Journal: eLife

Article Title: Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae

doi: 10.7554/eLife.02009

Figure Lengend Snippet: 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 indicated times. Panels ( I – K ) show quantification of immuno-EM values as labeling density (LD) normalized to the density in the ER, to avoid labeling variability across samples. ( L ) The amount of albumin or VSVG in indicated compartments were normalized to that present at time 0 in the ER and expressed as percentage. Values are mean ± SD from 30 stacks per time point, in three independent experiments for immuno-EM. Bar: 60 nm ( A ), 50 nm ( B , C , E , G ), 100 nm ( D , H ), 80 nm ( F ). DOI: http://dx.doi.org/10.7554/eLife.02009.003

Article Snippet: HepG2 human hepatoma and HeLa cells (both from ATCC) were grown in Minimum Essential Medium (MEM) supplemented with 10% foetal calf serum, glutamine and antibiotics.

Techniques: Infection, Blocking Assay, Labeling

Journal: eLife

Article Title: Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae

doi: 10.7554/eLife.02009

Figure Lengend Snippet: ( 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 against GFP (10 nm gold) and TGN 46 (5 nm gold; green arrows) ( A ). The albumin distribution depicted in ( A ) was quantified and the steady-state GFP-albumin in the cis -Golgi and trans -Golgi is shown as labeling density (LD) normalized to that of the ER ( B ). ( C – E ) Kinetics of secretion of GFP-albumin. HeLa cells expressing GFP-albumin (lanes 1, 3) and HepG2 cells (lanes 2, 4) were washed with serum free media and incubated at 37°C for indicated times in serum free media. The cell lysate immediately after wash ( C ) and media after 60 min of incubation ( D ) were resolved by SDS-PAGE and probed with anti-albumin antibody. Quantification of the secreted protein (as % total) shows that albumin and GFP-albumin are released into the medium with similar kinetics ( E ). The minor low-molecular-weight form of GFP-albumin in the intracellular pool ( C , lower band in lane 1) is probably a misfolded form of the protein undergoing degradation, and it is not secreted ( D , lane 3). ( F–H ) Dynamics of GFP-albumin at steady-state. HeLa cells were transfected with GFP-albumin and examined using the FRAP approach ('Materials and methods'). ( F ) Entry of GFP-albumin into the Golgi area. The fluorescence in the whole Golgi area was bleached, and the recovery of fluorescence into the bleached area was monitored. ( G ) Exit of GFP-albumin from the Golgi area. The fluorescence of the whole cell less the Golgi area was bleached, and the loss of GFP-albumin from the Golgi area was monitored. ( H ) Diffusion of GFP-albumin along the Golgi ribbon. The fluorescence of a part of the Golgi area was bleached, and the recovery of fluorescence into the bleached region was monitored. The data expressed are in mean ±S.D from three independent experiments ( E ) or five independent experiments ( F–H ). Bar: 130 nm ( A ). DOI: http://dx.doi.org/10.7554/eLife.02009.007

Article Snippet: HepG2 human hepatoma and HeLa cells (both from ATCC) were grown in Minimum Essential Medium (MEM) supplemented with 10% foetal calf serum, glutamine and antibiotics.

Techniques: Transfection, Labeling, Expressing, Incubation, SDS Page, Molecular Weight, Fluorescence, Diffusion-based Assay

Journal: eLife

Article Title: Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae

doi: 10.7554/eLife.02009

Figure Lengend Snippet: 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 ( 35 S-methionine and cysteine) for 5 min and then chased for indicated times in cold media. At the end of the chase period the cells were lysed and VSVG or antitrypsin were immunoprecipitated and subjected to Endoglycosidase H digestion as indicated and resolved by SDS-PAGE followed by autoradiography. EndoHr–Endoglycodisase resistant, EndoHs–Endoglycosidase sensitive, immature–EndoHr form of antitrypsin that was not processed by trans-Golgi resident enzymes and mature–EndoHr form of antitrypsin processed by trans-Golgi resident enzymes. It is important to note here that the quantities of antitrypsin and VSVG present are very similar suggesting that the difference in the transport behavior of these proteins is not due to differences in their abundance. In addition, as mentioned in the text, the transport behaviors of antitrypsin and albumin are similar, reiterating further that the differences in the transport behavior between soluble secretory cargoes (albumin and antitrypsin) and VSVG/PC is possibly not due to the differences in their abundance. DOI: http://dx.doi.org/10.7554/eLife.02009.008

Article Snippet: HepG2 human hepatoma and HeLa cells (both from ATCC) were grown in Minimum Essential Medium (MEM) supplemented with 10% foetal calf serum, glutamine and antibiotics.

Techniques: Pulse Chase, Infection, Immunoprecipitation, SDS Page, Autoradiography

Journal: eLife

Article Title: Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae

doi: 10.7554/eLife.02009

Figure Lengend Snippet: 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 connection. Detail of A shown in B ; note the complexity of the convoluted connection (follow the arrow to identify the continuity). ( C ) A gallery of tomographic digital slices (panels 1–12) used to construct the model in ( A and B ) shows a convoluted intercisternal connection, with the small arrow following the connection, and the arrowheads showing the two cisternae that are connected. Note the complexity of the connection, which would be nearly impossible to detect in traditional thin sections. See for facilitated visualization of the continuity. Bar: 150 nm. DOI: http://dx.doi.org/10.7554/eLife.02009.009

Article Snippet: HepG2 human hepatoma and HeLa cells (both from ATCC) were grown in Minimum Essential Medium (MEM) supplemented with 10% foetal calf serum, glutamine and antibiotics.

Techniques: Tomography, Construct

Journal: eLife

Article Title: Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae

doi: 10.7554/eLife.02009

Figure Lengend Snippet: ( 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 . We sought to resolve this discrepancy between in vivo and in vitro data by examining the conditions of the in vitro preparation. COPI vesicles were purified exactly as described by Rothman et al. . Their study used the non-hydrolysable GTP analogue GTPγS to increase the yield of vesicles in vitro. It was later shown, however, that GTPγS also affects the sorting of cargo into vesicles . We thus examined the effects of GTPγS, and we show here that while COPI-coated vesicles that are formed in the presence of GTPγS contained significant levels of albumin, those that are formed in the absence of GTPγS were depleted of albumin, in line with our in vivo data. The exclusion of soluble proteins from vesicles has been reported before (e.g., of proinsulin from KDEL-receptor-containing vesicles; ); however, the mechanism of exclusion remains unclear. Experimental details: Golgi membranes were isolated from rat liver ('Materials and methods') and treated with ( A ) or without ( B and C ) GTPγS, as described by Rothman and colleagues . The Golgi membranes were then pelleted, and processed for cryoimmuno-EM and labeled with an anti-albumin antibody. After GTPγS treatment, albumin can be seen in COPI vesicular profiles (round, 50–60 nm in diameter) ( A , arrows), while in the absence of GTPγS, the vesicles (less numerous) were not labeled for albumin ( B and C ; arrows). In both cases, albumin was concentrated in large pleomorphic structures, which were probably cisternal remnants. ( D ) Quantification of the percentage of vesicles containing albumin from ( A ) and ( B ) expressed as mean ± SD (n = 3). ( E–F ) VSV-infected HepG2 cells at steady-state were labeled with antibody against VSVG according to cryo-immuno EM protocol ('Materials and methods'). ( E ) Lack of VSVG labeling within elongated tubule-like (arrow) and round vesicle-like (arrowhead) profiles. ( F ) Morphometric analysis shows that VSVG labeling density (mean ± SD; n = 30 stacks) is significantly less in tubules and vesicles than in cisternae. Bar: 120 nm ( A and E ); 90 nm ( B and C ). DOI: http://dx.doi.org/10.7554/eLife.02009.012

Article Snippet: HepG2 human hepatoma and HeLa cells (both from ATCC) were grown in Minimum Essential Medium (MEM) supplemented with 10% foetal calf serum, glutamine and antibiotics.

Techniques: In Vivo, In Vitro, Purification, Isolation, Labeling, Infection