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First published online 3 May 2005
doi: 10.1242/jcs.02358

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tGolgin-1 (p230, golgin-245) modulates Shiga-toxin transport to the Golgi and Golgi motility towards the microtubule-organizing centre

^{1 1}Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6082, USA
³ Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6082, USA
⁵ Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6082, USA
⁶ Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6082, USA
² Integrated Imaging Center, Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA
⁴ Institut Curie, CNRS-UMR144, F-75248 Paris, Cedex 75005, France

View larger version (63K): [in a new window]   Fig. 1. Depletion of tGolgin-1 from HeLa cells using siRNA. (A) The mRNA encoding tGolgin-1, showing the positions of the overlapping 21-mers used for siRNAs relative to the coding region and the 5' and 3' untranslated regions. See Table 1 for the sequences of the oligonucleotides. (B,C) Low-magnification (40x) IFM image of tGolgin-1 (B) and TGN46 (C) expression in HeLa cells transduced once 2 days earlier with siRNA-1. Cells were co-stained with antibodies against tGolgin-1 and TGN46, and with appropriate fluorochrome-conjugated secondary antibodies. Arrows denote cells with reduced tGolgin-1 expression. (D) Untransfected HeLa cells (control) or cells transduced once with siRNA-1 were analysed by IFM as in B,C. Cells were counted as positive or negative for tGolgin-1 expression as indicated and, within each population, cells were scored for a `normal' ribbon-like or a dispersed, punctate staining pattern for TGN46 in single plane images. The proportions of cells in each category are given from a representative experiment. Results with two control siRNAs and with siRNA-4 were similar to those obtained with untransfected cells (E). Whole-cell lysates from HeLa cells transduced twice with siRNA-1 or control siRNA were fractionated by SDS-PAGE and analysed by western blot for tGolgin-1 production. Only the portion of the gel corresponding to full-length tGolgin-1 (260 kDa) is shown; the rest of the lanes were blank.

View larger version (57K): [in a new window]   Fig. 2. Dispersed TGN46 is a function of tGolgin-1 depletion. (A-F) HeLa cells transduced once with siRNA-1 (A,B), siRNA-2 (C,D) or siRNA-3 (E,F) were fixed 2 days later and analysed by IFM for endogenous tGolgin-1 (A,C,E) and TGN46 (B,D,F). Stars denote cells without tGolgin-1 labelling and arrowheads denote cells with significant residual levels of tGolgin-1. (G,H) HeLa cells were co-transduced with siRNA-1 and plasmids encoding either siRNA-1-resistant mouse tGolgin-1 (G-I) or irrelevant HA-epitope tagged Rab27a (J-L), then fixed 2 days later and analysed by IFM using antibodies specific for human tGolgin-1 (G,J), for both human and mouse tGolgin-1 (H,K), and for TGN46 (I,L), and appropriate fluorochrome-conjugated secondary antibodies. Arrows (G-I) denote cells with negligible labelling for endogenous human tGolgin-1 but that are reconstituted with mouse tGolgin-1; notice the ribbon-like labelling of TGN46. Arrowheads (J-L) denote cells with negligible labelling for endogenous human tGolgin-1; notice the dispersed TGN46 labelling in most of these cells.

View larger version (135K): [in a new window]   Fig. 3. Dispersal of TGN and Golgi elements in cells depleted of tGolgin-1. HeLa cells transduced twice with siRNA-1 were fixed 1 day later. They were then either analysed by IFM using primary antibodies to TGN46 and to the indicated proteins, followed by the appropriate fluorochrome-conjugated secondary antibodies (A-K), or embedded in epon and analysed by electron microscopy. (C-K) tGolgin-1-depleted cells were identified based on the dispersed pattern of TGN46 staining. (K) A colorized overlay of the images shown in I,J to emphasize the polarity of the Golgi and TGN elements; the boxed region is magnified 2.5 times in the top right corner. (L-Q) Characteristic electron microcopy fields containing Golgi membranes in cells transduced with control siRNA (L) or siRNA-1 (M-Q). Notice the reduced size of the stacks in cells transduced with siRNA-1. Bar in Q (applies to L-Q), 0.2 µm.

View larger version (83K): [in a new window]   Fig. 4. Association of Golgi elements with microtubules in cells depleted of tGolgin-1. (A-F) HeLa cells transduced twice with siRNA-1 (A-C) or with dynamitin-p50 (D-F) were fixed 1-2 days later and analysed by deconvolution IFM using primary antibodies to TGN46 and -tubulin. (C,F) Overlays of the two labels. The boxed regions are magnified 2.5 times in the bottom or top right corner to emphasize the alignment of Golgi elements along microtubules. (G-R) HeLa cells were transfected with GalT-EGFP alone and left untreated (Ctl.; G-I) or treated with nocodazole (Noc.; J-L), or cotransfected with GalT-EGFP and dynamitin-p50 (Dyn-p50; M-O) or with GalT-EGFP and siRNA-1 (P-R). Cells were fixed, labelled with anti--tubulin and RRX-conjugated secondary antibodies, and analysed by deconvolution fluorescence microscopy. Colorized overlays of the two labels are shown on the right. (S-X) Untransduced cells treated for 2 hours with nocodazole (Noc.; V-X) or cells transfected with dynamitin-p50 (S-U) were fixed and analysed by deconvolution IFM using primary antibodies to TGN46 and giantin. (U,X) Overlays of the two labels; the boxed regions are magnified 2.5 times in the top right corners.

View larger version (90K): [in a new window]   Fig. 5. tGolgin-1 depletion mimics disruption of dynein-dynactin or microtubules in terms of Golgi element position, function and movement. (A-F) HeLa cells transduced with both siRNA-1 and plasmid encoding Sec13-EGFP (A-C) or Sec13-EGFP alone (D-F) were fixed 2 days later without (A-C) or with (D-F) treatment with nocodazole (Noc.) for 2 hours, and analysed by fluorescence microscopy after labelling with anti-TGN46 and RRX-conjugated secondary antibody. Individual labels are shown in A,B,D,E, and the overlay is shown in C,F; the boxed region in C is magnified 2.5 times in the bottom right corner. tGolgin-1-depleted cells were identified based on the labelling pattern for TGN46. Notice the accumulation of TGN46 labelling near sites labelled by Sec13-EGFP. (G-L) HeLa cells transduced with both siRNA-1 and plasmid encoding VSV-G/EGFP were incubated overnight at 39°C and then fixed either immediately (G,H) or after a 30 minute (I,J) or 60 minute (K,L) incubation at 32°C. Cells were analysed by fluorescence microscopy after labelling with anti-TGN46 and RRX-conjugated secondary antibody. tGolgin-1-depleted cells were identified based on the labelling pattern for TGN46. Notice the appearance of cell-surface labelling (particularly in surface spikes) for VSV-G/EGFP by 60 minutes. (M-P) HeLa cells stably expressing GRASP65-EGFP were transduced transiently with siRNA-1 (M) or dynamitin-p50 (n), treated with nocodazole for 2 hours (O), or exposed to brefeldin A for 1 hour, washed, recovered at 16°C for 3 hours and then released at 37°C (P). Cells were analysed by video microscopy and representative images were collected every 60 seconds (M-O) or 20 (P) seconds for 5 minutes. Images from all time points were overlaid, with the initial image labelled in white and all subsequent images labelled in red to indicate movement from the initial position. Notice that very few labelled elements in m-o showed significant movement over the 5 minute time course. (Q) Quantitation of Golgi element movement. 50-150 spots from at least five cells in each experimental sample represented in m-p were analysed for distance moved over the 5 minute time course. The mean, median, maximum (max) and minimum (min) values, and 25th and 75th quartile values for distance moved in each experimental set are shown. Pairs of value sets were analysed by Student's t test, and P values for each paired analysis are indicated.

View larger version (91K): [in a new window]   Fig. 6. The requirement for tGolgin-1 in microtubule minus-end-directed movement of pre-Golgi intermediates is indirect. HeLa cells transduced with Sec13-EGFP (A-F) or untransduced HeLa cells (G-J) were treated for 1 hour with 1 µg ml–1 brefeldin A (BFA) and then washed and incubated at 16°C for an additional 3 hours in the absence of BFA. Cells were then fixed immediately or after an additional incubation at 37°C for 15 minutes to 2 hours, and analysed by IFM after labelling with antibodies to the indicated proteins and appropriate fluorochrome-conjugated secondary antibodies. Only the overlaid images from both labels are shown. (A-F) Comparison of the ER exit site marker Sec13-EGFP and either Golgi (GM130; A-C) or TGN (TGN46; D-F) elements at all three time points. (G) Comparison of Golgi (GM130) and TGN (TGN46) elements at the 15 minute time point. (H-J) Comparison of tGolgin-1 and TGN (TGN46) elements at all time points. Boxed regions in the top right corner of A,B,D,E,G-I represent 2.5 times magnified images from the corresponding panels to emphasize the degree of colocalization. Notice that, whereas GM130 initially localizes to Sec13/EGFP-containing structures before migrating towards the MTOC, TGN46 and tGolgin-1 initially localize to separate structures that remain separate during transport.

View larger version (92K): [in a new window]   Fig. 7. Endosome-to-Golgi transport of STxB is inhibited by tGolgin-1 depletion. HeLa cells that were untreated (Ctl.), transduced with siRNA-1 or treated for 2 hours with nocodazole (Noc.) were incubated with Alexa-488-conjugated STxB on ice for 45 minutes, washed and then warmed to 37°C for 60 minutes. Cells were then fixed, labelled with antibodies to GM130 and RRX-conjugated anti-mouse immunoglobulin (A-I) or to TGN46 and Lamp1 followed by AMCA- and RRX-conjugated secondary antibodies (J-O), and analysed by fluorescence microscopy. siRNA-1-transduced cells were identified based on the dispersed staining pattern for TGN46. Panels show single staining for STxB (A,D,G,J,M), GM130 (B,E,H) or Lamp1 (K,N), or overlays of the two labels, as indicated (C,F,I,L,O). Boxed images were magnified two-fold in the top right corner in C,F,I,O to emphasize the degree of colocalization.

View larger version (103K): [in a new window]   Fig. 8. Accumulation of aberrant multivesicular structures in tGolgin-1-depleted cells. HeLa cells transduced twice with siRNA-1 were fixed 1 day later and processed for conventional electron microscopic analysis. Typical fields are shown from these cells containing aberrant vacuolar structures (*), which appear to be derived from multilaminar (A,B) or multivesicular (C) endosomes. Notice the peripheral Golgi mini stack (G) in A. m, mitochondria; n, nucleus. Bar, 1 µm.

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