Retrograde vesicles that travel backward through the Golgi bud off of a cisterna to transfer enzymes to younger cisternae. Figure 3: Cisternal maturation in Golgi of Saccharomyces cerevisiae Golgi cisternae were labeled with dyes to track their movement over time in individual yeast cells. The cycling of red and green colors reflects the transient expression of different proteins at the cisternae surface. Video courtesy of Dr. Benjamin S. Glick, University of Chicago.
Today most Golgi researchers agree that the evidence favors the cisternal maturation model Emr et al. Evidence in support of this model came from the laboratories of Benjamin Glick and Akihiko Nakano, who concurrently performed experiments that strikingly demonstrated the process of cisternal maturation. In a stunning visual assay, both labs used live-cell fluorescence microscopy to directly observe cisternal maturation in Golgi of Saccharomyces cerevisiae Baker's yeast Figure 3 Losev et al.
The Golgi of S. Instead of appearing as the typical stack of pita bread, in S. The individual cisternae are spread in an irregular manner throughout the cell. This unusual structure was ideal for using light microscopy to observe changes in the individual cisternae over time.
The vesicular transport model would predict that an individual cis cisterna would remain cis, with characteristic cis enzymes, over its entire lifespan. However, the cisternal maturation model would predict that a newly formed cis cisterna would eventually mature into a medial, then a trans cisterna, before breaking apart when its contents were packaged for their final destinations in the cell.
In their experiments the two research groups linked fluorescent proteins glowing green or red to the proteins present in different, individual cisternae of S.
The researchers designed their experiments to test the predictions of the vesicular transport and cisternal maturation models. If the vesicular transport model were correct, then the cisternae would be stable and maintain the same fluorescently labeled Golgi resident proteins over time.
In contrast, if the cisternal maturation model was not correct, then each cisterna would contain a changing set of Golgi proteins over time. In their experiments, the researchers created beautiful movies of the yeast and observed that the individual cisternae changed color over time.
After analyzing a variety of Golgi proteins, the researchers consistently observed changes in the protein composition of individual cisternae over time.
Their results provided strong evidence for the cisternal maturation model. Although researchers generally agree that the cisternal maturation model best fits the current data, there is still some debate over whether or not all cargo proteins take the same path.
Jennifer Lippincott-Swartz and her colleagues pioneered fluorescence methods to quantitatively measure the dynamics of cellular membranes, including the Golgi. Using these methods, they learned that some cargo proteins travel through the Golgi more slowly than the rates at which the cisternae mature Patterson et al. The researchers concluded that the cisternal maturation model could not accurately account for their data.
While they do not dispute cisternal maturation, they additionally proposed a model whereby a two-phase system of membranes determines which cargo proteins and Golgi enzymes must distribute themselves during transport. Complicating the situation further, at least some cell types have connections between different cisternae within the Golgi stack e.
For example, Luini and colleagues observed intercisternal continuities during waves of protein traffic in mammalian cells Trucco et al. Many investigators will continue to investigate and refine these new models over time. While some aspects of protein transport through the Golgi are better understood than they used to be, there are still many unresolved issues surrounding the specifics within different organisms.
Moreover, questions remain about the unifying characteristics that are shared between all Golgi. A recent gathering of prominent Golgi researchers identified several important questions to be addressed in the future, including:.
The structure of the Golgi apparatus varies in different cell types. The dispersed nature of Golgi cisternae in the yeast Saccharomyces cerevisiae allowed researchers to resolve individual cisternae. By observing fluorescently labeled proteins that normal reside within different cisternae, researchers found convincing evidence that the Golgi cisternae change over time, supporting the cisternal maturation model of protein movement through the Golgi apparatus.
However, there is clearly much left to discover about the Golgi. Alberts, B. Molecular Biology of the Cell, 5th ed. New York: Garland Science, Becker, B. The secretory pathway of protists: Spatial and functional organization and evolution. Microbiological Reviews 60 , — Anterograde transport of algal scales through the Golgi complex is not mediated by vesicles.
Trends in Cell Biology 5 , — doi: Bonfanti, L. Procollagen traverses the Golgi stack without leaving the lumen of cisternae: Evidence for cisternal maturation. Cell 95 , — doi Emr, S. Journeys through the Golgi — Taking stock in a new era. Journal of Cell Biology , — doi: Farquhar, M. The Golgis apparatus: years of progress and controversy. Trends in Cell Biology 8 , 2—10 doi: Glick, B. The curious status of the Golgi apparatus.
From there they are modified and packaged into vesicles for distribution. Proteins targeted to organelles such as the endosome, cellular membranes, or for extracellular secretion, must be modified. The modification is necessary for the correct delivery of the protein to its final cellular location. The modification occurs when specific sugar molecules are added to a core oligosaccharide that is attached to the protein. These sugar complexes are the signal often required to direct the protein to its final destination.
One example of this, is mannose 6-phosphate. We focus here on the delivery of a hydrolase enzyme to the endosome. Hydrolases are enzymes that degrade other molecules. The endosome is an organelle that contains molecules to be degraded. Other key components include the M6P receptor protein. First, the hydrolase is delivered from the ER to the Golgi Apparatus via a vesicle.
While it is being transferred through the ER and cis-cisterna of the Golgi apparatus, modification of the sugar core oligosaccharide begins. The term for this process is glycosylation. Here we show two steps involved in the production of the mannose 6-phosphate signal. In humans, defects in Golgi glycosylation can lead to specific diseases. Once the hydrolase reaches the trans-golgi cisterna the mannose 6-phosphate signal has been completed. A major controversy in the field of membrane traffic is how cargo progresses through the Golgi complex.
Here the authors report a very important finding-namely that a soluble cargo and a large aggregate cargo collagen traverse a single Golgi stack at different rates. By itself, this finding would be inconsistent with simple cisternal maturation in that a means to traverse the Golgi without any cisternal movement is possible.
The work is carried out to a high standard but the manuscript needs reworking and certain parts need to be modified as described below. Figure S4 should really be a main figure combined with Figure 4a-b , while the rest of Figure 4 could be moved to a new figure. The modelling sits at the end, and should be better integrated. One option would be to bring the simulation in earlier and explain what predictions the different models make.
The data can then be presented in light of the simulation, commenting of which model provides the best fit to the experimental observations. Some of the EM is fantastic but other images could be a lot better.
Detailed suggestions follow here:. For this conclusion the machinery mediating cisternal continuity formation would need to be inactivated, and further experiments done showing i cisternal continuities are lost and ii transport ceases. The major question left unresolved by this work is the mechanism by which cisternal continuities between adjacent Golgi compartments form and resolve. A further question is how this occurs without collapse and mixing of the two compartments to generate one larger compartment.
Please acknowledge and discuss. They refer to Figure 1j -m, however this fails to convincingly show accumulation of albumin in the Golgi region marked by either GM or TGN The diffuse staining, possibly ER as the authors mention, does not seem to be reduced and accounts for the bulk of the signal while only a small proportion overlaps with Golgi markers. This suggests that albumin trafficking is not particularly efficient and only a subpopulation is being transported to the Golgi.
These conditions are used for the immunoelectron microscopy, so this is a problem for all the data shown. The reviewers would prefer to see more examples of the triple labeling shown in Figure 1a-f than that shown in j-q. In panel 2a a whole cell is shown, while for the other condition 3min at 32C only a magnified region of the Golgi is shown. The figure order should be changed. Was a Golgi marker used to define the cis cisternae? If yes, then a clearer image with highlights for the different markers as nicely done in Figure 1a-f should be shown.
The data in Figure 3 are also far from compelling evidence that albumin rapidly fills the entire Golgi stack while PC moves more slowly. The images in Figure 3 are not shown at the same scale.
This isn't entirely clear and the figure legend is confusing - it would be better if the scale was marked in the figure and not appended to the legend.
Panels 3a-c show whole cells, while 3d-i are Golgi only however at different scales. It would be better if cells were shown at one magnification while enlarged Golgi was shown at another standardized scale to allow simpler comparison of the different images and between figures.
The example in Figure 4a-c is not an unambiguous connection and the yellow labeled cisternae in Figure 4c appears to have a clear membrane bilayer in the region said to form a join. I have a similar problem with Figure 4i , apparently showing a continuity labeled for albumin that lies adjacent to a Golgi stack but isn't definitely connected to it. This isn't well integrated with the rest of the work.
The assumptions are not properly described in the text, and exactly what testable predictions the modeling generates is unclear. Why does VSV G not diffuse through cisternal continuities as rapidly as soluble cargo? From what I can tell the modeling is a simplification of the 3D membrane architecture of the Golgi to 2D, so surface area to volume ratio is not correctly accounted for.
My concern is that albumin levels are much higher than the other markers, and this may explain some of the differences seen. Have the authors considered the issue of whether fixation or cryoEM processing is fast enough to have full confidence in the 2 minute time point versus 5 minutes, for example? It does not change their story but is an important issue to control for experimentally.
If the compartment can fill in 2 minutes and the ER has little albumin, why does the compartment empty so much more slowly? Addition of cycloheximide and determination of the efflux rate would be informative here. How can TGN accumulation be explained?
Is the albumin binding to lipidic particles in the Golgi to slow its exit? Please comment on this in relation to use of vesicles versus simple diffusion. It may be hard to reconcile this view with the multi-step glycosylation at work for certain cargos. It would be important to know whether glycosylation kinetics fits with the observed transport kinetics. The alpha1-antitrypsin reporter chosen in this study may be an adequate example.
The use of lectin may be helpful to carry out a rough analysis. However, it seems to me that the export will then be very slow if no additional hypothesis is used to impose that the diffusion is biased in the anterograde direction. Otherwise, the cargo will always be homogeneously distributed in all cisternae and emptying the Golgi would be very slow. The authors mention pH gradient but without really using this as driving force.
In addition, not all cargo may be sensitive to the pH gradient that exists in the Golgi. The authors should take this in account in their simulations and try to estimate the exit time e. They should discuss this point. These values may be used in the simulation to see what level of diffusion bias one has to add to the diffusion model to explain the observed kinetics. It may also enable to detect differences of diffusion in Golgi sub-domains. It is clear that retention at 15 degree is already not occurring in the same sub-compartment for the different cargos and this difference may explain part of the downstream behaviour reported here.
Carrying out a photo-bleaching experiment to compare the kinetics of intra Golgi diffusion of small and large cargos may help to control for this in physiological conditions. We thank the reviewers for the appreciation of our work and their constructive criticism.
We have done this and modified the manuscript appropriately see new Figure 4. We have sought to modify the manuscript to take into account the perception of the reviewers that the models should be better integrated.
In order to achieve this, we need to consider that the scope of our models is limited. The models only aim to assess the equilibration rates of small soluble cargoes across a closed system with a stack-like geometry, and in particular to compare two possibilities: a cargo equilibration through the stack by diffusion via continuities and b equilibration via shuttling vesicles.
Importantly, the models do not aim to simulate the entire traffic process through the Golgi including cargo arrival, departure and intra-Golgi concentration steps. Given these limitations, we feel that it would be difficult for a reader to grasp the significance of these models without first providing the appropriate experimental background.
Rather, we think that the best way to use the models in this manuscript is to go through the following logical steps:. The experiments also establish an upper bound of 2 minutes, as the time required for albumin passage from the first to the last compartment. We therefore propose to improve on the original scheme, rather than to drastically change it.
In the revised version, we seek to achieve a better integration of the models by making reference to the original data on which the parameters of the models were based on, and by clarifying the logical flow that was adopted. However, if the editor and the reviewers strongly prefer to stick to their original advice to restructure the manuscript and bring the simulation in earlier, we will find a way to do so.
We agree with comment of the reviewers that the mechanistic question is not fully resolved, although a certain amount of information is available on the mechanism of formation and fission of the intercisternal tubules. The issue of coexistence of intercisternal connections and cis-trans polarity of the Golgi apparatus is not without antecedents.
They refer to Figure 1j-m , however this fails to convincingly show accumulation of albumin in the Golgi region marked by either GM or TGN As suggested by the reviewers we have now included more images of cryo-immunogold microcopy that define the distribution of albumin across the Golgi at high resolution see new Figure 1.
The rest of the time course is presented in Figure 1—figure supplement 1 where one can see that by 5 min the ER has already emptied to a large extent, indicating that the transport of albumin is remarkably efficient. Thus, under the conditions used for the experiments, the albumin transport appears to be efficient.
We have clarified the text in this regard. We have removed the Concanamycin data as suggested by the reviewer. Since one of the latter questions see point 10 raised by the reviewers directly refers to the role of pH in the albumin transport we had to retain the reference to this data and we have mentioned it as data not shown.
We have changed the images in Figure 2 , which now show enlarged Golgi in all conditions, so that they are now uniform. We have also changed the order of the figures so that PC localization by EM is presented before that of albumin. We apologize for not being clear with the image. A Golgi marker was indeed used to define the cis-Golgi in Figure 2f.
While PC can be identified morphologically by the presence of aggregates now indicated by an asterisk , the cis-Golgi was identified by using GM as a marker black dots labeled by immuno nanogold technique. The gold particles showing the presence of GM are now indicated by arrows. We have not done cryo-immunogold labeling usually used to label two or three markers simultaneously here, since PC can easily be identified morphologically and only single labeling for the cis-Golgi marker was required.
The experiments in Figure 3 complete the experiments described in Figures 1 and 2 , where we show that under synchronized conditions albumin traverses the Golgi stack faster than VSVG or PC. In other words, the experiments in Figure 3 are meant to examine whether the observed fast kinetics of albumin transport might be limited to the synchronization conditions Figures 1 and 2 or they are observed also at steady state.
The results show that, under steady state conditions, the newly recovered fluorescence of albumin immediately after photobleaching , that represents newly arrived cargo from the ER at the Golgi apparatus, can be seen to spread throughout the Golgi as determined from the overlap of albumin with both GM and TGN46 , while in the case of PC, it is mainly restricted to the cis side of the Golgi.
We think that such an overlap is fairly compelling evidence that the albumin traverses the Golgi stack faster than PC even under steady state conditions. Moreover the use of Golgi ministacks where the separation between the cis and trans Golgi is always clearer than that seen with the intact Golgi ribbon provides a further convincing argument that even under steady state conditions the albumin traverses the Golgi stack faster than VSVG or PC.
In addition, to confirm the above light microscopy data with a high resolution EM-based approach, we used a method based on photo-oxidation of GFP coupled to FRAP to unambiguously identify the newly arrived cargo, represented by the photoconversion product marked by arrows in Figure 3o-r. As can be seen from the figure in the case of albumin the photo-conversion product is present throughout the Golgi already at 2 min after bleaching, suggesting rapid transport across the stack consistent with all of the previous data , while it is restricted to the cis side in the case of VSVG and PC, which migrate through Golgi more slowly.
Regarding quantification, due to intrinsic limitations of the technique, the density of the photo-conversion product cannot be quantified in a meaningful way. However, as requested by the reviewer, we have now added quantifications by measuring the percentage of cells where the Golgi was completely filled with GFP-albumin and cells where GFP-albumin is present only at the cis side see Figure 3s.
We apologize for not having been clear about the description of the experiments represented in Figure 3o-r. We have rewritten the corresponding figure legend to make it clearer.
This isn't entirely clear and the figure legend is confusing — it would be better if the scale was marked in the figure and not appended to the legend. We apologize for the confusion. We have not marked the scale in this figure to maintain uniformity with other figures. We again apologize for not being clear with the image in Figure 4c.
The image is one virtual section from the tomogram shown in Figure S4 now Figure 4c. When seen in the context of the whole panel of successive images the reviewer can clearly see that the intercisternal connection is a valid one.
To avoid any confusion, we have removed panel 4c and shifted Figure S4 to the main figures. The new figure Figure 4 contains the whole set of virtual sections from the tomogram making it easier to visualize the rather tortuous intercisternal connection. We also note that most connections are fairly complex and require an analysis of several successive virtual sections, which explains why they are rarely detected by casual observers.
Regarding Figure 4i now Figure. As discussed in the text, these linear continuities are rare in thin sections. We have now added arrows to facilitate the appreciation of the continuity. Following the arrows one can clearly see the luminal continuity between the two non-adjacent cisternae that the tubule connects. Part of our response to these comments can be found above.
Basically, the two models, one based on equilibration via continuities and one on equilibration via shuttling vesicles, are simple, and are designed only to assess whether the equilibration rates of albumin across the stack are compatible with a continuity-based, or with a vesicle—based mechanism, or with both. Here, we will consider the above comments one by one. The parameters that were used for the modeling were not described in detail in the main text to enhance the readability of the manuscript.
Nevertheless, detailed descriptions of the necessary parameters are in the figure legends, and are now clearly referred to in the main text, and the scripts used for the modeling are also provided as supplementary material. Additionally, the technical aspects of the modeling and the assumptions made are now described in detail in the Methods section. The assumptions of the model are recapitulated briefly below:.
As detailed above, the use of modeling is restricted to testing which of the two models diffusion based or vesicle transport based can be reconciled with the experimental data. To this end we judge that a reader will be unable to grasp the significance of these models without first providing the appropriate experimental background. Thus we have followed the logical progression of first describing the experimental data an upper bound of 2 minutes for albumin equilibration through the stack , then building computational models of the two competing hypothesis transport based on diffusion of cargo or transport mediated by vesicles and finally concluding that only the diffusion based model is compatible with the experimental data.
In the revised version, in order to better integrate the models, while describing them we make reference to the original data on which the parameters of the models are based on, and also clarify the logical flow that was adopted.
For the purpose of this study, the models aim only to assess whether the equilibration rates of albumin across the stack are compatible with the continuity— or the vesicle—based model, or with both. In the revised version, we have clarified the limitations of the models and the logical flow that was adopted. Regarding VSVG, information that is not often appreciated but which emerges from the literature, and that has been briefly discussed in our manuscript, is that VSVG can behave in two different modes: it can equilibrate rapidly through the stack like albumin or it can behave like PC, depending on the synchronization conditions.
We have modified and extended these comments in the Discussion section. We agree with the reviewers that diffusion in 3D generally differs from diffusion in 2D. In the present situation, however, the 2D assumption perfectly matches the biological system.
We have additionally performed the same comparison for a system with large cisternae cisternae diameter of nm and keeping the other parameters same as before:. The results for the two system sizes are therefore very similar. We believe this justifies our simplification to the 2D case as used in the manuscript. This is now mentioned in the Methods section of the revised manuscript. We now state:. To address this point, we refer the reviewers to the new Figure 3—figure supplement 2 where we now show biochemical pulse-chase analysis of antitrypsin and VSVG in HepG2 cells under conditions similar to that used in Figure 1 transport assay monitored by EM.
As noted in the text, the transport kinetics of antitrypsin are indistinguishable from those of albumin. The conditions of immunoprecipitation were such that the proteins were completely depleted.
One can notice that the amounts of antitrypsin and VSVG are similar in terms of radioactive counts or here intensity of the protein bands , suggesting that the antitrypsin is not present in much higher levels than VSVG.
Therefore, the difference between their kinetic behaviors is not due to the different abundance of VSVG and antitrypsin and, by extension, of albumin.
We have now included the following phrase in the legend to Figure 3—figure supplement 2 : 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. How do they get there?
There are three main destinations for biochemicals released from the trans Golgi network: 1 inside the cell to the lysosomes; 2 the plasma membrane and 3 outside of the cell. In each case the destination is clearly linked to function. Using the food supermarket analogy, all the biochemicals transported away from the trans Golgi network have labels and barcodes built into them.
They are all packed in vesicles and the construction of the vesicle or vessel is largely related to the vesicle contents, its destination and end use. Animal cells contain many lysosomes and it is in these structures that some life expired organelles and other materials are digested see item CU9 about lysosomes.
Vesicles containing biochemicals for continuous secretion flow to and fuse with the plasma membrane. This group of secretions will contribute to the biochemicals of the extracellular matrix, act as chemical signals to other cells, and provide proteins for the repair and replacement of the plasma membrane.
This constitutive or continuous secretory pathway is also the default pathway. Products from the Golgi apparatus not labelled for other routes use this line. They move from the trans Golgi network TGN towards the plasma membrane but accumulate in number before reaching the membrane. Certain triggers will make the vesicles fuse with the plasma membrane and release their contents in regulated bursts from the cell surface.
Insulin release is an example of this when it is triggered by a rise in blood glucose level. Food intake is similar in that it triggers the release of mucus and digestive enzymes into the alimentary canal. These fragments are divided more or less evenly between the daughter cells. A new Golgi apparatus can only grow from a fragment of Golgi apparatus from the previous cell, so there is therefore the potential for a new Golgi apparatus to grow from each small fragment. However, if there are no fragments there will be no Golgi apparatus.
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