Arrival of fluorescent secretory cargo molecules during the early-to-late Golgi transition. (A) Visualizing traffic of the cargo to maturing cisternae. Cells expressing the APVNTT-DsRed-Express2-FKBP^RD(C22V) cargo together with GFP-Vrg4 (early Golgi) and Sec7-HaloTag (late Golgi) were grown to mid-log phase, treated with nocodazole for 2 h, labeled with JF₆₄₆, and imaged by 4D confocal microscopy. SLF was added for 3 min to enable early Golgi cisternae to become loaded with cargo. At that point, the cargo fluorescence in an entire daughter was bleached by illuminating with the laser at maximum intensity for ∼40 s. Then individual cisternae in the daughter were tracked as in Fig. 6 A by following the Golgi markers, which were not affected by the bleaching. Shown are projected Z-stacks from the representative data in Video 6. The top row is the complete projection, and the bottom row is the same projection after editing to highlight the cisterna that was tracked. Scale bar, 2 µm. (B) Quantification of the Golgi markers and the fluorescence recovery of the cargo during typical maturation events in a daughter after cargo bleaching. The first graph represents the cisterna tracked in A, and the second graph represents another typical cisterna tracked in a similar video. These data from two videos are representative of four total videos analyzed. As a control, when both the mother and daughter were bleached, no fluorescence recovery of the cargo was seen (not depicted).