosome morphology using time-lapse microscopy. Y. pseudotuberculosis or 4.5-µm beads were added to control (shScr) or Cdc42 (shCdc42)-depleted COS1 cells expressing Lyn-GFP and imaged every 15–20 s. At each time point, a bright-field and a GFP image were collected. Numbers indicate time (in minutes). Bars, 5 µm. (E) Transferrin receptor (TfR) localizes to large nascent phagosomes. COS1 cells were challenged with 1.5 (top)- and 4.5 (bottom)-µm beads and fixed, and endogenous TfR was detected using an anti-TfR antibody. In merged images, extracellular particles and TfR are pseudocolored red and green, respectively. Arrowheads point to phagosomes magnified in insets to emphasize the localization pattern. (F) Rab11 localizes to large nascent phagosomes. COS1 cells expressing Myc-Rab11 were challenged with 1.5 (top)- and 4.5 (bottom)-µm beads, and Myc-Rab11 was detected using an anti-Myc antibody. Bar, 10 µm; applies to E also. (G) Quantification of E and F. At least 50 phagosomes per condition were scored by eye for TfR or Rab11 recruitment. (H) The extent of TfR recruitment to phagosomes was determined by immunostaining for TfR and quantifying mean pixel intensity for ≥20 small and large phagosomes (Iphagosome). TfR staining at a nearby cytosolic region, devoid of attached particles, was determined also (Icytoplasm). Extent of recruitment is presented as the quantity Iphagosome/Icytoplasm. (I and J) Inactivation of Cdc42 or NWASP leads to reduced transferrin efflux. COS1 cells expressing dominant-negative Cdc42 and control cells (I) and NWASP+/+ and NWASP−/− MEFs (J) were pulsed with fluorescent transferrin and chased in its absence. Remaining transferrin amounts were quantified microscopically. Error bars represent SEM. **, P < 0.01; ***, P < 0.001.
