A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis (original) (raw)
Figure 2
Dynamin was recruited at the time of CCV formation.
(A) Portion of a NIH-3T3 cell co-transfected with TfR-phl (left) and Dyn1-mCherry (Dyn1, centre), observed with TIR-FM at pH 7.4. Dynamin1 was colocalized with a subset of TfR-phl patches (yellow dots in the merged image, right). (B) An example scission event. At time 0, a CCV was detected in the image at pH 5 (black arrowhead). Dynamin1 was recruited transiently, with a peak at time −4 s. (C) Fluorescence measurements (dark green, TfR7; light green, TfR5; red, dynamin1) corresponding to the event displayed in (B). The dots correspond to the images shown. Vertical blue line shows time = 0 and horizontal lines show fluorescence = 0. (D) Average fluorescence for TfR7, TfR5, and dynamin1 for the events detected in the cell shown in (A) (n = 290). Black lines represent the median and 95% confidence limits for random fluorescence measurements (see Materials and Methods for calculation). (E) Data as in (C) pooled for eight cells (1,297 events). Averages of fluorescent traces of terminal (light blue) and non-terminal events (magenta) for TfR7, TfR5, and dynamin1. Note the overlap of curves before time = 0. (F) Histogram of peak dynamin1 recruitment for individual events. (G) Average dynamin1 (red, eight cells) and dynamin2 (purple, six cells) fluorescence curves normalised to the randomized measures. au, arbitrary units.