(A) Maximum projections from 31-z stack widefield immunofluorescence images of wild-type cells, probed with anti-Myo1 (left panel) or anti-Myo1S742* phosphospecific (right panels) antibodies, illustrate that S742 phosphorylated Myo1 localizes to cortical foci (scale bar, 5 µm). (B) An example relative-intensity trace of a single mNeongreen.Myo1 endocytic event. Linear fitting (grey lines, 60 points) was used to find the maximum gradient for both the rising and the falling slope. The intercept with zero intensity level was used to calculate Tstart, Tend, and subsequently, the duration of the event Tdur. See detailed description in the Materials and methods section. Insert: an arrow highlights the analyzed endocytotic event (5 × 5 pixels area). (C) Averaged profile for individual Myo1 (blue) and Cam1 (red) membrane-association events, synchronized relative to Tstart and Tend. Dotted lines show fitted rising (Myo1, 537 AU/sec; Cam1, 1073 AU/sec) and falling (Myo1, 567 AU/sec; Cam1, 1028 AU/sec) gradients. (D) An example fluorescence trace from simultaneous two-color imaging of a Myo1 (blue line) and Cam1 (red line) membrane-association event observed in mNeongreen.myo1 cam1.mCherry cells is consistent with the relative intensities and timings observed using single-fluorophore strains. (E) Averaged intensity trajectories of individual Myo1 (blue line) and Myo1.S742A (red line) endocytosis events from TIRFM imaging of mNeongreen.myo1 and mNeongreen.myo1.S742A cells, respectively.