Volume 6 issue 12 December 2005
E-cadherin transport from the TGN in tubulovesicular carriers is selectively regulated by golgin-97
John G. Lock, Luke A. Hammond, Fiona Houghton, Paul A. Gleeson and Jennifer L. Stow

Figure 1. Ecad-GFP expression and exocytosis in HeLa cells. HeLa cells, denoted by DAPI nuclear labeling, have no expression or labeling of E-cadherin (A). Cells fixed 24 hours after transfection show Ecad-GFP concentrated at cell-cell contacts, and also present in the perinuclear region (B; images intensity inverted). Confocal imaging and 3D reconstruction show that Ecad-GFP (green) in the perinuclear region of transfected cells coincides with immunolabeling of the Golgi protein GM130 (red) (C - see Movie 1). Additional Ecad-GFP is visible in vesicular structures (1-3 mm in diameter) peripheral to the Golgi complex. Scale bars, 10 µm.

Figure 2. Analysis of Ecad-GFP-positive PGC budding from the Golgi complex. Live imaging of HeLa cells 3 hours after transfection shows Ecad-GFP associated with pleiomorphic PGCs (A-D) and spherical carriers (A, B and F-K), budding from the Golgi complex. Detachment of tubular PGCs is associated with elastic extension away from the Golgi complex prior to fission (A; black arrows). Budding is highly dynamic, as demonstrated by changes in sequential images captured 6 seconds apart (B and enlarged, Sobel-filtered images – C, D). The elasticity of budding membranes in the direction of force application is shown in a region cropped from a budding domain of the TGN (E - red rectangle, panel F and Movie 2 and Movie 3). Enlarged images (intensity based color display pallet; timepoints in sec) show a PGC (white arrow) budding from the TGN (white double arrows). The TGN source membrane is dragged and distorted by the PGC as it moves in the direction of force application (designated by red dotted arrow). In G, 3 key frames highlight the movement and deformation of the TGN membrane (white arrows) as it deforms during PGC detachment (14.8 sec) and then undergoes elastic recoil. Panel H displays each of these key frames as red, green and blue components of an RGB image, respectively. Panel I is a pixel intensity-based contour map of panel H. Scale bars, A, B, E – 10 µm, C, D – 2.5 µm, F – 1 µm, G-I – 2 µm.

Figure 3. GFP-tagged GRIP domains of p230/golgin-245 and golgin-97 are associated with dynamic TGN tubules. Live imaging of GFP-p230/golgin-245-GRIP in HeLa cells 3 hrs after transfection, shows labeling on dynamic tubular extensions of the TGN (A). Viewed at higher magnification in selected frames from a time series, one large tubule (~6 µm length, 300 nm diameter – solid arrowheads) extends away from the TGN before fragmenting simultaneously at two points of reduced fluorescent intensity (double arrowheads), giving rise to two separate fragments (solid arrowhead and empty arrowhead) which then constrict over 120 sec (see Movie 4). A GFP-golgin-97-GRIP labeled tubule extends from the TGN and detaches (see frames 14.5 {double arrow head} and 134.5 {arrow head and tail}) (B). Single arrowheads (solid and hollow) demarcate the positions of fragmented tubules (see Movie 5). Scale bars, A, C, D-10 µm, B-5 µm.

Figure 4. p230/golgin-245 and golgin-97 proteins and GRIP domains on different TGN subdomains. Cells were co-transfected with combinations of full-length golgin-97 and p230/golgin-245, or with the labeled GRIP domains of golgin-97 and p230/golgin-245. Cells were fixed at 48 h and imaged via high-resolution confocal microscopy. Full-length GFP-golgin-97 and myc-p230/golgin-245 have largely distinct distributions on the TGN (A; R value = -0.48). Co-expressed golgin-97 constructs alternatively tagged with either GFP or myc display almost perfect colocalization (B; R value = 0.52). Co-expressed CFP-p230/golgin-245-GRIP and YFP-golgin-97-GRIP also label distinct membrane domains although some colocalization was observed (C). The cell imaged in C was optically sectioned and 3D reconstructions were generated, using both surface rendering (D) and projection algorithms (See Movie 6 and Movie 7, respectively). A fluorescence intensity linescan profile (E) was also generated along the red line indicated in the merged panel of C, and demonstrates the independent distributions of each fluorescent population (blue and pink arrows in C and E designate corresponding structures and fluorescence peaks containing significant levels of only one fluorophore). Colocalisation in images of cotransfected cells was quantified using Pearson's correlation coefficient. Positive and negative overlap is shown for various fluorescent populations (F). Scale bars - 10 µm.

Figure 5. Ecad-YFP is not associated with CFP-p230/golgin-245-labeled tubules in live cells. Live epifluorescence dual-color imaging was performed on HeLa cells expressing both Ecad-YFP (red) and CFP-p230/golgin-245 (green) (A), with YFP/CFP image pairs captured at a rate of 2.5 pairs/sec, via sequential excitation of YFP and CFP. Image pairs cropped from the region of interest (white box in A) show a fragment of TGN from which two dynamic, CFP-p230/golgin-245-labeled tubules extend (white arrows in B; timepoints in sec). Edge contouring simplifies comparison of the fluorescent populations, and shows that Ecad-YFP coincides with CFP-p230/golgin-245 on the TGN but not in tubule extensions (C ). Four-dimensional dual color (linear unmixed) confocal imaging demonstrates the budding of an Ecad-YFP carrier in the absence of a GFP-p230-GRIP tubule as displayed in a surface rendered 3D image sequence (E – time in seconds – see Movie 8) derived from the region of interest (white box in transparency rendered image D – see Movie 9). Scale bars, A – 10 µm, B, C – 3 µm, D – 10 µm, E – 2 µm.

Figure 6. Ecad-YFP-positive PGCs associate with CFP-golgin-97-GRIP-labeled tubules . Live dual color epifluorescence imaging of Ecad-YFP (red) and CFP-golgin-97-GRIP (green). A single frame from an image sequence (2.5 image pairs/sec, 40 sec duration) shows Ecad-YFP is present in peripheral puncta and as well as colocalizing with CFP-golgin-97 at the TGN (A). A co-labeled fragment of TGN (from solid white box in A) gives rise to a CFP-golgin-97-GRIP-labeled tubule into which a bolus of Ecad-YFP is transferred. This Ecad-YFP bolus traverses the length of the tubule before expulsion from the tubules tip as shown (B; see Movie 10). A graphical representation outlining fluorescent populations through this sequence demonstrates the dynamic association of Ecad-YFP-PGCs and CFP-golgin-97-GRIP-labeled tubules (C). A second example of a CFP-golgin-97-GRIP-labeled tubule with Ecad-YFP at its tip is enlarged from the dotted box in A (D). 4D confocal imaging of another cell further demonstrates the association of budding Ecad-YFP with dynamic GFP-golgin-97-GRIP tubules (E and F). Colocalisation (yellow) between Ecad-YFP (red) and GFP-golgin-97-GRIP (green) was observed in a single 3 section transparency image (E – see Movie 11). 3D surface rendering of the region of interest (white box) at each timepoint demonstrates the extension of a GFP-golgin-97 tubule containing a population of Ecad-YFP at the leading tip of the tubule (F – time in seconds - see Movie 12). The Ecad-YFP population detaches when the tubule reaches maximum extension. Scale bars, A – 10 µm, B, C – 2 µm, D – 1 µm, E – 5 µm, F – 2 µm.

Figure 7. YFP-TNFα a associates with GFP-p230-GRIP-labeled tubules and not GFP-golgin-97-GRIP-labeled tubules. 4D confocal imaging, spectral unmixing and reconstruction of co-expressing cells reveals that TNFα a -YFP (red) and Ecad-GFP (green) exit the TGN in distinct carriers (A and B, respectively – white arrows denote carriers – time in sec). Discrete labeling of detaching carriers was observed in 90.3% of cases (n=62). Further imaging of YFP-TNFα a and GFP-GRIP domains shows YFP-TNFα a exiting the TGN in the absence of GFP-golgin-97-GRIP labeled tubules (D; white arrows denote YFP-TNFα a cargo in TGN – yellow arrows denote detached carrier - see Movie 13) despite colocalizing throughout much of the TGN (C - see Movie 14). In contrast, although YFP-TNFα a colocalizes similarly with GFP-p230-GRIP in the TGN (E - see Movie 15), it also exits the TGN in GFP-p230-GRIP-labeled tubules (F; white arrows denote YFP-TNFα a cargo in TGN – yellow arrows denote detached carrier - see Movie 16). All panels represent composites of at least three confocal sections (0.8 µm/section) and are displayed as top down transparency views (A, B, C, E) or surface rendered three/quarter views (D, F). Scale bars, A, B – 1 µm, C, E – 5 µm, D, F – 2 µm.