Characterization of the wasA knockout cells and analyses of clathrin-mediated endocytosis. (A) Top: Design of the wasA knockout construct. Bottom: Targeted clones were confirmed by PCR. (B) The indicated cells were plated clonally with bacteria (K. aerogenes) on standard medium agar for 5 d. Scale bar, 5 mm. (C) Localization of GxcM-RFP and GFP-ArpC4 in wasA⁻ cells. Scale bar, 5 μm. (D) Left: Projection length (Lp) of WT and wasA^– cells determined by micropipette aspiration using a constant pressure of 500 Pa for 5 min. Right: Quantitative analysis of the projection lengths of probed cells. Data were from two independent experiments; the scatter plot shows data points with mean ± SEM; n, number of cells analyzed. (E) Left: Localization of GFP-CLC and RFP-ArpC4 in WT, fbp17⁻, racC⁻, and wasA⁻ cells imaged by TIRF microscopy; scale bars, 5 μm. Right: Time-lapse imaging of clathrin vesicle internalization; scale bars, 1 μm. Images were acquired every 2–4 s for 8 min. In WT, fbp17⁻, and racC⁻ cells, recruitment of ArpC4 to clathrin pits coincides with their internalization. In wasA⁻ cells, clathrin pits fail to recruit ArpC4 and persist for hundreds of seconds. (F) Quantification of the lifetime (time between appearance and disappearance from the TIRF field of view) of clathrin-coated pits (CCPs) in WT, fbp17⁻, and racC⁻ cells. Data were from at least two independent experiments; the scatter plot shows data points with mean ± SEM; n, number of CCP analyzed. In wasA⁻ cells, the lifetime could not be accurately determined because it was often greater than the length of the time-lapse videos.