Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae
- Fondazione IFOM, Istituto FIRC di Oncologia Molecolare (IFOM-IEO Campus), Italy
- Consorzio Mario Negri Sud, Italy
- Institute of Protein Biochemistry, Consiglio Nazionale Delle Ricerche (CNR-IBP), Italy
- Telethon Institute for Genetics and Medicine (TIGEM), Italy
- Center for Anatomy and Cell Biology, Medical University of Vienna, Austria
- FEI Company, Netherlands
- Saarland University, Germany
Figures
Figure 1 with 1 supplement
Kinetic patterns of synchronized transport of albumin and VSVG through the Golgi stack.
VSV-infected HepG2 cells were synchronized according to the CHX/32-15°C protocol ('Materials and methods'). Following release of the 15°C block, the cells were examined by immuno-EM (A–H) at the …
Figure 1—figure supplement 1
Kinetic patterns of synchronised transport of albumin and VSVG through the Golgi stack as determined by immunofluorescence.
VSV-infected cells were synchronised according to the CHX/32-15°C protocol ('Materials and methods'). Briefly, the cells were treated with CHX for 3 hr, to clear the secretory pathway of all cargo. …
Figure 2
Kinetic patterns of synchronized transport of albumin and PC-I through the Golgi stack.
Human fibroblast cells were microinjected in the nucleus with cDNA for albumin and incubated for 2 hr before further treatments. Transport was synchronized according to the CHX/32-15°C protocol and …
Figure 3 with 2 supplements
Kinetic patterns of transport of GFP-albumin, VSVG-GFP and PC-III-GFP through the Golgi stack under steady-state conditions.
HeLa cells were transfected with GFP-albumin (A–K) or PC-III-GFP (L–N). After 16 hr of transfection, the Golgi area was bleached, and entry of these cargoes from the unbleached periphery (ER) into …
Figure 3—figure supplement 1
Localization, transport behavior, and dynamics of GFP-albumin at steady-state.
(A and B) Intra-Golgi distribution of GFP-albumin at steady-state. HeLa cells were transfected with GFP-albumin, kept for 24 hr at 37°C, and then fixed and labeled for immuno-EM with an antibody …
Figure 3—figure supplement 2
Kinetics of antitrypsin processing by Golgi enzymes reflects its fast kinetics of transport.
Transport of antitrypsin (A) and VSVG (B) along the secretory pathway was monitored by radioactive pulse chase assay. HepG2 cells infected with VSV was pulsed with radioactive aminoacids (35S-methion…
Figure 4
EM tomography facilitates the visualization of convoluted intercisternal tubules.
HepG2 cells were high-pressure frozen and prepared for EM tomography ('Materials and methods'). (A and B) Tomographic model of a stack from a 200-nm-thick section containing an intercisternal …
Figure 5 with 4 supplements
Albumin distribution in Golgi cisternae, vesicles and tubules in HepG2 cells.
(A–D) Albumin and VSVG distribution in the ER, Golgi stack and TGN (A and B, immuno-EM; C and D, immunofluorescence). White arrows in (A and B) indicate TGN46 labeling. For quantification see (G–J). …
Figure 5—figure supplement 1
Distribution of cargoes in COPI vesicles.
(A–D) As noted in the main text, Golgi vesicles appear depleted of albumin in vivo, while vesicles prepared in vitro have been reported to contain albumin (Malhotra et al., 1989). We sought to …
Figure 5—figure supplement 2
Gallery of cryo-immuno-gold EM images indicating the presence of albumin in intercisternal tubules.
HepG2 cells were labeled for albumin according to the cryo-immuno EM protocol ('Materials and methods'). Black arrows in all images indicate the convex sides of the intercisternal tubules; black …
Figure 5—figure supplement 3
Presence of albumin in intercisternal tubules revealed by serial sectioning followed by cryo-immuno-gold EM and DAB photooxidation followed by tomography.
(A) Serial sectioning by cryo-immuno EM indicates the presence of albumin in intercisternal tubules. HepG2 cells were labeled for albumin according to the cryo-immuno EM protocol ('Materials and …
Figure 5—figure supplement 4
Presence of albumin in intercisternal tubules revealed by cryo-immuno EM followed by tomography.
(A–C) HepG2 cells were labeled for albumin according to the cryo-immuno EM protocol ('Materials and methods'). Here, thick sections (200 nm) were used instead of the usual thin (70 nm) sections. The …
Figure 6 with 2 supplements
Computational simulations of intra-Golgi transport of albumin by diffusion via intercisternal tubules.
(A) The Golgi stack was modeled as a system of six circular cisternae connected in series by five (one per pair of cisternae) vertical cylindrical tubules. (B) The same stack drawn in a ‘distended’ …
Figure 6—figure supplement 1
Computational simulation of the intra-Golgi equilibration of albumin argues against the classic vesicular transport model.
To simulate albumin transport as mediated by vesicles traveling between adjacent cisternae, the geometry of the Golgi cisternae was set to the same parameters as in the standard configuration used …
Figure 6—figure supplement 2
Computer-simulated model of the formation of a pH gradient between continuous cis- and trans-Golgi cisternae.
(A) The model is based on three proton-handling components in the stack (the size and geometry of which are as defined in the legend to Figure 6): proton pumps located in the trans-most cisterna …
Videos
Video 1
A tomographic reconstruction of the Golgi stack shown in Figure 4.
Scripts used to simulate the transport of albumin across the Golgi stack (Supplement to Figure 6):
Tables
Table 1
Simulated intra-Golgi transport of albumin by diffusion, via intercisternal continuities, occurs in the timescale of seconds
| Length of tubule (nm) | Diameter of tubule (nm) | Number of cisterna | Time needed for 90% equilibration (s) |
|---|---|---|---|
| 100 | 30 | 6 | 14.9 |
| 100 | 60 | 6 | 7.8 |
| 100 | 120 | 6 | 6.8 |
| 50 | 30 | 6 | 11.4 |
| 150 | 30 | 6 | 19.1 |
| 100 | 30 | 4 | 6.4 |
| 100 | 60 | 4 | 3.4 |
| 100 | 120 | 4 | 3.0 |
| 50 | 30 | 4 | 5.0 |
| 150 | 30 | 4 | 8.4 |
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The size and geometry of the Golgi stack used for the simulations is defined in the legend to Figure 6. The variable parameters used for the simulation are: length of tubules (from 50 to 150 nm), diameter of tubules (from 30 to 120 nm), and number of cisternae (between 4 and 6). The tubules here refer to the intercisternal tubules connecting two cisternae of a Golgi stack. The time needed for 90% equilibration of albumin across the Golgi stack under varying combinations of the indicated parameters was computed. As can be seen from the data, equilibration across the cisternae happens in seconds across all the conditions.
Table 2
Simulated intra-Golgi transport of albumin mediated by vesicles, predicts extremely fast turnover of cisternal rims
| Vesicle diameter (nm) | 50 | 60 | 70 | |||
| Number of cisternae | 4 | 6 | 4 | 6 | 4 | 6 |
| Steps for 90% equilibration | 19,617 | 44,230 | 11,352 | 25,595 | 7149 | 16,120 |
| Rim turnover time (s) | 0.576 | 0.255 | 0.829 | 0.368 | 1.129 | 0.500 |
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The size and geometry of the Golgi stack and vesicles used for the simulations are defined in the legend to Figure 6—figure supplement 1. The variable parameters used for the simulation are: diameter of vesicles (from 50 to 70 nm) and number of cisternae (between 4 and 6). Albumin transport is considered to proceed stepwise, where each step is defined as one vesicle detaching from each cisterna and fusing with an adjacent one. For the calculations presented here, the albumin concentration in the vesicles is considered to be 20% of that present in the cisterna (see Figure 5 and also legend to Figure 6—figure supplement 1). The number of steps required to achieve 90% equilibration of albumin across the stack was computed and the time required for a single turnover event of the cisternal rim (rim turnover time) was calculated as described in the legend to Figure 6—figure supplement 1. The rim turnover time varied from 0.25 to 1.12 s or in other words, the rim turns over from 4 to 1 times per second, depending on the condition used for the simulation. The results presented here are for the scenario 'a' (discussed in Figure 6—figure supplement 1) and the results are very similar even in the case of scenario 'b'.
Additional files
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Supplementary file 1
Diffusion of albumin across the Golgi stack stable intercisternal tubules.
- https://doi.org/10.7554/eLife.02009.021
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Supplementary file 2
Diffusion of albumin across the Golgi stack via flickering intercisternal tubules.
- https://doi.org/10.7554/eLife.02009.022
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Supplementary file 3
Transport of albumin across the Golgi stack by vesicles.
- https://doi.org/10.7554/eLife.02009.023
