Figure 5

The N-terminus and the C-terminus of GMAP210 bind different regions of the Golgi ribbon. (A to C) Nter-DsRed (1–375 aas) and GFP-Cter (1778–1979 aas) constructs were co-transfected in RPE1 cells and their distribution analysed by confocal microscopy (A) or deconvolution (B, C). Merged images of the two constructs (A) or of the two constructs with giantin (B) or with endogenous GMAP210 (C) immunolocalisation are shown. High magnifications of the selected regions in (A) and (B) show individual labellings and the merge. Arrows indicate identical locations for each panel. (D) RPE1 cells co-transfected with Nter-DsRed and GFP-Cter and treated with BFA. All GFP-Cter becomes cytosolic except at the centrosome (arrow). (E, F) Time-lapse analysis of RPE1 cells transiently transfected with Nter-DsRed and GFP-Cter in control conditions or after nocodazole treatment. Selected frames of Movies 3 (control) or 4 (nocodazole) (see Additional files 3 and 4, respectively) are shown at the same time points. At right, enlarged views of selected areas are shown. (G) RPE1 cells transfected with a truncated mutant consisting of both N- and C-termini expressed in fusion (Nter-Cter-GFP, G1) and double labelled for endogenous GMAP210 (G2). A merged image is shown in G3. In (H), a Nter-Cter-GFP transfected cell was treated with nocodazole and triple labelled for endogenous GMAP210 and Golgin245. High magnifications of the selected region at right show individual labellings and the merge. Arrows indicate identical localisation of the truncated mutant and the endogenous protein in isolated Golgi ministacks. Bars = 5 μm.