COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generate partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.DOI:http://dx.doi.org/10.7554/eLife.13232.001 Proteins play many important roles for cells, and these roles often require the proteins to be in particular locations in or around the cells. A set of cell compartments called the Golgi packages certain proteins into bubble-like structures called vesicles to enable the proteins to be used elsewhere in the cell or released to the outside of the cell, in a process called the secretory pathway. The operation of the secretory pathway requires the Golgi compartments to be continually remodeled.Proteins and other materials can be ferried between the compartments of the Golgi by another type of vesicle. These vesicles are coated with a group, or complex, of proteins called COPI, which forms a curved lattice around the vesicles and helps them to capture the materials they will transport. However, it is not clear whether COPI is also involved in remodeling of the Golgi compartments.Papanikou, Day et al. addressed this question using a technique called the “anchor-away method” combined with microscopy to study COPI in yeast cells. The yeast were genetically engineered so that COPI activity was effectively shut down in the presence of a drug called rapamycin. The experiments show that COPI is involved in the early stages of remodeling the Golgi compartments, but not the later stages. This finding supports the emerging view of the Golgi as a self-organizing cellular machine, and it provides a framework for uncovering the engineering principles that underlie the secretory pathway.DOI:http://dx.doi.org/10.7554/eLife.13232.002 Some trCOPI also plays a role in intra-Golgi traffic, but the evidence is open to multiple interpretations. Initially the proposal was that COPI vesicles carry secretory cargoes forward from one Golgi cisterna to the next in a “vesicle shuttle” . After tcis face of the Golgi, then move through the stack to the trans face, then finally peel off to become secretory vesicles  compartment, COPI vesicles bud to remove resident Golgi proteins, thereby helping to drive the Golgi-to-TGN biochemical conversion  strain showed a pronounced growth defect . T2098L The next step was to test whether growth was inhibited when Sec21 was anchored away. Rpl13A-FKBPx2 served as an anchor on ribosomes. When this anchor was present in a rapamycin-resistant strain encoding wild-type Sec21, rapamycin addition had no effect on growth, as expected . By contA similar analysis was performed using the mitochondrial outer membrane protein OM45  as an anTo test this hypothesis, we added a double FRB tag to Sec21 while increasing the number of FKBP domains on OM45 to four. These changes were predicted to increase the functional affinity of the anchoring interaction at mitochondria. Indeed, with a strain expressing OM45-FKBPx4 and Sec21-FRBx2, rapamycin completely blocked growth . We alsoPichia pastoris  of the ER protein Sec71 fused to GFP . Sec71TMas faint . The rerreported , althougrer1△ strain  mutant is not. With regard to anchors, our initial trials employed an FKBPx2-tagged version of the plasma membrane protein Pma1 , but strIt should be noted that “anchor-away” is something of a misnomer here because the anchored COPI remained associated with Golgi membranes. Thus, when OM45-FKBPx4 was used as the anchor, entire Golgi compartments apparently became tethered to mitochondria. Such an effect is not unexpected because Golgi cisternae are mobile in the yeast cytoplasm . Despitesec21-3 cells at 37°C were the α-factor and carboxypeptidase Y precursors  to select for double-crossover replacement of FPR1. A similar method was used to delete the RER1 gene by replacing it with a hygromycin resistance cassette from pAG32 .DNA manipulations were simulated and recorded using SnapGene software . Annotated sequence files for 34 of the plasmids used in this study are included as a zip archive  were used to colorize and merge the images, adjust brightness, and create average projections. Correction for exponential photobleaching was performed with an ImageJ plugin (http://cmci.embl.de/downloads/bleach_corrector). Editing and quantitation of 4D data sets was performed using custom plugins for ImageJ. These plugins allowed a hyperstack to be hybrid median filtered, converted to a montage time series, edited to remove extraneous fluorescence signals, converted back to a hyperstack, and quantified to measure red and green fluorescence intensities. A zip archive Some of the static confocal images and all of the 4D confocal data were deconvolved using Huygens software . For the strain expressing GFP-Vrg4 and Kex2-mCherry, a single pass with a 2D hybrid median filter  was perf600 = 0.5–0.8) in SD medium. Prior to labeling, the cells were collected on a bottle-top filter, washed with SD lacking methionine (SD – Met), then resuspended in SD – Met at a concentration of 5 OD600 units/mL. The concentrated cells were incubated for 30 min. For pulse labeling, 25 μCi of TRAN35S-LABEL  was added per OD600 unit of cells, and the cells were incubated for 10 min. A 30-min chase was initiated by adding a 10x solution to give a final concentration of 5 mM unlabeled methionine plus 2 mM unlabeled cysteine. All of these manipulations were carried out with constant aeration at 30°C, except in the case of Cells were grown to log phase . This mixture was incubated for 5 min at 60°C. TCA-precipitated material was collected by centrifugation for 5 min at full speed  in a microcentrifuge, then solubilized by vigorous vortexing in 50 μL of SDS-PAGE sample buffer supplemented with 0.1 M dithiothreitol and 50 mM Na+ PIPES, pH 7.5. The sample was incubated for 30 min at 60°C, followed by a 3-min spin at full speed in a microcentrifuge to remove insoluble material.To analyze cellular proteins, the cell pellet was resuspended in the original volume of medium, and then TCA was added to a final concentration of 10%. This mixture was incubated for 5 min at 50°C followed by 5 min on ice. TCA-precipated material was collected by centrifugation for 5 min at 3000 rpm (1000xg) in a microcentrifuge, then resuspended in SDS-PAGE sample buffer supplemented with 0.1 M dithiothreitol and 50 mM Na600 units of cells. SDS-PAGE was performed with Mini-PROTEAN TGX Tris/glycine 4–20% gradient gels using the Precision Plus Protein Dual Color Standards molecular weight markers . Gels were dried, and radioactive signals were detected using a Storm 860 molcular Imager .Each gel lane was loaded with a sample of secreted or cellular proteins corresponding to 0.25 ODA 100-mL culture of untreated or rapamycin-treated yeast cells was grown in SD to mid-log phase at 30°C with shaking. Cells were then concentrated by vacuum filtration using a 0.22-μm bottle-top filter . The cell paste was transferred to planchettes , cryo-fixed using a Bal-Tec HPM 010 high-pressure freezing machine , and placed immediately into cryo-tubes containing a frozen cocktail of 0.1% uranyl acetate in anhydrous acetone. Samples were freeze substituted at -80°C for 48–60 hr in an EM AFS2 freeze substitution device (Leica). The temperature was then raised to -50°C and the samples were washed three times with acetone, followed by successive increasing overnight infiltrations with Lowicryl K4M resin , followed by three incubations of 1 hr each with 100% resin. Infiltrated samples were placed in molds and polymerized with ultraviolet light at -50°C for 13 hr. To preserve fluorescence, the plastic blocks were stored at -20°C prior to sectioning. Sections of 300–1500 nm were cut with an EM UC6 ultramicrotome (Leica) and placed on 200 mesh carbon-formvar coated London-Finder copper grids .+-HEPES, pH 7.5 was inverted onto the grid. The coverslip was immediately sealed with wax. Imaging was performed with an LSM 710 confocal microscope. Dual-color Z-stacks were captured with a 1.4-NA 100x Plan-Apo oil objective using a pinhole of 1.2 Airy units and with voxels ranging in each dimension from 0.30 to 0.43 µm. The grid was then retrieved for analysis by electron microscopy.For fluorescence microscopy, a grid was placed on a glass slide with the resin side up, and a 22x22 mm No. 1.5 glass coverslip with a 10-μl droplet of 500 mM NaFor transmission electron microscopy (TEM) as well as TEM tomography and scanning transmission electron microscopy (STEM) tomography, images were collected on a Tecnai G2 F30 electron microscope  with a Schottky field-emission gun operating at 300 kV. Grids were prepared for either TEM or STEM tomography by floating each side of the grid for 10 min on a 10-μl drop of 15 nm colloidal gold bead solution . The samples were then stained for 8–15 min with 2% uranyl acetate, and placed into a Model 2040 dual-axis tomography holder . For TEM tomography, 300–400 nm sections were analyzed using Serial EM  to colle review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the decision was reached after discussions between the reviewers. Based on our discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife. While the reviewers lauded the high quality of your study, the consensus view was that the advances that it presents do not carry the significance required to merit publication in eLife. In addition, as described in the comments from Reviewer 3 (point 2), your argument that Kex2 recycles within the Golgi, rather than from the endosome to the Golgi, is undermined by a published report . The reviews are included in their entirety below.Thank you for choosing to send your work entitled "COPI selectively drives maturation of the early Golgi" for consideration at Reviewer #1:This is an investigation of the role of COPI in the secretory pathway. The authors have optimized the anchor-away technique as a means to rapidly inactivate COPI function in yeast cells in order to assess the consequences to the maturation of Golgi cisternae and to secretion. They report that the transition from an early cisterna, marked by GFP-Vrg4, to a late cisterna, marked by Sec7-GFP, fails upon inactivation of COPI. In contrast, a maturation event monitored by Kex2-GFP dynamics is not affected, leading the authors to conclude that COPI is required solely for recycling of early Golgi proteins.This is a carefully executed, nicely presented study and I don't have any serious technical concerns, as much of the paper is an extensive validation of the anchor away approach. The significance of the major points of the work, however, is lessened by several factors.1) Prior reports  implicated a role for COPI in Golgi localization of multiple early/medial Golgi residents in yeast cells via recycling through the ER. Although this paper more directly visualizes a role for COPI for recycling of an early Golgi resident (Vrg4), the advance presented here is modest and questions regarding the role of COPI recycling pathway remain to be addressed. Does COPI direct Vrg4 to the ER, or to an earlier Golgi compartment? Related, Rizzo et al. (2013) provided evidence that an artificial Golgi resident based on mannosidase II (MAN-FM) recycles via COPI vesicles/tubules from the trans Golgi cisterna of HeLa cells. Is Vrg4 recognized by COPI?2) As the authors point out, it has been established that multiple late Golgi proteins (including Kex2) rely on AP-1/clathrin to maintain Golgi residence, and that COPI localizes predominantly to early Golgi compartments, so the finding that COPI inactivation does not prevent removal of Kex2 from the hybrid Golgi compartment is not unexpected. Further, I don't find the argument that Kex2 recycles within the Golgi (rather than from endosomes to the Golgi) to be sufficiently substantiated.3) This work confirms, and modestly elaborates, that secretion of some proteins continues in COPI temperature sensitive mutants , which was shown many years ago (as the authors point out).Reviewer #2:In this study, the authors test the effects of functional removal of the COPI machinery on protein secretion and cisternal maturation in yeast. Previous studies with thermosensitive COPI mutants had indicated that COPI ablation inhibits the secretion of certain proteins, but not of others, and that it slows down, but does not block, cisternal maturation.They now use a potentially 'cleaner' and more radical approach based on anchoring away specific COPI subunits to revisit the same issues. Using this technique, they find that secretion is strongly inhibited, although to different extents for different proteins, and that the maturation process and the organization of the Golgi are markedly altered. In control cells, early cisternae lose their residents to newly forming cis elements and simultaneously acquire trans Golgi markers, which they later loose, presumably during formation of secretory post Golgi carriers. In cells where COPI is inactivated by the anchoring away approach, early and late Golgi residents merge  into a hybrid compartment, where they behave differently: the cis residents become unable to cycle and remain stably in this compartment  while trans residents can still cycle on and off the hybrid Golgi body though with delayed kinetics (suggesting that they cycle via a COPI- independent mechanism).The authors interpret these data in the framework of the cisternal maturation scheme: because of the loss of COPI, the maturation process is inhibited at the cis side and cis-cisternae are 'stabilized'; they can thus serve as 'acceptors' of the trans residents, resulting in a hybrid Golgi. This hybrid compartment can still absorb input from the ER and release secretory material towards the surface, albeit at slow rates.What are the new and important statements made by this study? One is that COPI is essential for recycling of cis residents. While the role of COPI in recycling from the Golgi to the ER was a relatively well established fact, this was not the case for the intra-Golgi stage. The other is that the cycling of trans Golgi residents is COPI-independent, and this represents a surprise. The mechanism of cisternal maturation is obviously an important issue in membrane biology and these findings represent a potentially significant advance.The study is technically very thorough and the factual statements are convincing and well supported. We feel, however, that it has limitations, in that it leaves some essential question open and inconsistencies unresolved. Certain key issues should be addressed by suitable arguments and/or by additional experiments.The main problem relates to the difference between the previous results based on thermosensitive mutants and the present ones based on the anchoring away approach. The anchoring method, as noted by the authors, results in the adhesion of the whole Golgi cisternae to the mitochondria, rather than in the 'removal' of the COPI machinery from Golgi cisternae. The results of this are hard to predict and may not always be congruent with the specific inactivation of the concerned proteins. It is conceivable that the strong block in recycling of the cis-Golgi residents might be due to extensive proximity/adhesion between Golgi and mitochondrial membrane, rather than to a specific block of the COPI function. This possibility undermines the author's central conclusions about the role of COPI in the recycling of cis residents and should be appropriately discussed or addressed experimentally. Some relevant questions and considerations and some possible approaches are suggested below:1) A re-examination of the ultrastructure of the Golgi-mitochondria contact due to anchoring might help. How extensive are the contacting surfaces? Does the morphology of these contacts justify the above doubts about the effects of anchoring on recycling?2) Could the anchoring of the Golgi membranes to mitochondria through a COP-unrelated cis-Golgi protein reproduce the recycling block?3) The authors obtain similar results by anchoring COPI to ribosomes, which might be less likely than mitochondria to severely obstruct membrane exchanges at the Golgi surface. Exploiting and characterizing the ribosome data at the ultrastructural level might help resolve the issue.4) Another approach might be to consider that while early Golgi markers are blocked, late markers keep cycling. How are they disposed with respect to the contacting Golgi and mitochondrial surfaces?Reviewer #3:This study from the Glick lab uses the anchor-away technique to inactivate the COPI complex in order to examine its role in Golgi maturation. The authors begin with a thorough analysis of the anchor-away method for inactivating COPI proteins. The analysis is aided by comparisons to anchor-away inactivation of the COPII complex.The most surprising finding is that secretion is not completely blocked when COPI is inactivated. Anchoring-away different COPI subunits results in similar secretion phenotype. The secretion phenotype also appears identical when comparing COPI inactivation by anchor-away versus ts-mutant (sec21-3). The authors show that anchor-away is preferable to thermosensitivity because at permissive temperature the sec21-3 mutant appears compromised (Golgi morphology defects), meaning that indirect effects may be observed when using this mutant.Anchor-away is used to show that Golgi dynamics are dramatically altered upon inactivation of COPI, although post-Golgi recycling appears to still occur. In wild-type cells, it is shown that the Kex2 transmembrane protein, a cargo that cycles between the Golgi and endosomes, normally appears at Golgi compartments after the peak of the Vrg4 transmembrane protein, a marker for early Golgi compartments. Kex2 remains present while Sec7 appears at that compartment, and Kex2 then leaves the compartment before the Sec7 signal completely disappears. However, in COPI-inactivated cells there are two major differences: First, the morphology of the compartments are quite different, with early and late markers showing partial colocalization to larger "hybrid" compartments. Second, in mutant cells maturation is altered so that compartments labeled with an early marker (Vrg4) persist for a longer time while later markers/cargos (Sec7 and Kex2) appear to cycle in and out of the "hyrbrid" compartment.eLife. However, I have a couple of major concerns that need to be addressed before I would consider the manuscript to be suitable for publication:Given these results, the authors conclude that COPI is important for maturation and recycling of the early Golgi, but dispensable for late Golgi function. I agree that this overall conclusion is generally supported by the data, and I think this is a very important result that warrants publication in a journal like 1) One potential concern is that the anchored-away COPI proteins are still associated with the Golgi. Although cell growth is blocked by anchoring COPI, given the resolution of the growth curve data it is a formal possibility that partial COPI activity remains, and growth may not halt until after 1 or 2 more cell divisions. This might affect the interpretation of the data , if partial COPI activity remains. The authors imply that because the sec21-3 mutant displays an identical secretion phenotype to the anchor-away COPI phenotype, it is likely that both methods result in complete inactivation of COPI. However, I think the authors need to demonstrate that the Golgi-ER COPI trafficking pathway is completely blocked by anchor-away inactivation of COPI. There are various "retrieval assays" in the literature. Another option would be to test by microscopy whether a protein that normally cycles between the ER and Golgi (i.e. a p24 protein or other cycling ER cargo receptor) is now exclusively found in the Golgi.2) In the second half of the Discussion, the authors propose that AP-1 traffics Kex2 directly from late Golgi to early Golgi compartments. This would be a fairly radical idea, and would upend the current model that AP-1 traffics Kex2 from the Golgi to endosomes, and then Kex2 is retrieved from endosomes back to the Golgi by other machinery. Yet the only data provided specifically in support of this new model is the statement "our video microscopy revealed that most and perhaps all of the Kex2-labeled compartments acquire Sec7, suggesting that Kex2 recycles from older to younger cisternae within the Golgi". I disagree with both the data and interpretations contained in this statement. First, by watching their videos it appears that there are a few smaller Kex2 structures that may disappear without acquiring Sec7. Second, if we assume the first part of the statement is correct, this observation is consistent with Kex2 being retrieved from endosomes (where it may reside for only a few seconds), via vesicles, to Golgi compartments that have not yet acquired Sec7. To my knowledge, the precise Golgi compartment that fuses with endosomally-derived vesicles has not been firmly established in yeast, but the Nakano lab found that Ypt6 precedes Sec7 at the Golgi (PNAS 2013). As Ypt6 (together with the GARP complex) is primarily responsible for tethering/fusing vesicles from endosomes, this suggests Kex2 retrieval from endosomes to the Golgi occurs at a stage of Golgi maturation just prior to acquisition of Sec7. Therefore, it seems unfounded to propose a role for AP-1 in recycling cargos from late Golgi to early Golgi compartments.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your revised article has been favorably evaluated by Randy Schekman (Senior editor) and three reviewers, one of whom is a Member of our Board of Reviewing Editors. The manuscript has been improved but there are some remaining minor issues that the reviewers feel should be addressed before acceptance, as outlined below:Thank you for resubmitting your work entitled "COPI selectively drives maturation of the early Golgi" for further consideration at The reviewers recommend that you address the following points with revisions to the text:1) Reviewer 1 and Reviewer 2 raise the issue that the anchor away approach that you employed results in different outcomes for Golgi residents when compared to the sec21-3 mutant and a Vps74/Golph3 mutant. They feel that this should be directly addressed in the Discussion. The comments from Reviewer 1 and 2 (below) elaborate this point.2) Since the arrested cisterna ('hybrid compartment') appears to remain competent to receive ER-derived material, and to export material, a brief discussion of the implications of this with regard to interpreting the results from both a cisternal maturation mechanism and a stable compartment mechanism of transport should be included in the Discussion. The comments from Reviewer 2 are most relevant for this point.3) Can the authors clarify in the text which of the studies cited in the Discussion (tenth paragraph) demonstrated that AP-1 was involved in Kex2 trafficking and describe the Kex2-localization phenotype(s) seen previously by others in AP-1 mutant strains?The entire reviews from each of the referees are included below to help you appreciate their suggestions. Addressing any other points raised in the reviews is left to your discretion.Reviewer #1:In light of the observation that the anchor away approach results in entire COPI-coated cisternae to be associated with mitochondria, a major point of discussion amongst the reviewers during review of the earlier version of the manuscript regarded the question, does the technical approach (anchor away) impinge on cisterna function independently of COPI? It is puzzling to me that in the sec21-3 mutant (permissive temperature) GFP-Vrg4 transits the Golgi and is sorted to the vacuole, but it remains in place (in the hybrid compartment) in anchor away cells. The reason may lie in incomplete inactivation of COPI in the sec21-3 mutant (the figure shows permissive temperature), but difference in outcome is striking and begs resolution. The only way that I can think of that might meaningfully address this would be to compare results with 'inert' anchor away targets. What, for example, happens when if FRB is appended to a Golgi resident , so that the cisterna is addressed to the mitochondrion, but COPI is not manipulated? Despite this shortcoming, I think that in its present form the manuscript makes an important contribution to the field, even though the message, as summarized in the title, may be open to continuing debate.Reviewer #2:In this study, the authors test the effects of inactivating the COPI complex on cisternal maturation and protein secretion in yeast. Previous studies with thermosensitive COPI mutants had indicated that COPI inactivation inhibits the secretion of many but not all proteins (Gaynor and Emr), and that it slows down, but does not block, cisternal maturation as visualized by live microscopy (Matsuura-Tokita and Nakano).The authors attribute these inconclusive results to the fact that the available ts mutants can be either still partially active (Matsuura-Tokita and Nakano) or, when fully inactive, can markedly perturb the morphology of the Golgi (this manuscript), making it impossible to monitor directly the maturation process by live microscopy.To resolve these uncertainties, they use the anchoring-away technique applied to specific COPI subunits. They find that COPI inactivation results in complete block of the recycling of the cis Golgi marker Vrg4, which now permanently labels the cis cisternae. This indicates that Vrg4 normally recycles through COPI. Moreover, they find that trans-Golgi residents (Sec7 and Kex2) partially merge with these stabilized cis cisternae, forming hybrid cis/trans Golgi bodies; and also that these trans markers cycle on and off the hybrid structures (though with somewhat delayed kinetics). This suggests that these markers cycle via a COPI-independent mechanism.The study is technically very thorough and the factual statements are convincing and well supported. My comments will therefore concern only the novelty and the significance of the results.eLife. They also raise the following questions and comments.As the authors emphasize, their data are the first direct demonstration that COPI is required for cisternal maturation. Evidence that COPI is required for maintaining the localization of Golgi residents (via Vps74/Golph3) has been available for some time , but these previous observations are compatible both with the stable cisternae model and the cisternal maturation model. Thus, while not unexpected, these results are an important step towards establishing the molecular mechanism of cisternal maturation, and in principle deserve publication in a major journal such as The stabilization of cis Golgi markers in cis cisternae probably results from the specific type of COPI inactivation that is induced by the anchoring-away approach, which causes COPI to remain bound to the cis Golgi and so possibly to retain cis markers in the cis cisterna. However, the stabilization of the cis Golgi it is not the only possible outcome of COPI inactivation, under the maturation model. One might imagine, for instance, that if COPI were inactivated in a different way, e.g., by rapid degradation, this might cause cis markers to move forward along with cargo proteins to reach the vacuole or the plasma membrane. This is actually what happens when the COPI-bound adaptor Vps74 is removed: the Golgi enzymes leave the Golgi and move to the vacuole (Tu-Banfield). This apparent discrepancy might disconcert the reader. I therefore feel that the above point should be discussed, even though it does not in my view undermine the conclusion that COPI is needed for cis Golgi maturation.The authors appear to interpret the data in the framework of the cisternal maturation scheme. However, the observation that the hybrid compartment can still absorb input from the ER and release secretory material towards the surface, albeit at slow rates, is difficult to reconcile with the maturation model. Since the recycling of cis markers is inhibited, and the formation of a new cis cisterna  requires such recycling, a logical suggestion deriving from these data would be that the hybrid Golgi incorporates cargo and membrane from the ER by a mechanism different from cisternal maturation. In my view, these results are consistent with the idea that the main intra-Golgi transport mechanism is cisternal maturation, but they also suggest the existence of secondary mechanisms that are used by some protein classes, and/or of backup mechanisms used by the system when maturation fails. Again, I feel that, in order to avoid confusing the reader in a field that is already rich in controversies, the authors should discuss these issues, which, incidentally, are referred to also in the Introduction.As noted, we do not have comments worthy of mention regarding the (high) technical quality of the figures and in general of the data.Reviewer #3:This work convincingly clarifies the role of COPI, providing further evidence for a primary function in retrograde trafficking that is important for Golgi maturation. I think the authors now do a good job of explaining why some cargos are more sensitive to COPI inactivation than others, and how a basal level of secretion can be supported in the absence of COPI function. Furthermore, they have clarified the itinerary of Kex2. Therefore, I am in support of publication.My only remaining concern is a relatively minor one, which I would expect could be addressed through small changes in the text: Can the authors clarify in the text which of the studies cited in the Discussion (tenth paragraph) demonstrated that AP-1 was involved in Kex2 trafficking and describe the Kex2-localization phenotype(s) seen previously by others in AP-1 mutant strains? In other words, what is the existing evidence in the literature that led to the model of AP-1 functioning in Kex2 traffic between the Golgi and endosomes? Furthermore, can the authors briefly discuss the established role of AP-1 in mammalian cells , and whether the authors think AP-1 has similar or different functions in yeast? I think explicitly addressing these points will help readers to better understand the insights from the current data in light of previous studies. [Editors’ note: the author responses to the previous round of peer review follow.]An issue with the initial submission was that we got ahead of ourselves by building the interpretations on additional data from ongoing work. The COPI results have inspired us to explore the relationship between endosomes and the Golgi, and we have obtained evidence that yeast cells lack an early endosome compartment that is distinct from the TGN. That story is still coming together, but last year we published a crucial result by showing that compartments previously termed early endosomes are actually identical to the Sec7-labeled TGN . As described below, an implication is that many late Golgi proteins likely recycle within the Golgi. This unconventional idea is justified more thoroughly in the revised manuscript and is presented as a working hypothesis.The revision also includes two new data figures. First, as suggested by a reviewer, we confirm that our COPI inactivation protocol inhibits Golgi-to-ER recycling. Second, because our inferences about Kex2 trafficking were a concern for the reviewers, we quantitatively characterize Kex2 localization and dynamics. The major contributions in the revised manuscript are as follows:This advance answers a central and long-standing question in the field.• We use 4D microscopy of GFP-Vrg4 to provide clear evidence that Golgi maturation requires COPI. Yeast 4D microscopy is the only method available for directly visualizing cisternal maturation. • We show that when COPI is functionally inactivated, the yeast Golgi reorganizes into hybrid structures that remain dynamic with respect to the cycling of late Golgi components. This result can explain the puzzling earlier observation, reproduced and extended here, that the yeast secretory pathway remains partly operational in the absence of COPI function.• We provide a carefully controlled analysis of the anchor-away method, which has attracted considerable interest. Our data document the effects of various parameters as well as strengths and limitations of the method.• We provide evidence that Kex2 cycles within the Golgi rather than between endosomes and the Golgi as is generally assumed. Although this conclusion will benefit from further validation, the present results establish Kex2 as a model protein for studying COPI- independent intra-Golgi recycling.Reviewer #1:[…] This is a carefully executed, nicely presented study and I don't have any serious technical concerns, as much of the paper is an extensive validation of the anchor away approach. The significance of the major points of the work, however, is lessened by several factors.1) Prior reports  implicated a role for COPI in Golgi localization of multiple early/medial Golgi residents in yeast cells via recycling through the ER. Although this paper more directly visualizes a role for COPI for recycling of an early Golgi resident (Vrg4), the advance presented here is modest and questions regarding the role of COPI recycling pathway remain to be addressed. Does COPI direct Vrg4 to the ER, or to an earlier Golgi compartment? Related, Rizzo et al. (2013) provided evidence that an artificial Golgi resident based on mannosidase II (MAN-FM) recycles via COPI vesicles/tubules from the trans Golgi cisterna of HeLa cells. Is Vrg4 recognized by COPI?The papers mentioned by the reviewer are relevant and we now cite them in the Introduction. But while a role for COPI in the recycling of resident Golgi proteins has some experimental support, the COPI data from both yeast and mammalian studies have been confusing. For example:• Yeast COPI subunits were originally implicated in anterograde ER-to-Golgi transport. That interpretation is no longer favoured, probably because the early papers reported indirect effects.trans-Golgi cisternae as predicted by the maturation model, but a 2013 paper by Lavieu et al. (published in eLife) concluded that mammalian Golgi cisternae do not mature. The discrepancies between those parallel studies have yet to be resolved.• Rizzo et al. did indeed provide evidence in 2013 that a resident Golgi protein could recycle from mammalian • Gaynor and Emr (1997) reported that COPI is dispensable for the secretion of some yeast proteins. To our minds, that paper has long been the most baffling in the field, because the experimental data were strong and yet the conclusion was seemingly at odds with all extant models for Golgi traffic.Video microscopy of the yeast Golgi is currently the only direct assay for cisternal maturation, and our results with GFP-Vrg4 are the first direct evidence that maturation requires COPI. We believe that this advance is more than modest. Others in the field apparently agree that this sort of analysis is crucial because the Nakano lab is also trying to characterize how COPI inactivation affects Golgi dynamics.Finally, an interaction of Vrg4 with COPI was nicely shown by Abe et al. (2004), and that evidence was one of our motivations for using Vrg4 as an early Golgi marker. The reviewer points out that we cannot say with certainty that Vrg4 recycles within the Golgi rather than returning to the ER. However, when we inactivated COPII by the anchor-away method, GFP- Vrg4 showed no ER accumulation see , implyin2) As the authors point out, it has been established that multiple late Golgi proteins (including Kex2) rely on AP-1/clathrin to maintain Golgi residence, and that COPI localizes predominantly to early Golgi compartments, so the finding that COPI inactivation does not prevent removal of Kex2 from the hybrid Golgi compartment is not unexpected. Further, I don't find the argument that Kex2 recycles within the Golgi (rather than from endosomes to the Golgi) to be sufficiently substantiated.We got ahead of ourselves in the initial submission by inadequately justifying the proposal that Kex2 recycles within the Golgi. That limitation has been overcome with the new The COPI results inspired us to consider the possibility that late Golgi membrane proteins undergo intra-Golgi recycling. That idea has received surprisingly little attention. While pursuing it, we obtained evidence that yeast cells lack an early endosome compartment that is distinct from the TGN. Although that story is still coming together, last year we published a crucial result by showing that compartments previously termed early endosomes, as judged by labeling with newly internalized FM 4-64, are actually identical to the Sec7-labeled TGN .If we provisionally accept that there is no yeast early endosome distinct from the TGN, then a simple argument can be made:• AP-1 has been shown to mediate recycling of a subset of late Golgi membrane proteins in yeast;• AP-1 has been shown to localize to the TGN. Our ongoing work confirms data from the Payne lab indicating that AP-1 is found on Sec7-labeled compartments;• The implication is that AP-1 mediates recycling of a subset of late Golgi proteins from the TGN to earlier Golgi compartments.In light of this working hypothesis and the data presented here, a reasonable supposition is that Kex2 undergoes AP-1-dependent and COPI-independent intra-Golgi recycling. We are excited about this unconventional model, which is shown in the new 3) This work confirms, and modestly elaborates, that secretion of some proteins continues in COPI temperature sensitive mutants , which was shown many years ago (as the authors point out).sec21-3 mutation was unable to block COPI function completely. But after optimizing the anchor-away method, we concluded that yeast can indeed secrete without functional COPI. That result urgently needed an explanation.The reason for our emphasis on the Gaynor and Emr (1997) paper is that it was the most careful prior analysis of the role of yeast COPI in Golgi traffic, and it was apparently inconsistent with cisternal maturation. At first we suspected that even the strong Our data now provide a plausible explanation. When yeast COPI is inactivated, the Golgi forms a hybrid structure that can presumably continue to receive input from the ER, and late Golgi components of this structure continue to cycle in a manner consistent with ongoing secretory activity. We have therefore addressed the primary argument against an involvement of COPI in yeast Golgi maturation.Reviewer #2:[…] The study is technically very thorough and the factual statements are convincing and well supported. We feel, however, that it has limitations, in that it leaves some essential question open and inconsistencies unresolved. Certain key issues should be addressed by suitable arguments and/ or by additional experiments.The main problem relates to the difference between the previous results based on thermosensitive mutants and the present ones based on the anchoring away approach. The anchoring method, as noted by the authors, results in the adhesion of the whole Golgi cisternae to the mitochondria, rather than in the 'removal' of the COPI machinery from Golgi cisternae. The results of this are hard to predict and may not always be congruent with the specific inactivation of the concerned proteins. It is conceivable that the strong block in recycling of the cis-Golgi residents might be due to extensive proximity/adhesion between Golgi and mitochondrial membrane, rather than to a specific block of the COPI function. This possibility undermines the author's central conclusions about the role of COPI in the recycling of cis residents and should be appropriately discussed or addressed experimentally. Some relevant questions and considerations and some possible approaches are suggested below:1) A re-examination of the ultrastructure of the Golgi-mitochondria contact due to anchoring might help. How extensive are the contacting surfaces? Does the morphology of these contacts justify the above doubts about the effects of anchoring on recycling?2) Could the anchoring of the Golgi membranes to mitochondria through a COP-unrelated cis-Golgi protein reproduce the recycling block?3) The authors obtain similar results by anchoring COPI to ribosomes, which might be less likely than mitochondria to severely obstruct membrane exchanges at the Golgi surface. Exploiting and characterizing the ribosome data at the ultrastructural level might help resolve the issue.4) Another approach might be to consider that while early Golgi markers are blocked, late markers keep cycling. How are they disposed with respect to the contacting Golgi and mitochondrial surfaces?S. cerevisiae are mobile, and nothing should prevent anchored COPI from interacting with and capturing mobile cisternae. Fortunately, all of our data, including the new We were not overjoyed to learn that when COPI was anchored to mitochondria, the Golgi cisternae were anchored as well. This experiment arguably falls in the “one control too many” category. Yet the Golgi-mitochondria association is not surprising in hindsight because Golgi cisternae in We believe that the concerns and suggestions from the reviewer are addressed by the ribosomal anchoring data. If mitochondrial anchoring had specific effects, then ribosomal anchoring should have different effects, but we saw essentially identical results with the two types of anchor.A more extensive analysis of the Golgi-mitochondria association could be carried out. However, that approach is very labor-intensive, and we are unsure how much more insight would be gained.We hope the reviewer will appreciate that our characterization of COPI anchoring to mitochondria is an attempt to be thorough in exploring the strengths and limitations of the anchor-away method. In the end, our judgment is that this method provides an efficient way to inactivate COPI.Reviewer #3: 1) One potential concern is that the anchored-away COPI proteins are still associated with the Golgi. Although cell growth is blocked by anchoring COPI, given the resolution of the growth curve data it is a formal possibility that partial COPI activity remains, and growth may not halt until after 1 or 2 more cell divisions. This might affect the interpretation of the data , if partial COPI activity remains. The authors imply that because the sec21-3 mutant displays an identical secretion phenotype to the anchor-away COPI phenotype, it is likely that both methods result in complete inactivation of COPI. However, I think the authors need to demonstrate that the Golgi-ER COPI trafficking pathway is completely blocked by anchor-away inactivation of COPI. There are various "retrieval assays" in the literature. Another option would be to test by microscopy whether a protein that normally cycles between the ER and Golgi (i.e. a p24 protein or other cycling ER cargo receptor) is now exclusively found in the Golgi.600 is common for sec mutants at the nonpermissive temperature as the cells become denser, and a similar effect could explain the small increase in OD600 after anchoring COPI.We note that a gradual increase in ODThe reviewer points out that we cannot fully exclude the possibility that COPI retains residual activity. We have done our best by anchoring different COPI subunits alone or in combination, and by varying the rapamycin concentration and the duration of rapamycin treatment. No matter how hard we tried to inactivate COPI, secretion persisted. Those results fit with the data from Gaynor and Emr (1997), who used the strongest available COPI thermosensitive mutants. Ultimately, we concluded that yeast cells can almost certainly continue to secrete at some level in the absence of functional COPI.Given the importance of this point, an additional control is merited. Thanks to the reviewer for the excellent suggestion of examining Golgi-to-ER retrograde traffic. The Sec71TMD-GFP construct from Sato and Nakano proved to be a suitable marker because it localizes to the ER by COPI-dependent recycling of the Rer1 retrieval receptor. In the new 2) In the second half of the Discussion, the authors propose that AP-1 traffics Kex2 directly from late Golgi to early Golgi compartments. This would be a fairly radical idea, and would upend the current model that AP-1 traffics Kex2 from the Golgi to endosomes, and then Kex2 is retrieved from endosomes back to the Golgi by other machinery. Yet the only data provided specifically in support of this new model is the statement "our video microscopy revealed that most and perhaps all of the Kex2-labeled compartments acquire Sec7, suggesting that Kex2 recycles from older to younger cisternae within the Golgi". I disagree with both the data and interpretations contained in this statement. First, by watching their videos it appears that there are a few smaller Kex2 structures that may disappear without acquiring Sec7. Second, if we assume the first part of the statement is correct, this observation is consistent with Kex2 being retrieved from endosomes (where it may reside for only a few seconds), via vesicles, to Golgi compartments that have not yet acquired Sec7. To my knowledge, the precise Golgi compartment that fuses with endosomally-derived vesicles has not been firmly established in yeast, but the Nakano lab found that Ypt6 precedes Sec7 at the Golgi (PNAS 2013). As Ypt6 (together with the GARP complex) is primarily responsible for tethering/fusing vesicles from endosomes, this suggests Kex2 retrieval from endosomes to the Golgi occurs at a stage of Golgi maturation just prior to acquisition of Sec7. Therefore, it seems unfounded to propose a role for AP-1 in recycling cargos from late Golgi to early Golgi compartments.We are indeed proposing a fairly radical idea, and many in the field would likely have responded with similar skepticism to our initial submission. The revised manuscript tackles these issues with additional data and discussion. Our analysis began with the perception that current ideas about Kex2 trafficking are murky:• One model is that Kex2 traffics in a similar manner as the vacuolar hydrolase receptor Vps10, by traveling to prevacuolar endosomes with the aid of Gga1/2 and then recycling to the Golgi. But this idea is puzzling because unlike Vps10, Kex2 has no obvious reason to visit prevacuolar endosomes;• Another model is that Kex2 recycles from an early endosome to the Golgi with the aid of AP-1. But the molecular identity of this putative early endosome is unclear. Moreover, AP-1 localizes to the TGN.Two results from our work support a simpler model in which Kex2 recycles within the Golgi. First, as shown in the new Prompted by the reviewer’s comment, we carefully reexamined Our tests cannot rule out very transient passage of Kex2 through an endosomal compartment, and follow-up work will tackle this question with additional experiments. However, given the data presented here and the relative weakness of the published evidence supporting other views of Kex2 trafficking, we think it is justified to use Kex2 as a model late Golgi transmembrane protein for studying cisternal maturation.If Kex2 and other late Golgi proteins recycle within the Golgi, candidate tethers include Ypt6 effectors. GARP functions in endosome-to-Golgi traffic but could also function in intra-Golgi recycling. Importantly, another Ypt6 effector is Sgm1, and its mammalian homolog is TMF, which was recently implicated in the intra-Golgi recycling of late Golgi proteins . These points are now emphasized in the Discussion.[Editors' note: the author responses to the re-review follow.]The reviewers recommend that you address the following points with revisions to the text:1) Reviewer 1 and reviewer 2 raise the issue that the anchor away approach that you employed results in different outcomes for Golgi residents when compared to the sec21-3 mutant and a Vps74/Golph3 mutant. They feel that this should be directly addressed in the Discussion. The comments from Reviewer 1 and 2 (below) elaborate this point.After prolonged treatment with rapamycin, GFP-Vrg4 shows progressive accumulation at the vacuolar membrane and plasma membrane, as would be expected given the block in recycling. This finding is now stated in the Results section as “data not shown”. Because there is actually no discrepancy between the expected and observed results, we chose not to elaborate in the Discussion.2) Since the arrested cisterna ('hybrid compartment') appears to remain competent to receive ER-derived material, and to export material, a brief discussion of the implications of this with regard to interpreting the results from both a cisternal maturation mechanism and a stable compartment mechanism of transport should be included in the Discussion. The comments from reviewer 2 are most relevant for this point.The last paragraph of the Discussion has been rewritten to address this issue more directly. We propose that Golgi compartmentation and COPI-driven cisternal maturation are aspects of the same phenomenon. Cisternal maturation is only needed, and can only occur, in the context of a compartmentalized Golgi. When Golgi compartmentation is lost, the yeast secretory pathway apparently operates in an alternative mode in which a single hybrid compartment receives COPII vesicles and produces secretory vesicles.3) Can the authors clarify in the text which of the studies cited in the Discussion (tenth paragraph) demonstrated that AP-1 was involved in Kex2 trafficking and describe the Kex2-localization phenotype(s) seen previously by others in AP-1 mutant strains?We had accidentally omitted the Abazeed and Fuller reference, which provides indirect evidence that AP-1 influences the localization of Kex2. That oversight has been corrected, and the discussion of AP-1 has been modified to emphasize that a role for AP-1 in Kex2 recycling is currently speculative.The entire reviews from each of the referees are included below to help you appreciate their suggestions. Addressing any other points raised in the reviews is left to your discretion.Reviewer #1: In light of the observation that the anchor away approach results in entire COPI-coated cisternae to be associated with mitochondria, a major point of discussion amongst the reviewers during review of the earlier version of the manuscript regarded the question, does the technical approach (anchor away) impinge on cisterna function independently of COPI? It is puzzling to me that in the sec21-3 mutant (permissive temperature) GFP-Vrg4 transits the Golgi and is sorted to the vacuole, but it remains in place (in the hybrid compartment) in anchor away cells. The reason may lie in incomplete inactivation of COPI in the sec21-3 mutant (the figure shows permissive temperature), but difference in outcome is striking and begs resolution. The only way that I can think of that might meaningfully address this would be to compare results with 'inert' anchor away targets. What, for example, happens when if FRB is appended to a Golgi resident , so that the cisterna is addressed to the mitochondrion, but COPI is not manipulated? Despite this shortcoming, I think that in its present form the manuscript makes an important contribution to the field, even though the message, as summarized in the title, may be open to continuing debate. As described above, we now state that GFP-Vrg4 accumulates in post-Golgi compartments after prolonged treatment with rapamycin.Reviewer #2:In this study, the authors test the effects of inactivating the COPI complex on cisternal maturation and protein secretion in yeast. Previous studies with thermosensitive COPI mutants had indicated that COPI inactivation inhibits the secretion of many but not all proteins (Gaynor and Emr), and that it slows down, but does not block, cisternal maturation as visualized by live microscopy (Matsuura-Tokita and Nakano).The authors attribute these inconclusive results to the fact that the available ts mutants can be either still partially active (Matsuura-Tokita and Nakano) or, when fully inactive, can markedly perturb the morphology of the Golgi (this manuscript), making it impossible to monitor directly the maturation process by live microscopy.To resolve these uncertainties, they use the anchoring-away technique applied to specific COPI subunits. They find that COPI inactivation results in complete block of the recycling of the cis Golgi marker Vrg4, which now permanently labels the cis cisternae. This indicates that Vrg4 normally recycles through COPI. Moreover, they find that trans-Golgi residents (Sec7 and Kex2) partially merge with these stabilized cis cisternae, forming hybrid cis/trans Golgi bodies; and also that these trans markers cycle on and off the hybrid structures (though with somewhat delayed kinetics). This suggests that that that these markers cycle via a COPI-independent mechanism.The study is technically very thorough and the factual statements are convincing and well supported. My comments will therefore concern only the novelty and the significance of the results.As the authors emphasize, their data are the first direct demonstration that COPI is required for cisternal maturation. Evidence that COPI is required for maintaining the localization of Golgi residents (via Vps74/Golph3) has been available for some time , but these previous observations are compatible both with the stable cisternae model and the cisternal maturation model. Thus, while not unexpected, these results are an important step towards establishing the molecular mechanism of cisternal maturation, and in principle deserve publication in a major journal such as eLife. They also raise the following questions and comments. The stabilization of cis Golgi markers in cis cisternae probably results from the specific type of COPI inactivation that is induced by the anchoring-away approach, which causes COPI to remain bound to the cis Golgi and so possibly to retain cis markers in the cis cisterna. However, the stabilization of the cis Golgi it is not the only possible outcome of COPI inactivation, under the maturation model. One might imagine, for instance, that if COPI were inactivated in a different way, e.g., by rapid degradation, this might cause cis markers to move forward along with cargo proteins to reach the vacuole or the plasma membrane. This is actually what happens when the COPI-bound adaptor Vps74 is removed: the Golgi enzymes leave the Golgi and move to the vacuole (Tu-Banfield). This apparent discrepancy might disconcert the reader. I therefore feel that the above point should be discussed, even though it does not in my view undermine the conclusion that COPI is needed for cis Golgi maturation.This point is important, but as described above, there is actually no discrepancy because anchoring COPI eventually leads to the appearance of GFP-Vrg4 in the vacuolar membrane and plasma membrane.The authors appear to interpret the data in the framework of the cisternal maturation scheme. However, the observation that the hybrid compartment can still absorb input from the ER and release secretory material towards the surface, albeit at slow rates, is difficult to reconcile with the maturation model. Since the recycling of cis markers is inhibited, and the formation of a new cis cisterna  requires such recycling, a logical suggestion deriving from these data would be that the hybrid Golgi incorporates cargo and membrane from the ER by a mechanism different from cisternal maturation. In my view, these results are consistent with the idea that the main intra-Golgi transport mechanism is cisternal maturation, but they also suggest the existence of secondary mechanisms that are used by some protein classes, and/or of backup mechanisms used by the system when maturation fails. Again, I feel that, in order to avoid confusing the reader in a field that is already rich in controversies, the authors should discuss these issues, which, incidentally, are referred to also in the Introduction.The arguments here are somewhat subtle. As described above, we modified the Discussion in an attempt to clarify our ideas about how the Golgi operates in unperturbed cells and in cells with inactivated COPI.Reviewer #3:This work convincingly clarifies the role of COPI, providing further evidence for a primary function in retrograde trafficking that is important for Golgi maturation. I think the authors now do a good job of explaining why some cargos are more sensitive to COPI inactivation than others, and how a basal level of secretion can be supported in the absence of COPI function. Furthermore, they have clarified the itinerary of Kex2. Therefore, I am in support of publication. My only remaining concern is a relatively minor one, which I would expect could be addressed through small changes in the text: Can the authors clarify in the text which of the studies cited in the Discussion on (tenth paragraph) demonstrated that AP-1 was involved in Kex2 trafficking and describe the Kex2-localization phenotype(s) seen previously by others in AP-1 mutant strains? In other words, what is the existing evidence in the literature that led to the model of AP-1 functioning in Kex2 traffic between the Golgi and endosomes? Furthermore, can the authors briefly discuss the established role of AP-1 in mammalian cells , and whether the authors think AP-1 has similar or different functions in yeast? I think explicitly addressing these points will help readers to better understand the insights from the current data in light of previous studies.As described above, we added the relevant reference about AP-1 and Kex2, and revised the accompanying text to emphasize that an involvement of AP-1 in Kex2 recycling needs further investigation. We also now briefly discuss how studies of mammalian AP-1 are potentially consistent with our model for AP-1-dependent recycling in yeast.


Aurora A has been extensively characterized as a centrosomal kinase with essential functions during cell division including centrosome maturation and separation and spindle assembly. However, Aurora A localization is not restricted to the centrosomes and compelling evidence support the existence of specific mechanisms of activation and functions for non-centrosomal Aurora A in the dividing cell. It has been now well established that spindle assembly involves an acentrosomal RanGTP-dependent pathway that triggers microtubule assembly and organization in the proximity of the chromosomes whether centrosomes are present or not. The mechanism involves the regulation of a number of NLS-containing proteins, generically called SAFS (Spindle Assembly Factors) that exert their functions upon release from karyopherins by RanGTP. One of them, the nuclear protein TPX2 interacts with and activates Aurora A upon release from importins by RanGTP. This basic mechanism triggers the activation of Aurora A in the proximity of the chromosomes potentially translating the RanGTP signaling gradient centered on the chromosome into an Aurora A phosphorylation network. Here, we will review our current knowledge on the RanGTP-dependent TPX2 activation of Aurora A away from centrosomes: from the mechanism of activation and its functional consequences on the kinase stability and regulation to its roles in spindle assembly and cell division. We will then focus on the substrates of the TPX2-activated Aurora A having a role in microtubule nucleation, stabilization, and organization. Finally, we will briefly discuss the implications of the use of Aurora A inhibitors in anti-tumor therapies in the light of its functional interaction with TPX2. Drosophila  network disappears and highly dynamic MTs organize the bipolar spindle, the molecular machine that provides the support and forces for chromosome segregation. The progression and coordination of the events that drive spindle assembly and culminate with the birth of two daughter cells rely on complex regulatory networks involving several kinases. One of them is Aurora A , a kinasosophila . In highosophila . Aurora osophila –5.The potential link between the Aurora kinases and tumor initiation and/or development has fueled the interest in understanding their function and regulation over the last years. Indeed, Aurora A gene is located in a region of chromosome 20 that is frequently overexpressed in human cancers , 7, and Although the three Aurora kinases share a conserved catalytic domain, a few critical amino acid substitutions in their catalytic domains confer activator specificity. Moreover, divergent N- and C-terminal domains provide specificity at least in part through protein–protein interactions and distinct subcellular localizations during mitosis. While Aurora B and C localize to the kinetochores and the anaphase central spindle as part of the chromosomal passenger complex , Aurora in vitro assays showed that PKA phosphorylates Aurora A on at least three residues, including Thr288   , 37. DesXenopus) .Xenopus laevis)   contribu laevis) , other a laevis) .The conformational change induced by the binding of TPX2 to Aurora A results in the change in position of Thr288 that moves it into a buried position inaccessible to inactivating phosphatases . TherefoFinally, TPX2 protects Aurora A from degradation that occurs under normal conditions at the end of mitosis through the cdh1 activated APC/C proteasome pathway . CertainXenopus Aurora A phosphorylates TPX2 on three serine residues   , 45. In d Ser94) . Howeverd Ser94)  while ckTPX2 and Aurora A both perform essential functions during cell division although not all of them are dependent on their interaction. Aurora A null mouse embryos, similar to TPX2 ablation, are embryonic lethal failing to undergo the morula-blastocyst transition due to defects in mitosis –51.Xenopus egg extract and in mammalian cells TPX2 is essential for acentrosomal MT assembly driven by the chromosome-dependent RanGTP pathway in M phase  are cytosolic. This promotes the formation of a RanGTP gradient centered on the chromosomes that has been directly visualized in extracts –58 and iextracts , 60. In extracts ]. In mamextracts –64. One omosomes , 65. ThiXenopus egg extract -binding protein 1] as well . The bintpx2 knockout mice display severe developmental defects and embryonic lethality  ((TPXL-1) . Althoug(TPXL-1) .These data suggest that different evolutionary modules may exist to control the localization and activation of the Aurora kinases during cell division. Vertebrates seem to have developed a unique module to integrate the control of localization and activation of Aurora A by the chromosomal RanGTP-dependent pathway through a single interacting protein, TPX2.Aurora A and TPX2 are overexpressed in several types of tumors and have been implicated at different levels in cancer. Although the mechanism underlying the role of TPX2 and Aurora A in tumorigenesis may be at least in part independent, there are data to suggest a role for the complex. TPX2 was initially identified as a proliferation marker with a potential role in human cancer . It is iXenopus (Interestingly, both TPX2 and Aurora A genes are located on chromosome 20q, whose amplification is found in tumors and moreover, co-expression of TPX2 and Aurora A has been observed in some tumors . For insXenopus .Some data suggest that the increased levels of Aurora A in various tumors may be the consequence of protein stabilization rather than gene amplification. Indeed, phosphorylation of Aurora A Ser51 inhibits its degradation via the cdh1 activated ubiquitin ligase APC/C at the end of mitosis and Aurora A constitutively phosphorylated at Ser51 was shown to be present in neck and head cancer tissues with Aurora A overexpression . AlthougThe clear implications of Aurora A in cancer have promoted the intensive search for small molecule inhibitors for their potential therapeutic use . Some ofAll authors listed, have made substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


WHO recommends that stavudine is phased out of antiretroviral therapy (ART) programmes and replaced with tenofovir (TDF) for first-line treatment. In this context, the Integrated HIV Care Program, Myanmar, evaluated patients for ART failure using HIV RNA viral load (VL) before making the change. We aimed to determine prevalence and determinants of ART failure in those on first-line treatment.Patients retained on stavudine-based or zidovudine-based ART for >12 months with no clinical/immunological evidence of failure were offered VL testing from August 2012. Plasma samples were tested using real time PCR. Those with detectable VL>250 copies/ml on the first test were provided with adherence counseling and three months later a second test was performed with >1000 copies/ml indicating ART failure. We calculated the prevalence of ART failure and adjusted relative risks (aRR) to identify associated factors using log binomial regression.Of 4934 patients tested, 4324 (87%) had an undetectable VL at the first test while 610 patients had a VL>250 copies/ml. Of these, 502 had a second VL test, of whom 321 had undetectable VL and 181 had >1000 copies/ml signifying ART failure. There were 108 who failed to have the second test. Altogether, there were 94% with an undetectable VL, 4% with ART failure and 2% who did not follow the VL testing algorithm. Risk factors for ART failure were age 15–24 years  compared to 25–44 years and previous ART in the private sector  compared to the public sector.This strategy of evaluating patients on first-line ART before changing to TDF was feasible and identified a small proportion with ART failure, and could be considered by HIV/AIDS programs in Myanmar and other countries. The scale up of antiretroviral therapy (ART) in low- and middle-income countries (LMIC) in the last decade has been a remarkable public health success. By 2013, an estimated 11.7 million people living with HIV (PLHIV) were receiving ART, representing 36% coverage of the 32.6 million PLHIV in these countries  who were tested for HIV RNA viral load and these were all included in the analysis. The median (IQR) duration of ART was 28 (16–35) months. Most patients were aged 25–64 years, most had received secondary school education, and two thirds had never consumed alcohol. Nearly one quarter had been initially treated with antiretroviral drugs in the private sector before enrolling in the Integrated HIV Care Program. d4T-based ART was being taken by 82% and AZT-based ART by 18% of patients. At the time of registration for ART, 65% of patients were in WHO Clinical Stage 3 or 4 and 50% had a CD4 cell count <200 cells/uL. Just over 40% of patients had an opportunistic infection, of whom half had TB.The management and results of viral load screening are shown in Baseline characteristics associated with ART failure are shown in This is the first report from Myanmar of a strategy of viral load screening to assess whether patients on d4T-based or AZT-based first line ART for over 12 months and who had no clinical or immunological evidence of ART failure could safely change to a TDF-3TC-EFV first line regimen or needed switching to second line therapy. The majority of patients who were enrolled in the study (94%) completed the viral load screening algorithm and had no evidence of virological failure. A small proportion (4%) had viral load failure and needed to be switched to second line therapy, with the main risk factors being young age of 15–24 years (aRR-2.4) and previous HIV treatment in the private sector (aRR-1.6).The strengths of this study were the large number of patients being evaluated for viral load and the implementation of this activity within the context of a national program. Results can therefore be generalized to a wider population. Baseline data collection was adequate with only a few missing results. We also used STROBE guidelines and sound ethics principles for the conduct and reporting of this observational study ,10.There were four main limitations. First, resource constraints meant that some eligible patients were not tested. Second, there were many patients on AZT-based ART who were not given the opportunity of viral load testing due to decisions by clinicians to maintain them on this therapy. Although we do not have precise quantification of why this was done, there are some possible reasons. There were large stocks of AZT in the pharmacies at the time and some clinicians felt these should be used before changing to TDF. The 2013 WHO Guidelines moreover emphasized the phasing out of d4T, and while TDF was recommended as the preferred first-line option, AZT was also mentioned as an alternative option –13, and The main risks for viral load failure were young age between 15–24 years and previous treatment in the private sector. Adolescents (defined as 10–19 years) are a vulnerable group that have been identified as being at particularly high risk of poor adherence to treatment, loss to follow-up and treatment failure –16 and tThere are some important implications from this study. First, there are good reasons to change to a TDF-based regimen in line with the policy decision in Myanmar. The drug is patient friendly through its once daily dosing, there are few adverse side effects, few drug-drug interactions, the chances of accumulation of drug resistance are lower compared with other nucleoside reverse transcriptase inhibitors, and the drug has dual action against Hepatitis B ,19,20,21Based on this experience, we would suggest that this strategy be considered in other HIV/AIDS programmes in Myanmar and other countries, subject to sufficient human and financial resources. Finally, if this strategy is taken up it will be important to ensure adherence to protocol and prevent decisions on treatment being made based on one viral load test as happened in a few patents in our study.In conclusion, we were able to enroll nearly 5000 patients with HIV infection and on d4T-based or AZT-based ART for at least 12 months into a strategy of viral load testing before considering a change to TDF-based ART. The large majority of these patients had no evidence of ART failure and could be safely changed to a first-line TDF-based ART regimen. However, a small proportion did have evidence of ART failure and needed to be identified and switched to second-line therapy. Little is known about the effect of HIV-1 resistance mutations present at the time of regimen switch on the response of second line therapy. A point of care genotypic resistance test coupled with VL would avoid the logistical challenges and delays associated with centralized VL testing, but this of course requires resources. This should prevent them accumulating drug resistance mutations and improve their long-term prognosis. This strategy could be considered by HIV/AIDS programmes in Myanmar and other countries.S1 Dataset(DTA)Click here for additional data file.


Studies from the USA suggest that rates of pediatric bipolar disorder have increased since the mid-90s, but no study outside the USA has been published on the rates of pediatric bipolar disorder. Further, it is unclear whether an increase in rates reflects a true increase in the illness or more diagnostic attention. Using nationwide registers of all inpatients and outpatients contacts to all psychiatric hospitals in Denmark, we investigated (1) gender-specific rates of incident pediatric mania/bipolar disorder during a period from 1995 to 2012, (2) whether age and other characteristics for pediatric mania/bipolar disorder changed during the calendar period (1995 to 2003 versus 2004 to 2012), and (3) whether the diagnosis is more often made at first psychiatric contact in recent time compared to earlier according to gender. Totally, 346 patients got a main diagnosis of a manic episode (F30) or bipolar affective disorder (F31) at least once during the study period from 1995 to 2012. For both sexes, annual rates of mania/bipolar disorder two to four doubled during the study period (0.001% before year 2004 to 0.002%–0.004% in 2010). Median age at the index diagnosis was very similar during the two calendar periods  indicating that the diagnosis of mania/bipolar disorder was not made earlier in the recent calendar period. Similarly, there were no differences between early versus late in the study period in the fractions of first contact diagnosis of mania/bipolar disorder diagnoses, the contact number at which patients got the diagnosis or the duration from first psychiatric contact to the diagnosis of mania/bipolar disorder. The rate of diagnosis of mania/bipolar disorder increased from 1995 to 2014, which did not seem to be explained by more diagnostic attention. An influential paper by Wozniak and colleagues raised in 1995 the question of whether pediatric bipolar disorder was being overlooked in clinical practice  gender-specific rates of incident pediatric mania/bipolar disorder during a period from 1995 to 2012, (2) whether age and other characteristics for pediatric mania/bipolar disorder have changed during this calendar period, and (3) whether the diagnosis is more often made at first psychiatric contact in recent time compared to earlier according to gender.The Danish Psychiatric Central Research Register (DPCRR) is nationwide with registration of all psychiatric hospitalizations in Denmark for the 5.3 million inhabitants  that can be logically checked for errors, so it can be established with great certainty if a patient has had contact to psychiatric service previously, irrespective of changes in name, etc.No private psychiatric inpatient hospitals or department are in operation in Denmark; all are organized within public services and reporting to the DPCRR.The Medicinal Product Statistics contains data on all prescribed medication purchased at pharmacies from January 1, 1995 and onwards  or inpatient  with at least one main diagnosis of mania/bipolar disorder  during the study period from January 1, 1995 to December 31, 2012. Patients with psychiatric contacts prior to 1995 and back to 1970 were excluded.Aim 1. Annual rates of incident pediatric mania/bipolar disorder in children and adolescents were calculated from 1995 to 2012 separately for boys and girls using the entire Danish population of less than 19-year-olds as denominator.Aim 2. The study period was divided into two 9-year periods: 1995 to 2003 and 2004–2012. Patients belonged either to the first or to the second group depending on the date of their first diagnosis of mania/bipolar disorder (the index diagnosis). Characteristics of patients during the two periods were compared.In Table Aim 3. For each calendar year, the fraction of first contact diagnosis of mania/bipolar disorder diagnoses out of all contacts diagnoses of mania/bipolar disorder was calculated for boys and girls.P < 0.05 was used to indicate statistical significance.Categorical data were analyzed with chi-square test, and continuous data were analyzed with the Mann-Whitney test for two independent groups. Totally, 346 patients got a main diagnosis of a manic episode (F30) or bipolar affective disorder F31) at least once during the study period from 1995 to 2012. Figure  at leastAmong the total 346 patients, 111 patients (32.1%) got the main diagnosis during the calendar period 1995–2003 whereas the 235 remaining patients (67.9%) got the diagnosis in the years 2004 to 2012. Table As can be further seen from Table More psychiatric comorbid diagnoses were made during 2004–2012 than during 1995–2003, 21.7% versus 11.7%, but the type of psychiatric comorbid diagnoses did not differ significantly between the two calendar periods.N = 635) divided by person years (177.28 person years). Similar, the incidence rate of lithium as first drug was 0.4 per person year corresponding to 40 patients who got lithium per 100 person years, which was calculated as the number of patients who got lithium (N = 46) divided by the person years (105.81) for the drug.During the recent calendar period, lithium was prescribed more rarely after the index diagnosis in general and as first drug and conversely, antipsychotics and anticonvulsants were used more often. For example in the calendar period 1995–2003, the rate of lithium treatment after the index diagnosis was 3.6 prescriptions per person year, which was calculated as the number of lithium prescriptions from the index diagnosis to the end of the calendar period/19th birthday  indicating that the diagnosis of mania/bipolar disorder was not made earlier in the recent calendar period. Similarly, there were no differences between early versus late in the study period in the fractions of first contact diagnosis of mania/bipolar disorder diagnoses, the contact number at which patients got the diagnosis or the duration from first psychiatric contact to the diagnosis of mania/bipolar disorder. Indeed, it is unlikely that increased diagnostic attention in primary care would not have led to a change in these parameters in psychiatric health care service.The study included a Danish nationwide sample of all in- and outpatients below 19 years of age with an outpatient or inpatient psychiatric hospital contact with a diagnosis of mania/bipolar disorder in psychiatric settings. In Denmark, doctors are obliged to make a diagnosis when a treatment period is terminated, i.e. at discharge from hospital or at the end of an ambulatory treatment period, and all diagnoses are reported to the Danish Psychiatric Central Research Register.Overall, our study including a Danish sample confirm the increase in rates of inpatient psychiatric hospitalization  found in recent studies might suggest  are less than 19 years old), and further, the population is ethnically and socially homogeneous and with a very low migration rate. The entire population (approximately 100%) of patients treated in psychiatric settings in a whole country during in- or outpatient settings was included. Psychiatric care is well developed in Denmark, so persons with mania or bipolar disorder can easily come in contact with psychiatric community centers or hospitals. Also, as psychiatric treatment in Denmark is free of charge, the study is not biased by socioeconomic differences.It should be noted that the study included patients who have passed the threshold for treatment to psychiatric outpatient settings (psychiatric ambulatories and community centers) or to psychiatric hospitalization, only. Although the vast majority of children and adolescents with bipolar disorders are treated in these hospital settings as in- or outpatients, some patients with milder types of the illness may be treated within private psychiatric practice. Such patients are not included in the study, as private psychiatric practice does not report to the DPCRR. General practitioners do not treat children and adolescents with bipolar disorder in Denmark. It cannot be excluded that including milder cases of bipolar disorder into the sample might have resulted in other findings.It should be noted that the diagnosis of bipolar disorder in ICD-10 includes both bipolar disorder I and bipolar disorder II but does not discriminate between the two subtypes, as ICD-10 bipolar disorder is defined as a disorder with at least two mood episodes among which at least one is a hypomanic or a manic episode. It is most likely that the majority of the 346 patients in the present study suffered from bipolar disorder, type I, as patients were included via their contact to hospital psychiatric settings. It is most likely that patients with bipolar disorder, type II were not included as also reflected in the low incidence rates of 0.001% to 0.004% compared with 12-month prevalence rates for mania with and without depression of 2.2% and 1.3% from a recent population-based US survey of 13–18-year-old adolescents  and that only 25% of the patients were below 16.2 years of age when the diagnosis of mania/bipolar disorder was made for the first time reflecting that child bipolar disorders are rarely diagnosed in Denmark.Frequent comorbid conditions in bipolar disorder such as anxiety disorders, hyperkinetic disorders, and substance use may complicate the diagnostic process. It should be stressed that the present study focused on the main diagnoses that are given for the main illness leading to investigation and treatment. The main diagnoses are given according to the diagnostic hierarchy in ICD-10 giving priority to diagnoses with lower ICD-10 codes (World Health Organisation In Denmark, annual rates of mania/bipolar disorder two to four doubled for both sexes during a study period from 1995 to 2012, and this increase did not seem to be explained by increased diagnostic attention.