Figure 4

The C-terminal region of GMAP210 targets the cis-Golgi compartment and the centrosome. (A, B) RPE1 cells were transfected with the C-terminal domain of GMAP210 (aa 1778–1979) in fusion with GFP, fixed and double labelled for GM130 and endogenous GMAP210 (A) or for the centrosomal protein ninein and GMAP210 (B). Merged images are shown in A2 and B2. Arrows indicate the centrosome. (C) A merged image of GMAP210-depleted cells transfected with the GFP-Cter construct and stained for GM130 and endogenous GMAP210. ND-NT indicates a non-depleted non-transfected cell, D-NT a depleted non-transfected cell and D-T, a depleted and transfected cell. (D to F) Confocal images showing the distribution of GFP-Cter fusion protein with respect to the cis-Golgi marker GM130 (D), the medial Golgi marker CTR433 (E) or the trans-Golgi marker Golgin 245 (F). Corresponding fluorescence intensity profiles are shown at right. (G) A GFP-Cter transfected cell treated with nocodazole and labelled for GM130. Enlarged views of single labellings are shown at the bottom. (H) Live imaging of a GFP-Cter transfected cell treated with BFA. BFA was added to a final concentration of 2.5 μg/ml at time 5.0 seconds and images were taken with 0.2-second intervals. Selected frames of Movie 2 (in Additional file 2) at indicated time points are shown. Note that GFP-Cter rapidly dissociated from membranes after BFA addition whereas fluorescence at the centrosome decreased only slightly (arrow). Bars = 5 μm.