FIGURE 2.
ERdj4sfGFP is a dynamic ER protein. A, FLIP experiment of an MDCK cell transiently expressing ERdj4sfGFP. The cell was repeatedly photobleached in the ROI (white box), and images are collected between bleaches. If the fluorescent proteins are mobile and can pass through the ROI, all fluorescence will be depleted. B, FRAP of U2OS cell transiently expressing ERdj4sfGFP. The cells were photobleached a single time in the ROI (white box), and recovery of the signal was monitored over the indicated times. Scale bars, 10 μm. C and D, FRAP Deff values with ERdj4sfGFP, VSV G-GFP, or ER-RFP transiently transfected in MDCK (left) or U2OS cells (right). Each dot represents the Deff value for a single cell and solid lines indicate mean Deff values. Translational inhibition was performed with 0.2 μm pactamycin (Pact) for 1 h to deplete cells of nascent substrates for ERdj4. The mean Deff value ± S.E. is reported over each column. The dashed line in C was added to emphasize the lack of overlap between the Deff of a well established integral membrane protein (VSV G) and the reported transmembrane protein ERdj4 (plus pactamycin (Pact)). The overall higher D values in U2OS cells likely reflect either decreased ER tortuosity (58) and/or decreased viscosity, because molecular diffusion is dependent on environmental viscosity (59).