Persistence of the fluorescent secretory cargo in maturing Golgi cisternae. (A) Visualization of the cargo in maturing Golgi 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, labeled with JF₆₄₆, and imaged by 4D confocal microscopy while flowing in medium containing SLF. Shown are projected Z-stacks from the representative data in Video 5. The top row is the complete projection, the second row is the same projection after editing to highlight the two cisternae that were tracked, and the remaining three rows are the individual channels for the edited projection. Scale bar, 2 µm. (B) Quantification of the fluorescence intensities of Golgi markers and cargo during typical maturation events. The graphs represent the cisternae tracked in A, with the signals normalized to a maximum value of 1.0. (C) Average cargo signal change during the early-to-late Golgi transition. For each of 19 separate maturation events, fluorescence was quantified over a period of 90 s with Z-stacks collected every 3 s. Normalization of a trace was performed by defining 1.0 as the average of the first six fluorescence values for Vrg4 or the cargo, or as the average of the last six fluorescence values for Sec7. Traces were aligned at the midpoints of the Vrg4-to-Sec7 transitions, and the normalized fluorescence signals were averaged. The shaded borders indicate SEM.