Quantitatively probing the budding yeast CME pathway through systematic imaging. (A) Illustration of key steps in the budding yeast CME pathway. Mechanisms of site initiation, transition from the early, variable lifetime stages to the later, more regular lifetime stages, and initiation of actin assembly are poorly understood. Diagram adapted from Tonikian et al., 2009. (B) Top: Montage of Las17-GFP and Abp1-RFP recruitment to an endocytic site viewed en face by live two-color TIRFM of budding yeast. Bottom: Centroid position tracks in the x and y dimensions for the Las17-GFP (green) and Abp1-RFP (magenta) spots depicted in the montage. •, beginning; x, end. See Video 1. (C) Fluorescence (Fluor.) intensity vs. time for Las17-GFP and Abp1-RFP spots depicted in B. Time points of note and key measurements recorded are indicated by dotted lines and arrows, respectively. (D–F) Box-and-whisker plots of the time from the appearance of the indicated GFP-tagged query protein to the appearance of Ede1-RFP (D), Sla1-mCherry (E), and Abp1-RFP (F). Red lines are median values, boxes indicate interquartile range, and whiskers indicate full range of the data. Colored text identifies each protein imaged as a component of the early, coat, WASP/myosin, actin, or scission modules according to the color scheme below.