Figure 8. Inter-Golgi exchange of anterograde cargo at 37°C.
(A) HeLa cells expressing ss-GFP-FM4-CD8 and GST-PKD(DI) were mixed and fused with cells expressing ST-RFP, GST-PKD(DI) and VSV-G. Prior to fusion, cells were incubated at 37°C for 15 min in the presence of the disaggregating drug and CHX. Cells were monitored by video confocal microscopy at 32°C in the presence of the disaggregating drug and CHX. Graphs show quantification of both markers over time for Golgi 1. Results are representative of two independent experiments. (B) 1 hr post-fusion, cells were fixed and prepared for immunofluorescence against GST to assess the presence of the PKD(DI). Note that the confocal micrograph showed the very same field of cells (shown in A) after fixation. (C) PKD(DI) inhibits plasma membrane targeting of ss-GFP-FM4-CD8. HeLa cells expressing ss-GFP-FM4-CD8 alone (upper panel) or with GST-PKD(DI) (lower panel) were incubated for 1 hr at 37°C in the presence of the disaggregating drug. Non-permeabilized cells were incubated on ice and incubated with an anti-GFP antibody (detected with a secondary antibody labeled with Alexa-546) to assess for cell surface exposure of ss-GFP-FM4-CD8. Then, cells were fixed, permeabilized and processed for immunofluorescecne against GST (using a secondary antibody labeled with Atto-647).