FIGURE 2.

Cargos localize to distinct Golgi compartments and respond to temperature block differently. HeLaM cells were transiently transfected with plasmids directing the expression of CD8-furin or CD8-M6PR. The next day (∼16 h), cells were fixed and labeled for CD8 and TGN46 or giantin. Stacks of images were collected using wide field imaging and were deconvolved, as described under “Experimental Procedures.” A, deconvolved images were imported into Imaris, and two isosurfaces were defined: one using the cargo (CD8-furin or CD8-M6PR) and the other using a TGN or Golgi marker (TGN46 or giantin). Total cargo fluorescence was calculated for each cell, and then the amount of cargo fluorescence within the Golgi marker isosurface was determined. The values shown indicate the percent of total cargo intensity found within the Golgi marker isosurface, and bars indicate S.E. (n = 5). Student's t test was used to compare 37 °C to 20 °C for each cargo/Golgi marker pairing. Asterisks indicate p < 0.01. Results are typical of at least three experiments. B, isosurfaces were generated for the indicated cargo and markers, as described under “Experimental Procedures.” The cargo was falsely colored with a heat map indicating the amount of Golgi marker found within the cargo isosurface. The color range of intensities is shown as a bar on the right. C, cells expressing either CD8-M6PR or CD8-M6PRΔC were fixed and stained for CD8 and the indicated Golgi marker. Single-plane, confocal images were collected, and the amount of Golgi marker signal that is also positive for cargo was calculated using Mander's coefficients. A Student's t test was used to compare the overlap of each cargo with the Golgi marker indicated. An asterisk indicates statistical significance (p < 0.01).