Inter-Golgi transport mediated by COPI-containing vesicles carrying small cargoes
- Yale University School of Medicine, United States
- Yale University, United States
- Technische Universität München, Germany
Figures
Figure 1
General procedure.
HeLa cells co-expressing a GFP-labeled Golgi localized protein and VSV-G are mixed with HeLa cells expressing a RFP-labeled Golgi localized protein. Cell fusion is triggered by acidic exposure of …
Figure 2
Inter-Golgi transport of Golgi resident glycosyl-transferase proteins.
HeLa cells expressing either GT-GFP and VSV-G, or ST-RFP were mixed and fused by acidic exposure (1 min, pH 5) and then monitored by confocal video-microscopy at 20°C. Cells were treated with CHX …
Figure 3 with 1 supplement
Inter-Golgi transport of small anterograde cargo.
(A) HeLa cells expressing either ssGFP-FM4-CD8 or ST-RFP (+VSV-G) were mixed and fused. Before fusion, cells were incubated at 20°C for 2 hr in the presence of CHX (100 μg/ml) and AP21998 (500 nM) …
Figure 3—figure supplement 1
Inter-Golgi transport is microtubule independent.
HeLa cell expressing GT-GFP (+VSV-G) and ST-RFP were mixed and fused. When required, nocodazole (2.10−3 μg/ml) was added 2 hr before the fusion and was maintained during the time course of the …
Figure 4 with 1 supplement
ER as a minor source of anterograde cargo during inter-Golgi transport.
(A) FRAP performed on Golgi within fused or single cells treated with H89 (50 μM). Top panel, Golgi 1 (green) from a single cell and Golgi 2 (green and red) from fused cells 30 min post-fusion in …
Figure 4—figure supplement 1
Inter-Golgi transport does not involved transit through endosomes.
HeLa cells expressing (A) Rab7-GFP (+VSV-G) or ssDsRed-FM4-CD8 (+VSV-G), (B) ssGFP-FM4-CD8 (+VSV-G) or Rab5A-RFP (+VSV-G), (C) ssGFP-FM4-CD8 (+VSV-G) or ssDsRed-FM4-CD8 (+VSV-G), were incubated at …
Figure 5 with 1 supplement
Diffusible inter-Golgi transport intermediates.
(A) 30 min post fusion, the entire volume a dikaryon (ER and acceptor Golgi 2) was photobleached except for the Golgi 1 that remains the only source of fluorescence. Small diffusing fluorescent dots …
Figure 5—figure supplement 1
ARF1Q71L abolished fluorescence recovery in the inter-Golgi zone and at the Golgi.
Cells expressing either ssGFP-FM4-CD8 or ARF1Q71L–DsRed (+VSV-G) were incubated at 20°C for 2 hr in the presence of CHX (100 µg/ml) and AP21998 (500 nM) to trigger the release of the cargo from the …
Figure 6 with 1 supplement
Role of ARF1 and ε−COP on inter-Golgi transport.
(A) Mixed and fused HeLa cells expressing GT-CFP (+VSV-G), GT-YFP (+VSV-G) or ARF1Q71L–DsRed, in presence of CHX. Graphs show the fluorescence intensity over time for each fluorescent marker within …
Figure 6—figure supplement 1
ARF1 WT does not prevent inter Golgi exchange of small anterograde cargo.
Mixed and fused HeLa cells expressing either ssDsRed-FM4-CD8 (+VSV-G) or ARF1WT-GFP in the presence of AP21988 and CHX at 20°C. The graphs show the fluorescence intensity overtime of each marker for …
Figure 7 with 2 supplements
Inter-Golgi transport intermediates are compatible with COPI vesicles.
(A) In vitro prepared COPI vesicles labeled with Alexa488 were attached to glass coverslips. Immunofluorescence against COPI was performed, and samples were imaged by both confocal and STED …
Figure 7—figure supplement 1
Inter-Golgi carriers are COPI positive and COPII negative.
(A) Cells expressing CD8-GFP-FM4, ST-RFP and VSV-G were incubated at 20°C in the presence of AP21998 and CHX for 2 hr, fused, and fixed after 30 min. Immunofluorescence was performed against β-COP …
Figure 7—figure supplement 2
Discriminating tubules from vesicles with STED.
Vesicles and tubules with different dimensions simulated in a set of different 3D orientations to represent an isotropic distribution (supplementary method section). Pictures illustrate the object …
Figure 8
Inter-Golgi exchange of anterograde cargo at 37°C.
(A) HeLa cells expressing ss-GFP-FM4-CD8 and GST-PKD(DI) were mixed and fused with cells expressing ST-RFP, GST-PKD(DI) and VSV-G. Prior to fusion, cells were incubated at 37°C for 15 min in the …
