Traffic of the fluorescent secretory cargo to Golgi compartments. (A) Concentration of the cargo in a nascent Golgi cisterna that formed near an ERES. A strain expressing the APVNTT-DsRed-Express2-FKBP^RD(C22V) cargo together with Sec31-HaloTag (ERES) and GFP-Vrg4 (early Golgi) was imaged by 4D microscopy during and after addition of SLF in a flow chamber. Depicted here is a representative example of the results, in frames from Video 4. The top row is the complete projection, the second row is the same projection after editing to highlight the tracked compartments, and the remaining three rows are the individual channels for the edited projection. Time zero represents ∼45 s after the cells were exposed to SLF. The solubilized cargo became concentrated in a structure that was near an ERES, and then GFP-Vrg4 arrived at the same structure. Scale bar, 2 µm. (B) Sequential appearance of the cargo in early and then late Golgi cisternae. Cells expressing the aggregated 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. Movies were analyzed by quantifying the percentages of the early and late Golgi cisternae that contained detectable cargo at the indicated time points. Data were taken from 27 cells in seven videos. Error bars represent SEM.