Figure 6.

VIP36-SP-FP and VSVG3-SP-FP cargo are found together in TCs frequently at earlier stages of transport, but infrequently at later stages. COS cells transiently coexpressing VIP36-SP-FP and VSVG3-SP-FP were incubated 6 to 12 h at 39.5°C to accumulate cargo in the ER and then shifted to 32°C for various times in the presence of 100 μg/ml cycloheximide. Movies were taken at frame rates of 3 to 10 s/frame (see online data for representative movies). In the movies, individual TCs, defined as globular structures in the cell periphery (arrowheads in Figure 5) were scored blind for detectable CFP signal, YFP signal, or both. CFP and YFP signals were subsequently assigned to VIP36-SP-FP and VSVG3-SP-FP. Based on the timing of VSVG3-SP-FP transport, cargo-containing TCs observed earlier than 20 min are pre-Golgi carriers, those later than 20 min are primarily post-Golgi carriers. We quantitated 218 TCs in 38 cells: 82 TCs in 23 cells before 20 min and 136 TCs in 15 cells after 20 min.