Formation of new Golgi cisternae near the ER in nocodazole-treated cells. (A) Tracking of a representative early Golgi cisterna. The early Golgi was labeled with GFP-Vrg4, and the ER lumen was labeled with E2-Crimson-HDEL (Strack et al., 2009b). From Video 3, the top row is a projection of optical sections from the center of the cell, the middle row is the same projection after editing to highlight the tracked cisterna, and the bottom row illustrates the path followed by the tracked cisterna. Scale bar, 2 µm. (B) Enlargement of the track from part A. Time is indicated by a gradient from blue to red. Scale bar, 2 µm. (C) Quantification of the distances between cisternae and the ER during the arrival (Start) and departure (End) of early and late Golgi markers. Strains contained E2-Crimson-HDEL, together with either GFP-Vrg4 (early Golgi) or Sec7-GFP (late Golgi). Cells were grown to mid-log phase, treated with nocodazole for 2 h, and imaged by 4D confocal microscopy. Measurements were derived from the daughters, which lacked perinuclear ER fluorescence. Each of the chosen cisternae experienced a transition in GFP-Vrg4 or Sec7-GFP labeling while near the center of the cell along the vertical axis. At the time of a transition, 8–12 optical sections from the center of the cell were projected, and the distance to the cortical ER was measured using the Line tool in ImageJ. The scatter plot for a transition shows the mean distance from the ER and the standard deviation. For each condition, data were taken from events in at least 30 different cells visualized in seven or more videos. To determine statistical significance, the Vrg4 datasets were compared using the Mann–Whitney U test.