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EMBO reports - Peer Review Process File - EMBOR-2013-38037 Manuscript EMBOR-2013-38037 Endosomal transport of septin mRNA and protein indicates local translation on endosomes and is required for correct septin filamentation Sebastian Baumann, Julian Konig, Janine Koepke and Michael Feldbruegge Corresponding author: Michael Feldbruegge, Heinrich-Heine University Dusseldorf Review timeline: Transfer date: Editorial Decision: Revision received: Accepted: 26 September 2013 26 September 2013 10 October 2013 14 October 2013 Editor: Esther Schnapp Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) Transfer Note: Please note that this manuscript was originally submitted to The EMBO Journal, where it was peer-reviewed. It was then transferred to EMBO reports and the original referees’ comments, as well as the authors’ responses, are shown below. Original referees’ comments and authors’ responses – EMBO Journal 26 September 2013 Referee #1: This manuscript describes the possibility of endosomal transport of septin (Cdc3) mRNA being coupled to protein synthesis. This finding would be significant since it is typically assumed that mRNA trafficking serves to deliver message to the site of synthesis. The data are very nicely presented and take advantage of high spatial and temporal resolution afforded by the experimental system. © European Molecular Biology Organization 1 EMBO reports - Peer Review Process File - EMBOR-2013-38037 The manuscript largely hinges on whether one accepts that endosomal Cdc3 is indeed translated during motility (i.e. on the endosomes) rather than the data reflecting delivery of entire translation systems (including ribosomes) to the ultimate site of synthesis. Unfortunately in this regard the key data are explained in a somewhat confusing manner. The key experiment hinges on mutants of Rrm4 that are not adequately explained for a general (or perhaps even specialist) audience. These key data are also spread between figures 3, 4 and S4. The paragraph on Page 8 relating to this must be explained in much greater depth. Essentially how do these experiments conclusively demonstrate that ongoing onendosome translation is occurring? It is my understanding that these mutants are defective in RNA binding and therefore support the requirement for Rrm4 in the endosomal localization of Cdc3 (as stated in the subheading) but do not definitively show that translation of the protein is occurring on the endosomes. We agree that we do not directly show Cdc3 translation on endosomes. However, such direct proof is not possible with current technology and therefore comparable information is missing in the majority of systems studying localized translation. For example, there is no direct proof that local translation takes place at synapses in dendrites. It was also never directly shown that ASH1 mRNA is translated at the pole of daughter cells in S. cerevisiae. It is only now that techniques for direct monitoring of sites of translation are emerging. Thus, in the field of localized mRNA translation most researchers use the following logic: (i) mRNA and translation product should co-localise at the same subcellular site. (ii) Without mRNA localisation the protein should not localise at the same destination. (iii) Active ribosomes need to be present for translation. We followed the same strategy that is generally accepted in the field and found that our results meet all three criteria. We apologize for the short explanations that were given on page 8 and we regret that in particular the full potential of the FRAP experiments was not described adequately. In order to improve the manuscript substantially we did the following: In Figure 4 we now show that mRNA and protein localise to identical shuttling endosomes and that the presence of septin protein on endosomes depends on the presence of mRNA. In Figure 5 we demonstrate that ribosomes are present on endosomes and this depends on the presence of mRNAs. We explained the mutants of Rrm4 in more detail and important results on these mutants are now included in Figure 4. In short, the two different mutations in the RNA-binding domain of Rrm4 both affect mRNA binding without altering the endosomal localization of Rrm4. We substantially improved the description of the FRAP experiments and provided additional data demonstrating that local translation at the growth poles does not contribute extensively to the assembly in septin filaments. To show this, we bleached the whole hypha and measured the recovery of cytoplasmic Gfp 15 minutes after bleaching. As expected, translation and maturation of Gfp takes far longer than the measured half lives of recovery (Figure 7F-G). Thus, delivery of mRNAs and ribosomes for translation at the growth pole appears very unlikely as an explanation for the in vivo data. Other points: Figure 5 shows robust (if delayed targeting of Cdc3 to the hyphal tip even in the presence of benomyl. This is somewhat in contrast to the clear effects of benomyl in Figure 3A. Can the authors provide greater clarity here. Fig 5 suggests that microtubule-based transport merely serves to enhance the efficiency of Cdc3 delivery to the tip where Figure 3 suggests that it is required. Hence, the statement on p12 that microtubule-based transport is "crucial" does not seem to be supported by the data in Fig 5. © European Molecular Biology Organization 2 EMBO reports - Peer Review Process File - EMBOR-2013-38037 We are sorry for this misunderstanding. The main difference between the results shown in Figure 3A-C and those of Figure 5 is that due to the experimental set-up the hyphae analyzed in FRAP experiments (Figure 5) are investigated for a prolonged time below a coverslip. In these experimental conditions the strong septin localization in filaments at the hyphal tip does not recover completely (Supplementary Fig. 5). Therefore, the data underestimate the recovery of septin filaments at the hyphal tip. Due to this small caveat we cannot make any conclusions on the percentage of recovery which is used to determine the immobile fraction of molecules in FRAP experiments. However, these data clearly show that there is a microtubule-dependent process involved. We explained this in more detail and included the following text “Note, that according to the experimental set-up hyphae were analysed for a prolonged time below the coverslip. Under these conditions the Cdc3G gradient did not recover to the same extend as observed at optimal growth conditions (Figure 3A and 3C)”. Furthermore, we replaced the word crucial. The text now reads “indicating that microtubuledependent transport is important for efficient accumulation of Cdc3G at the growth pole.” The data in Figure 3 describe cells grown at optimal growth conditions. The data clearly indicate that microtubule-dependent transport is needed to form the gradient of Cdc3 in filaments. Without microtubule-dependent transport septin filaments can still be formed, but we do not know whether these altered filaments are functional. On page 10 the authors mention that endosomal fusion and fission events would act in the distribution of Cdc3 after local translation on a subset of organelles. Why would this be necessary as opposed to distribution by diffusion? This could be tested by inhibition of homotypic endosomal fusion. What are the dynamics of Cdc3 on the endosome? The Cdc3 localization in Fig 3 onwards - what is the correlation between the distribution of Cdc3G and the assembled septin filaments? Does filament assembly occur on endosomes during transit or only upon delivery to the tip? This has some quite significant implications for the model. Unfortunately, we do not have the required molecular tools to inhibit homotypic endosomal fusion and it is also very difficult to determine the dynamics of Cdc3 on shuttling endosomes. We hypothesized that endosome-coupled translation of septin mRNA is important for the assembly of septin building blocks and therefore to assemble septin filaments. This assembly process is presumably more efficient than assembly in the cytoplasm and distribution by diffusion. This assumption is strongly supported by the observation that without mRNA transport (hyphae treated with benomyl), Cdc3G is still present in filamentous structures but also localizes to small circles that most likely reflect aberrant mR1 localisation. Also our experiments with Rrm4 mutants were very informative. In Rrm4 or RRM in Rrm4 expressing strains RNA binding of Rrm4 is clearly disturbed, but Rrm4 still localises at endosomes (and Rrm4 has no influence on endosome formation or shuttling). In these mutants cdc3 mRNA is translated in the cytoplasm but the protein is unable to localize on endosomes. Thus, without mRNAs on endosomes these endosomes are not capable of receiving septin Cdc3 suggesting that septin translated in the cytoplasm does not bind endosomes. Δ In the revised version, we now include data on a second septin Cdc12. This shuttles bidirectionally on microtubule-dependent units and localises in septin filaments. Importantly, in the absence of Cdc12, Cdc3 is unable to localize to endosomes and filament assembly is disturbed. These additional data further support our hypothesis that septin oligomer assembly initiates on endosomes. Minor points: © European Molecular Biology Organization 3 EMBO reports - Peer Review Process File - EMBOR-2013-38037 Gfp and Rfp should both be capitalized throughout. We follow the nomenclature in U. maydis because these heterologous proteins are expressed in the fungus. However, if required we can change this. Some consideration should be given to the ordering of the figures as often these are referred to out of sequence, detracting from readability. We addressed this issue. Referee #2: Previous studies performed in Ustilago by these authors demonstrated that cdc3 mRNA, which encodes a septin, interacts with an RNA-binding protein (RBP), Rrm4, using CLIP experiments. In addition, the authors demonstrated that Rrm4 is trafficked in hypha in a microtubule (MT)-dependent manner and co-localizes with endosomal markers, and that the transport of ribonucleoprotein particles, as designated using poly-A binding protein (Pab1), is disrupted in cells bearing a deletion or mutation in RRM4. In the current study, the authors now demonstrate that cdc3 mRNA and protein may be transported on particles that co-label with ribosomal proteins, Pab1, and FM4-64, and in a manner that is dependent upon MT and Rrm4 function. The results correlate with the delivery of Cdc3 protein via endosomes and the authors suggest that its translation on endosomes (instead of at the target site) may be a means of septin delivery. Two major problems with this work significantly lower the enthusiasm for this view. First, Cdc3GFP protein accumulates in the hyphal tip even after photobleaching in the presence of benomyl, which would inhibit both RNA and protein transport (Figure 5). Hence, Cdc3GFP recovery is likely to result from localized translation (which supports the more commonly held view that RNA trafficking leads to localized translation at the target site). This result runs counter to the idea of either endosome-based protein delivery or co-translational delivery, the latter being strongly hinted at (based upon correlative RNA/protein co-localization experiments). It is correct that in the absence of RNA and protein transport Cdc3G accumulates in filaments (Figure 3A-C). Thus, septin filaments can be formed with building blocks translated in the cytoplasm and distributed by diffusion. However, we do not know whether these filaments are functional. In wild type hyphae, Cdc3 forms a gradient emanating from the hyphal tip and microtubule-dependent transport is essential to establish this gradient. Unfortunately, as mentioned above we did not explain our FRAP experiments carefully enough. We can clearly exclude de novo protein synthesis by local translation of delivered mRNAs at the hyphal tip as a microtubule-dependent mechanism for formation of septin filaments at the growth pole. Importantly, photobleaching and measuring fluorescence recovery specifically at the hyhpal tip is a powerful approach to exclude the contribution of newly synthesized protein at this site. This is due to the fact that translation and maturation of Gfp takes more than 15 minutes (Figure 7F-G). Thus, instead of using translational inhibitors that globally affect translation and interfere with numerous biological processes within the cell we, by this means, can specifically exclude that newly translated Cdc3G contributes significantly to septin filament assembly in the analysed region. In addition, according to our state-of-the-art RNA live imaging we do not detect any accumulation of mRNAs at the hyphal tip. As mentioned above, the only caveat of our in vivo study is that the hyphae are studied at a prolonged time below a coverslip. Thus, we do not get a full recovery of Cdc3 filaments and therefore we underestimated the influence of microtubule-dependent transport under these conditions. © European Molecular Biology Organization 4 EMBO reports - Peer Review Process File - EMBOR-2013-38037 In the revised version we explain this more careful and include additional FRAP data showing that recovery of cytoplasmic Gfp cannot be detected 15 minutes after the whole hypha was photobleached (Figure 7F-G). Second, the authors suggest that translation occurs on the shuttling endosomes, yet the experiments needed to actually prove this have not been performed (see specific comments 9 & 10 below). Although co-translational delivery of some portion of Cdc3 protein via endosomes cannot be ruled out, it is still premature to make this conclusion. To prove the site of translation directly is a very difficult task and has not even been achieved for other systems (see above). Even if only a fraction of Cdc3 is translated on endosomes, the process is necessary to ensure efficient assembly. Although with the current state of the art we are not able to give direct proof, we still present an intriguing novel finding and this opens up new possibilities for septin biology and protein delivery by membrane trafficking. We respond to comments 9 and 10 in detail below. There is some precedent for protein translation occurring in transit in fungi, prior to localized translation at the target site, although this was not shown to involve membranes. ABP140 mRNA localization to the distal pole in S. cerevisiae involves translation of the amino terminal of the protein for directed RNA trafficking to occur in an actin-dependent fashion (Kilchert and Spang, EMBO 2011). However, much more work is required to show that cdc3 mRNA is translated on endosomes. The work by the Spang lab published in your journal is a great example. It shows that translation of ABP140 mRNA is needed for a novel pattern of protein localization at the distal pole of the mother cell. The most likely explanation is co-translational transport, which was stated in the title and we fully agree with this. However, the authors must admit that they never showed local translation of ABP140 mRNA directly. They used RNA live imaging to demonstrate movement but neither did they demonstrate that the mRNA is translated during transport nor did they show that local translation takes place at the distal pole. An alternative explanation for their findings would be that the first round of translation removes an inhibitor of mRNA transport and that mRNAs are transported subsequently. Moreover, they did not provide any evidence that this transport is biologically significant for the cell. Hence, we believe that our work is at least of the same scientific quality. Specific comments 1. Page 4, line 5 - Supplementary Figure 1 and Movie 1 describe the movement of Rrm4 and Pab1, yet it is not explained either in the text or legend how they are labeled in order to be visualized. This should be clarified for the reader. We clarify this issue in the figure legend of Supplementary Figure S1. The text now reads: “A hypha of strain AB33rrm4C/pab1G is depicted which expresses Rrm4 fused to mCherry and Pab1 fused to eGfp. Strains were generated by homologous recombination. Thus, functional fusion proteins are produced at endogenous levels (Baumann et al., 2012).” 2. Figure 1C - Why do cdc3Δ hypha switch to unipolar growth at later time points? What is the compensatory mechanism? Is there another septin at work? In rrm4Δ cells, why don't septa form around the initial cell? These are very important and interesting questions that we are currently trying to address. However, answering these questions is beyond the scope of this publication. 3. Figure 2 - Shows the movement of CDC3 RNA using the lambdaN/boxB in an Rrm4-dependent fashion. Where is the control of the lambda N reporter alone (i.e. without the boxB-tagged RNA)? How do the authors know that the native cdc3 RNA © European Molecular Biology Organization 5 EMBO reports - Peer Review Process File - EMBOR-2013-38037 associates with these endosomes (and that its localization to endosomes is not due to artifacts associated with presence of the RNA aptamer tag or LambdaN RNAbinding protein)? Localization of an RNA encoding a non-relevant protein would be helpful, likewise a FISH experiment for native cdc3 would also go far towards proving or disproving the point. In König et al. (EMBO J, 2009) we established the lambda N/boxB RNA live imaging technique for fungi. We showed that the Rrm4 target mRNAs rho3 and ubi1 are shuttling in particles along microtubules. This is dependent on Rrm4 and for the ubi1 mRNA we demonstrated that Rrm4 and ubi1 mRNA co-localize in vivo. We also showed that a control mRNA was transported less often and over shorter distances. Furthermore, we demonstrated that in a control strain expressing only the λN variant hardly any directed particles were observed, indicating that formation of particles was dependent on the presence of boxB binding sites (one particle in 30 hyphae). We verified our results by FISH experiments. These were not easy to perform in U. maydis and it took us a considerable amount of work to prove that in our system FISH and RNA live imaging are fully consistent. Therefore, we did not repeat all the controls for the current study. However, it is a fair criticism to assume that the modified λN* reporter has endosomebinding activity. Therefore, we analysed the amount of directed signals in strains lacking box B tagged mRNA. The text now reads: 16 “In contrast, in control experiments with hyphae lacking boxB -containing mRNAs we did 2 not observe directed movement of λN*G (13 hyphae analysed).” 4. Figure 2B - What do the authors mean by processivity (i.e. overall movement, the non-detached movement as typically used in the description of motor proteins?)? This is unclear. Likewise, text in legend should make clear that in B there are no errors bars - just min, max - otherwise it is very confusing to the reader. We used the term processivity because mRNAs and Rrm4 are co-transported on endosomes and these endosomes are transported by the processive movement of Kinesin3 type Kin3 and split dynein. However, we agree that this might be far-fetched for the reader of this publication. Therefore, we introduced the term “directed movement” like we did in the EMBO J. paper in 2009. We also explain the RNA live imaging data in a lot more detail, because it is a crucial part of the publication. The legend in the figure (Figure 2D) now indicates that error bars for the bar diagrams depicting velocity, directionality and the number of mRNPs is given as s.e.m. and the range of directed movement is given as a whisker diagram with median and min/max values. 5. Page 6, para 1, last line - It is written that cdc3 RNA colocalizes with Rrm4 on shuttling endosomes (Figure 2D). Since there is no endosomal marker in this experiment I don't see how one can say that. The results only correlate with previous localization experiments that used endosomal markers. In previous studies (Baumann et al. 2012, JCS) we have shown that Rrm4 is cotransported with distinct endosomes that can be stained with FM4-64, that co-localize with the SNARE Yup1 and that are transported by the action of Kin3 and split dynein. However, we never quantified the extent of co-localisation. In the revised version we now show dynamic co-localisation with Rab5a fused to mCherry (Figure 2A). The small G protein is a well-established marker for this endosomal compartment also extensively used in G. Steinberg’s group (University of Exeter). We determined that the vast majority of Rrm4 and Rab5a signals co-localise on shuttling endosomes. In fact, since Rrm4 in contrast to Rab5a is restricted to this membranous system, it can be considered as specific marker for this endosomal compartment. © European Molecular Biology Organization 6 EMBO reports - Peer Review Process File - EMBOR-2013-38037 6. Figure 2E shows statistics for the movement of RNA particles in different strains. However, some Rrm4 mutants shown are not discussed or mentioned either in the legend or the text until much later in the paper. This is confusing - they should be listed in the legend even if not discussed here. Or better yet, they could be removed altogether until they are discussed. We solved this issue. As suggested we mention the statistics on RNA particles of Rrm4 mutants when they are mentioned later (Figure 4I). 7. Page 6 last line - States that they "did not detect any deposition of cdc3 mRNAs" (i.e. by leaving some behind?) and that this is "contradicting the commonly held view of local translation upon mRNA delivery to the target site." This statement is erroneous - first, no examination of translation in this experiment was performed; Correct; we deleted the statement. However, in the revised version we include a Supplementary Figure S3 showing that it is possible to observe λN* signals that switch from directed to restricted movement or vice versa. These events might reflect deposition or loading events, respectively. Noteworthy, we did not observe these events taking place at the hyphal tip. This does not favor the classic view of mRNA transport followed by translation at the growth pole. Also, the signal intensity of λN*-labeled mRNA did not decrease after an mRNP reached the hyphal tip and shuttled backwards (see Figure 2C). Thereby, we can exclude that a subfraction of λN*-labeled mRNA is deposited. Moreover, we did not observe accumulation of mRNAs in our RNA live imaging experiments like it was reported in cases of mRNAs in other systems. second (and more importantly), how do the authors know that the lambdaN-GFP reporter would remain bound to a translating message (deposited or not) in the first place? In all likelihood, the many copies of the lambda-GFP reporter (which total 48 GFPs, if all the boxB binding sites are saturated) bound to the message would have to fall off for translation to occur (i.e. due to steric hindrance with the engaged ribosome). Thus, translating RNAs are likely to visually absent using this detection system and those RNAs that are observed are likely to be translationally repressed! Again, a FISH experiment to examine the localization of native mRNA might be useful before one can make any conclusions. These arguments are in contrast with the currently held view in the field of RNA transport using RNA live imaging. Beautiful work of the Singer lab using a transgenic mouse model showed that the presence of 24 YFPs, which are recruited to the 3'UTR, does not interfere with function of β-actin mRNAs. Also localized mRNAs were not considered translationally repressed (Lionnet et al. 2011, Nature Methods 8:165). In fact, the Singer lab is currently establishing a technique that makes use of the fact that phage proteins binding to heterologous binding sites in the ORF are removed during translation while those in the 3’ UTR are not removed (translational kick-off assay). We also now provide data that mRNA and protein co-localise on the same endosome suggesting that the boxB-containing mRNAs are translated (see below, Figure 4A). 8. Figure S2 - It is stated in the text that cdc3 mRNA and protein don't change drastically. Nevertheless, Cdc3 protein does go up significantly in the absence of Rrm4. Perhaps even the RNA as well (no qRT-PCR data shown). Does Rrm4 affect cdc3 translation? The main point of these experiments is that cdc3 expression is not drastically reduced in rrm4 deletion strains, which would interfere with the interpretation of the localization data and FRAP experiments. The text now reads “Importantly, the absence of Rrm4 did not result in a decrease of cdc3 mRNA or protein (Supplementary Figure S4A-B).” 9. Page 8, para 1, last line - "As co-localisation of mRNA and the encoded protein is indicative for the site of translation (St Johnston, 2005), the simultaneous presence of cdc3 mRNA and Cdc3 protein at shuttling endosomes strongly points towards its local translation on endosomes." There is no proof of this. © European Molecular Biology Organization 7 EMBO reports - Peer Review Process File - EMBOR-2013-38037 In order to prove it one would have to: 1) Co-localize both RNA and protein on the same exact endosome, which is never done in this paper; This was a very valuable suggestion. Although it was not an easy task, due to the dynamic movement and the sensitivity of the system, we succeeded in following the experimental approach suggested below (see point 9.4). 2) Perform this experiment with cycloheximide to show that the Cdc3 protein is not present upon treatment (i.e. that the block in translation blocks the appearance of the protein on endosomes; We also followed this excellent advice. Treating hyphae with cycloheximide did not influence endosomal localization of Rrm4. However, ribosomal proteins were no longer present on shuttling endosomes after translational inhibition. Importantly, endosomal movement of Cdc3 was inhibited and the gradient in septin filaments was lost. However, septin was still present in filaments. This fits perfectly to our data that translation on endosomes is needed for transport and the formation of gradients of septin filaments at the growth pole. 3) Perform the RNA/protein co-localization experiment with a control cdc3 RNA that lacks the start codons (to show that the RNA is there, but not the protein); We did think about such an experiment before. The problem with this approach is the following: if we offer an mRNA that is not translated due to the missing start codon, we cannot investigate the subcellular localization of the translation product. Therefore, we decided to choose the opposite approach. Removing the RNA-binding binding domain in Rrm4 removed the mRNA from the endosomes. Importantly, the translation product does no longer localize to the endosomes. This is an important finding: without mRNA on endosomes the translation product is in the cytoplasm, and is no longer present on endosomes. 4) Add RFP or mCherry in frame to the coding region of the boxB-tagged cdc3 message (in order to show RNA and protein co-localization upon translation). As indicated above, it was a very valid point and we achieved it. In the initial submission we were only able to show that cdc3 mRNA and Cdc3 protein localize to the same endosomal compartment. Now we show in Figure 4A co-localisation on the same endosome. This was a major step forward. 10. Figure 5 aims to show that endosomal septin transport is important for its accumulation in filaments and at the hyphal tip. Yet, FRAP experiments show a complete recovery of Cdc3GFP even when benomyl is used to uncouple microtubule-based transport. This shows that localized translation of cdc3 mRNA at the hyphal tip accounts for Cdc3 protein accumulation and obviates the transportbased model. The authors might suggest that the slower rate of recovery is proof of endosome-mediated protein delivery, but one could easily argue that it is the lessened delivery of RNA for additional localized translation. The experiment in 5A should be performed with added cycloheximide (together + benomyl) to help determine whether Cdc3 protein at the hyphal tip recovers via transport or localized translation. This could be an important experiment to do. Again we need to apologize but the FRAP experiments were not explained to the full extent. The data indicated relative fluorescence recovery (the plateau is set to 100%). The absolute values were given in Supplementary Fig. 6. As mentioned above, the Cdc3G fluorescence did not recover completely, because of the prolonged time below a coverslip. (Compare Figure 3 and 5 and see the Supplementary Fig. 5). © European Molecular Biology Organization 8 EMBO reports - Peer Review Process File - EMBOR-2013-38037 We can exclude the mechanism of RNA delivery and localized translation because the time for mRNA transport, translation and maturation of Gfp takes considerably longer than 6 minutes. Thus, microtubule-dependent mRNA transport in combination with tip localized translation does not contribute to the measured recovery. We did not perform these experiments with cycloheximide because it would be a very crude approach that inhibits translation of all proteins and alters numerous cell biological processes. Therefore, we favoured in vivo FRAP experiments of the hyphal tip because recovery of the green fluorescent protein takes longer than 15 minutes (Figure 7) and thus we can exclude de novo protein synthesis. Also, why not FRAP a lambdaN-GFP labeled cdc3-mCherry RNA (if created as suggested above in 9) and examine the recovery of both RNA and protein, both with and without cycloheximide and benomyl (alone or together). This could also be supportive of translation on endosomes if the signal for Cdc3 protein, but not cdc3 RNA, recovers in benomyl-treated, but not benomyl- and cycloheximide-treated cells. I approached my colleague Sebastian Baumann with a comparable idea. However, he could argue very convincingly that it is impossible to FRAP a single λN-Gfp-labelled mRNA that is moving at 2.5 µm per second on endosomes and even if he would succeed, he would never be able to trace it back again. Thus, we are facing the same problem as the majority of researchers studying mRNA transport and local translation: it is very difficult to show local translation directly. 11. Figure 6 - Why not do this with photoactivatable Cdc3 as well? We tried this experiment in strains overexpressing Cdc3Gpa but the signals of Cdc3Gpa on endosomes were not strong enough to perform these experiments in a comparable manner. Since Cdc3 and Rrm4 co-localize almost exclusively on endosomes, we believe it is fair to draw the conclusions using photoactivatable Rrm4. Reviewer 3 Baumann et al find that septin transcripts and proteins are transported on endosomes for delivery to hyphal tips in the fungus Ustilago maydis. Septin mutants have defects in polarized growth and the transcripts can be visualized as moving in a microtubule dependent fashion at velocities consistent with endosomes. There is some evidence that septin protein can also be found on the endosomes and that the septin transcripts and proteins are dependent on the RNA-binding protein Rrm4. The colocalization of ribosomes with Rrm4 spots supports that transcripts may be translated en route to the tip. This is an interesting finding that merits publication but I would like a few claims substantiated with better quantification, some interpretations moderated and ideally one other septin protein examined to assess whether septin complexes are forming on endosomes. 1. What proportion of Rrm4-decorated endosomes have septin transcript? We quantified the amount of Rrm4-decorated endosomes; the text now reads: “We detected 48 (+/- 8) Rrm4G-positive signals on shuttling units per hyphae (668 Rrm4G signals in 14 hyphae).” and “Detailed microscopic analysis revealed that about one directed 16 cdc3B mRNA particle was present per hypha (Figure 2D).” 2. What is the evidence that there is no pool of cdc3 transcript deposited at tips (mentioned at bottom of page 6). This is based on in vivo RNA live imaging, see referee #2 point 7. © European Molecular Biology Organization 9 EMBO reports - Peer Review Process File - EMBOR-2013-38037 3. Cdc3G is said to localize "almost exclusively" with Rrm4 endosomes-what is the actual proportion not co-localized with Rrm4? We quantified 91 Rrm4C-positive endosomes in six hyphae. 88 of these also carried Cdc3 (97%). See point 4. 4. What is the frequency of septin transcript and protein on anterograde vs retrograde endosomes? As quantified in Figure 2D there is a slight bias of septin mRNAs that are moving in the anterograde direction. The same holds true for Cdc3 protein on endosomes: 47 in anterograde direction and 41 in retrograde direction. The text now reads: “Consistently, Cdc3G co-localised almost exclusively with Rrm4positive endosomes (Figure 3F; 97% of endosomes contained both proteins, 91 Rrm4Cpositive endosomes in six hypha were quantified, 47 and 41 Cdc3G signals in anterograde and retrograde direction; Supplementary Movie S8).” 5. Regarding section, pg 9 "Rrm4 function is essential for correct formation of cdc3 filaments": beginning with the title, this section is over-interpreted. Rrm4 is important for a polarized distribution of Cdc3 protein but filaments are tough to see and confirm at this level. To clarify this point we can submit a high resolution image of the filaments in z-stacks. In our opinion, particularly in the maximum projection (Figure 3A, bottom) it is clearly visible that Cdc3G accumulates in filaments and that the staining is more pronounced at the hyphal tip. The FRAP data indicate a slower recruitment when endosomal targeting or motility is lost but this is not necessarily due to or assessing an "assembly" process. It is a measure of recruitment, diffusion, transport to this location. Diffusion and transport take place in the order of seconds. Thus, a recovery half life in the order of minutes indicates that the protein cannot be replaced by an incoming protein very quickly. This is indicative for the interaction of the protein with other macromolecular components such as other proteins. Comparable FRAP experiments were done for the analysis of septins in S. cerevisiae (Dobbelaere et al. Dev. Cell 2003; Caviston et al. MBC 2003; Li et al. JCS 2012). These authors concluded that the half live in the order of minutes indicates an assembly process. We used the same arguments for our study. In the revised version we include additional FRAP experiments verifying that recovery of cytoplasmic Gfp based on diffusion is in the order of seconds in our system as well (Figure 7D-E). 6. The paper would be strengthened by an analysis of at least one other septin protein that would allow conclusions about where heteromeric septin complexes are forming. It is unclear why Cdc3 would be transported and translated in this way where the other stoichiometric septins in the complex would rely on diffusion. Do any other septin-FPs display an endosomal transport-even if this has to be observed by overexpression it is worth a check because it is not well understood where, when and how septins oligomerize. Can you use FISH to readily assess the distribution of other septin transcripts without strain construction and see if they colocalize on any endosomes or are polarized for local translation at tips. As pointed out above, FISH is not a standard technique and difficult to perform. However, we are grateful for the idea of avoiding strain generation, which is tedious and takes two months to obtain the correct strain on average. Based on this valuable suggestion we studied a second septin, Cdc12, in more depth and the results are described above (see the last major comment of referee #1) and shown in Figure 8. © European Molecular Biology Organization 10 EMBO reports - Peer Review Process File - EMBOR-2013-38037 Revision comments and authors’ responses – EMBO Journal Referee #1: The revisions to this manuscript are most welcome and now greatly aid the readability and do go some way to supporting the authors' conclusions. As they themselves admit, there is no definitive experiment within the manuscript that septin translation does indeed occur on endosomes during their translocation. The point is also well made by the authors that this has not been demonstrated in other systems. The work on Cdc12 is a welcome addition but is unfortunately not supported by mRNA as well as protein localization. While I am reluctant to suggest too many additional experiments, the model that septin assembly happens on endosomes and that Cdc12 is required for stable association of Cdc3 means that the authors case would be supported much more strongly if they could demonstrate localization of Cdc12 mRNA to these motile endosomes. However, the more uniform distribution of Cdc12 along the hypha could mean that endosomal translation of Cdc12 is not necessary since a pool of the protein is available throughout the hypha. Therefore one could argue that this experiment would not provide a definitive answer to the question. Information on septin assembly is currently scarce and our findings that mRNA transport of septin mRNA and septin protein on endosomes is needed for correct subcellular localization of septins is a novel finding and a substantial contribution to the field of septin biology. In preliminary experiments we observed that, as expected, cdc12 mRNA is transported on Rrm4-positive endosomes. However, we agree that these further results do not provide the definite answer and are thus beyond the scope of this manuscript. I do find one statement in particular lacking in its scope. The authors state on page 15 that: "These data are consistent with our hypothesis that at least some steps in septin assembly take place on endosomes and this is a prerequisite for formation of functional septin filaments." While true, do these data not also support a model whereby septin assembly is required prior to endosomal loading? Translation of Cdc12 and Cdc3 after delivery could then augment this pool of assembled septin. We agree that we have to phrase this more carefully. Septin assembly can take place in the cytoplasm independent of endosomal localization because we observe the formation of higher order septin structures at septa and filaments in rrm4D mutants. However, for efficient formation of the Cdc3 septin gradient we need translation on endosomes. Please, keep in mind that without septin mRNA on endosomes, we do not observe septin protein on endosomes. Thus, cytoplasmic assembly of Cdc12 and Cdc3 is not sufficient to enter the endosomal compartment. Consistent with this view, we observe that in the absence of mRNA transport aberrant small septin rings are formed in the cytoplasm. To sum up, of course there are alternative explanations for our work, but we feel that our © European Molecular Biology Organization 11 EMBO reports - Peer Review Process File - EMBOR-2013-38037 hypothesis is very valid because it is consistent with the results presented. I do agree that the data are consistent with the authors' model but it is important to indicate throughout that there are alternative explanations for these data. In summary, I would be supportive of publication. We are very pleased that you support publication of the revised version. There also remain a few small typographical errors. Referee #2: General This is the revised version of a manuscript #84671 in which septin mRNA (cdc3) and protein (Cdc3) are proposed to undergo delivery to the hyphal tip in Ustilago maydis on endosome-like structures to ensure proper septin formation, and that the uncoupling of RNA delivery/association on these membranes (via the deletion/mutation of an RNA-binding protein Rrm4, for example) blocks efficient/normal unipolar growth. This reviewer had serious issues regarding the initial submission about whether the authors can argue that mRNA translation on endosomes indeed accounts for septin delivery to the hypha, based upon the original experimentation presented. The authors now have added additional experiments that they conclude supports their original hypothesis. The work is improved in many areas including its clarity and the added experiments alleviate some of the original concerns, i.e. about the identity of the Rrm4-associated membranes (i.e. co-localization of Rrm4 and Rab5a in new Figure 2A), or that it is now stated in the text that lambdaN-GFP does not show directed movement in the absence of boxB-labeled cdc3 RNA. Also, the imaging of cdc3 mRNA and Cdc3 protein within the same cell (Fig. 4A) is a necessary addition. However, additional controls (that might help the author's case) are still lacking (see specific comments). While the work is interesting, I am not convinced (as of yet) that their argument against a classical model for localized (cdc3 mRNA) translation as a source for Cdc3 protein (or for that matter, endosome-mediated protein delivery) is not valid in septin formation in Ustilago. Without being able to directly show where cdc3 mRNA translation is actually happening it is impossible to conclude that it occurs on endosomes (even if the RNA and ribosomes are present on the endosome membrane). And if it is just a suggestion, as the authors say in many places, then the significance of the work is lessened. While one cannot rule out translation on endosomes, it is just as likely that everything could be co-transported together (including any pre-synthesized/recycled Cdc3 protein that is endosomally targeted along with cdc3 RNA). Our improved FRAP experiments demonstrate that local translation at the hyphal tip of Cdc3G does not contribute to the formation of septin filaments at the hyphal tip. These in vivo data strongly argue against the proposed mechanism that formation of septin filaments is dependent on microtubule-dependent delivery of © European Molecular Biology Organization 12 EMBO reports - Peer Review Process File - EMBOR-2013-38037 mRNAs and their local translation at the hyphal tip. Ironically, while the authors state that cdc3 localization to endosomes is crucial for the presence of the protein there - in fact, their new data (Fig 4A) demonstrates that there are many more Cdc3 protein-labeled endosomes than cdc3 mRNA-labeled endosomes in the same cell at the same time. This would seem to preclude the idea that presence of the RNA precedes (or is causal to) the appearance of the protein on the endosomes. The new data are completely consistent with the data from our first submission. Already in the first submission we noted that there are only few mRNAs present on endosomes and that Cdc3 protein is present on substantially more endosomes. To solve this apparent contradiction we analysed the fission of endosomes using photoactivatable Rrm4. We demonstrate active fission and since the amount of Rrm4-positive endosomes does not increase over time it is logic that active fusion must take place at the same rate. Thus, our hypothesis that septin translation takes place on endosomes and is the entry point to this endosomal compartment is valid. Septin protein is then spread throughout the endosomal compartment by active fission and fusion. This is needed for efficient assembly of the gradient observed at the hyphal tip. If one would examine the localization of native cdc3 RNA (i.e. using single molecule FISH), instead of only the tagged RNA, it is possible that a better accounting of RNA localization could be made (see comment 5 below). Here, we can only repeat our arguments, which are still valid. We have shown in our previous publication (König et al. EMBO J. 2009) that our RNA live imaging data are fully consistent with FISH experiments and single molecule FISH experiments are very difficult to perform in U. maydis. We do not as yet have the numerous years of experience like in other model systems such as yeast and Drosophila. Moreover, experiments using cycloheximide to examine Cdc3 localization in hypha fail to consider RNA localization under these same conditions and, moreover, show no reduction in Cdc3 protein levels along the length of the hypha. The cycloheximide experiments must be carried out under conditions when translation is blocked but the protein is still present in comparable amounts. Otherwise, we would detect the decrease of septin protein on endosomes because the protein is simply degraded. On the other hand we have to treat the cells with cycloheximide for a certain amount of time because Cdc3 protein is present at the onset of the experiment. The additional control experiment is a good suggestion. However, it does not provide definite proof (see below). In addition, FRAP experiments with GFP-tagged Cdc3 show that at best a block in transport of protein/RNA only results in a two-fold change in recovery time vis a vis untreated cells - therefore, the entire raison d'etre for active targeted delivery of either RNA and/or protein is a matter of efficiency, but not of necessity. This lowers the overall enthusiasm for the study. Biology is quantitative and efficiency is as important as necessity. Referee #2 mentioned in the first response that the study by Kilchert and Spang (EMBO J., © European Molecular Biology Organization 13 EMBO reports - Peer Review Process File - EMBOR-2013-38037 2011) is a good example where authors demonstrated that translation takes place during RNA transport. Please note that in only about 50% of budding cells the ABP140 mRNA localizes to the distal pole and there is up to date no biological function assigned to this localization process. Overall, the work is too lengthy and ponderous, redundant in places, and does not prove the author's contention that either targeted septin delivery to the hypha is all that important (for proper hyphal formation/filamentation) or that septin RNA translation on endosomes actually occurs. Thus, the work might be more appropriate for a specialty publication if they cannot add more convincing data. Referee #2 did spend a lot of time and effort in our work and we acknowledge this. However, even after these two extensive rounds of criticism, the numerous results and in vivo data that we provided are still consistent with the hypothesis that translation takes place on endosomes. Alternative explanations, such as translation in the cytoplasm, fail to explain the observation that cdc3 mRNA must be present on endosomes for the localization of the Cdc3 protein on endosomes. Specific comments 1. Figure 2C - It appears that cdc3 mRNA does not accumulate at the hyphal tip in Figure 2C, as noted by the authors, although in Figure 2F there is certainly more concentrated GFP fluorescence there (i.e. being indicative of RNA). Which is it? Can a photo of the hypha shown in the kymograph in SF be provided? This is an important point in regards to the localized translation model for Cdc3 accumulation at the hyphal tip. There is no accumulation at the hyphal tip. Please watch Supplementary Movie S3. The figure in 2F is taken from the center of the filament and we can easily provide the picture. 2. While it is convincingly shown that cdc3 RNA shows directed movement on Rrm4-labeled membranes and, by correlation (with Rrm4) on Rab5a- and FM4-64 labeled membranes in a microtubule-dependent (benomyl-inhibited) manner, the work is still missing some negative controls. Although the authors mention in their rebuttal that they have used such controls in a previous study (Koenig et al 2009), they are still necessary for this work. For example, perhaps all RNAs undergo Rrm4-directed movement? If so, perhaps the level of specificity implied in the paper for cdc3 mRNA transport is overstated. We have shown in König et al EMBO J. 2009 that all mRNAs can be transported by the Rrm4 machinery. The control mRNA is transported less often and over shorter distances. We cannot repeat all our previous and already published experiments in every new manuscript. Our results are consistent with the microtubule-dependent movement of the poly(A) binding protein (see Supplementary Movie S1). Most endosomes are loaded with mRNA. Thus, there are numerous other mRNAs that are transported by this machinery for example ubi1, rho3 and cts1, and we never claimed that cdc3 mRNA is the only target. 3. The switching of markers between the different figures to show an © European Molecular Biology Organization 14 EMBO reports - Peer Review Process File - EMBOR-2013-38037 endosomal pattern of localization for Rrm4, and for cdc3 mRNA or Cdc3 protein, is a bit tedious. For example, Rrm4 is shown to co-localize with Rab5a (Fig. 2A), while tagged cdc3 mRNA is shown to localize with Rrm4 (Fig. 2F), and Cdc3 protein with FM4-64 (Fig 3F) and then with Rrm4 (Fig. 3G). Is cdc3 mRNA ever shown to associate with an endosomal marker directly, just via the presumed endosomal localization of Rrm4? Reading this comment it is absolutely clear that we will never convince Referee #2. The endosomal localization of Rrm4 is supported by numerous in vivo observations. The endosomal compartment that is positive for Rrm4 is well studied in Ustilago maydis. Gero Steinberg’s group in Exeter published a number of papers on this subject that show that these endosomes are positive for Rab5a, Yup1 and can be stained with FM4-64. The endosomes are transported to the plus ends of microtubules by Kin3 and to the minus end by split Dyn1/2 (Wedlich-Söldner et al. EMBO J. 2000; Wedlich-Söldner et al. EMBO J. 2002; Fuchs et al., Plant Cell 2006; Lenz et al. EMBO J. 2006; Schuster et al. EMBO J. 2011; Schuster et al. PNAS 2011). We recently published in the Journal of Cell Science that Rrm4 is transported by the same set of motors and that the mechanism of mRNA transport is co-transport of Rrm4 with Rab5a-positive endosomes. This is a novel mechanism of mRNA transport and we were first to identify the motor set that is necessary for microtubule-dependent shuttling of mRNPs: kinesin-3 type motor for plus-end movement and dynein for minus-end movement. This work was performed with the same high scientific quality as the results presented here, and there we also provided mainly in vivo data. Recently, evidence is accumulating that Kinesin-3 type motors are involved in neuronal transport of mRNAs (Charalambous et al. 2013 Cell Mol Life Sci; Lyons et al. 2009 Nat Genet). Thus, we believe that this mode of transport is not restricted to U. maydis. Here, we show that Rrm4 co-localizes with Rab5a in 81% of all cases. The amount of co-localization is underestimated because Rab5a is more difficult to detect than Rrm4 (Supplementary Movie S2). Thus, there is no doubt that Rrm4 localizes to Rab5a-positive endosomes and that Rrm4 is the better endosomal marker because it exclusively localizes to endosomes and not to additional membrane compartments like Rab5a (compare Supplementary Movies S1 and S2 for endosomal movement of Rrm4 versus Rab5a, respectively). 4. Cdc3 protein is enriched in the hyphal tips (Fig. 3A), although according to the kymograph in Figure 3F it is moving in different directions on the FM464-labeled membranes. Which is it? Where is a picture of the hypha from which the kymograph in Fig. 3F was made? It would be nice to know to where the particles actually localize therein. Likewise, the non-moving Cdc3 particle in Fig. 3G - where in the hypha is that? The tip? Again, a picture of the entire hypha should be included for reference. For a complete view, please watch our Supplementary Movie S6. Cdc3 shuttles on endosomes in both directions most likely for correct assembly of the septin subunits. According to our in vivo FRAP experiments microtubule-dependent transport is needed for efficient filamentation at the hyphal tip. The non-localizing © European Molecular Biology Organization 15 EMBO reports - Peer Review Process File - EMBOR-2013-38037 particles correspond to the accumulations at the cortex as can be seen in Figure 3A. 5. In my initial review, I argued that the translation of tagged cdc3 RNA might preclude its visualization due to the fact that the multiple lambda N RNAbinding proteins (with multiple GFPs) bound to the 3'UTR might require removal before translation termination could be completed. Therefore, translating cdc3 RNA at the hyphal tip (or wherever) might become "invisible" during the translation process, while the endosome-localized translation-repressed cdc3 RNA could remain visible. This might lead to authors to incorrectly conclude that RNA is not accumulating at the hyphal tip, and why a FISH experiment is called for. The authors argue that this experiment is difficult to perform, however, single molecule FISH, which gives very robust signals, should work well in this system and would go a long way to refute the idea that RNA accumulation and localized translation in the hyphal tip is not the mechanism for Cdc3 protein deposition. The authors also note in their rebuttal letter that the work of Singer and colleagues suggests that aptamer-tagged RNAs can be translated (and are functional). We do not argue against this idea at all. However, we do argue that it is completely unclear whether actively translating aptamer-tagged RNAs are still visible (i.e. by indirect fluorescence via a fluorescent RNAbinding protein). This has not been resolved. Again, single molecule FISH might help the author's case here, by localizing where endogenous native cdc3 mRNA is present within the cell under the different conditions examined (i.e. in wild-type cells, upon rrm4 deletion, upon benomyl addition, etc.). This assay would better account for where both translationally active and repressed cdc3 mRNA is present within the cell. There are currently no indications either by us or other groups that translation would remove RNA-binding proteins in the 3’ UTR during RNA live imaging. We already explained that FISH experiments are difficult to perform in our system. We tried hard to obtain good quality results but we failed. Our main problems are that firstly during the fixation process the hyphal filaments shrink and we lose spatial resolution. Secondly we observe spotty background signals so that we cannot differentiate between mRNA on endosomes and background. However, we do not observe accumulation of cdc3 mRNA at the hyphal tip as shown for ASH1 mRNA at the daughter cell pole in S. cerevisiae. Also in the work of Kilchert and Spang, 2011 the authors did not perform single molecule FISH. They only observe mRNA accumulating at the pole and never single molecules moving towards the pole. However, we are left with the following criticism. “Therefore, translating cdc3 RNA at the hyphal tip (or wherever) might become "invisible" during the translation process, while the endosomelocalized translation-repressed cdc3 RNA could remain visible.” Since we cannot provide in situ data, we tackled this by in vivo FRAP experiments. We now show that local translation of cdc3G mRNA at the hyphal tip cannot contribute to the observed accumulation of Cdc3G protein at the hyphal tip because translation and maturation of the Gfp fusion protein take too long. 6. Figure 4 - The live cell imaging of cdc3 mRNA and Cdc3 protein is a necessary addition to the paper, although it seems that only a few co-labeled particles track together in the kymograph (Fig. 4A), whereas numerous Cdc3labeled particles can be observed in the figure. Why is this? Doesn't this © European Molecular Biology Organization 16 EMBO reports - Peer Review Process File - EMBOR-2013-38037 imply that there are only a few cdc3 RNAs that are undergoing trafficking, whereas there are many more endosomes that are labeled with protein? If so, how does an endosome-based translational model account for that? The authors then state that the presence of cdc3 mRNA is crucial for the endosomal localization of Cdc3 protein (p. 11, line 3). Unfortunately, this causal statement is not supported by their own data (Fig. 4A), where clearly there are Cdc3-labeled endosomes that lack RNA. Thus, presence of the RNA on the endosome is not a pre-requisite for the protein to be associated therewith. While endosome fusion/fission events could be responsible for some of these effects, the evidence presented (Fig. 6) is incomplete and does not examine Cdc3 protein itself. As pointed out above, the results are consistent the results of our initial submission. For technical reasons we used Rrm4 and not Cdc3 because the Rrm4 is the superior endosomal marker. Even in strains strongly overexpressing photoactivatable Cdc3G we were unable to detect photoactivated protein. We strongly believe that our approach using Rrm4 as endosomal marker is valid because 97% of the endosomes contain both proteins. Thus, it is impossible that Rrm4 and Cdc3 would be separated during the fusion and fission process. Our observation that without mRNA Cdc3 protein cannot enter this endosomal compartment is a very important finding. However, once the protein is present on endosomes mRNA is no longer needed. We will revise this sentence to make this clear. 7. Figure 5F and G demonstrate that the treatment of hypha with cycloheximide alters the localization of Cdc3 protein. The results show a possible lack of shuttling of the protein, which the authors suggest is additional proof of translation on endosomes being needed for Cdc3 transport. There are problems with this experiment: 1) Cycloheximide treatment of the hypha is for nearly 4 hours and, thus, it is impossible to know how many other cellular processes are altered during this very lengthy period of treatment (i.e. most likely those related to cytoskeletal function/endosome movement, based upon the results); We agree that results using translational inhibitors have to be viewed critically. Therefore, we did not perform these experiments in the first place and only after Referee #2 suggested this type of experiment. The results have to be taken cautiously and can only support our strong in vivo data. Our main aim of this study was to show that the ribosomes on endosomes are translationally active. Therefore, we treated the cells for 80 minutes (not 4 hours) with translational inhibitor CHX. We observed the absence of ribosomes on endosomes. 2) Based upon the level of protein labeling, the levels of translated Cdc3 protein in the kymograph look about the same as in the untreated cells wouldn't one expect to see less signals on the endosomes in the presence of cycloheximide, unless they were already loaded with protein at the start of transport?; The cycloheximid treatment was started after hyphae were formed. We cannot treat yeast cells with CHX because we need transcriptional activation and translation of the transcription factor that activates filamentous growth. Thus, Cdc3G protein was present on endosomes at the onset of the experiment. To deplete this relatively stable pool of septins we had to treat for 3.5 hours. As expected we observe less septin protein on shuttling endosomes. © European Molecular Biology Organization 17 EMBO reports - Peer Review Process File - EMBOR-2013-38037 3) Finally, it's impossible to talk about RNA localization and translation on the endosome when no localization of RNA is actually performed in the same experiment, and since it appears that not all endosomes have RNA (Fig. 3G and 4A) it may be questionable what the source of protein was. As pointed out above this is a good control experiment. However, it still would not give a definite answer. If the septin mRNA is still transported but not translated this would support our hypothesis. If the septin mRNA is no longer transported on endosomes this would also support our hypothesis. Without mRNA on endosomes there is no septin protein in the endosomal compartment. 8. Figure 7A-C - If the recovery of Cdc3-GFP from FRAP in benomyl-treated wild-type cells (or in rrm4delta mutants, in which mRNA transport should be blocked entirely) is only twice the time of recovery in FRAP-treated wild-type cells, it says that there is either another delivery process for Cdc3 (i.e. cytoplasmic diffusion) or localized translation of cdc3 at the hyphal tip is operant (or perhaps both). The FRAP experiments clearly exclude local translation at the hyphal tip because translation and maturation are not fast enough to contribute to the measured recovery. We do not argue that translation only takes place on endosomes because we do see recovery in the absence of Rrm4. But endosomal transport of septin protein is needed for efficient assembly. The former certainly appears likely, given the recovery of GFP alone after FRAP (Fig. 7D). This result call into question the relevance of the findings to cell physiology - perhaps, as they authors suggest, that active cdc3 RNA/protein transport improves efficiency of the system, but since it isn't an absolute requirement it lowers the enthusiasm for the study. We disagree with the statement that a twofold difference is not relevant for cell physiology. 9. Finally, the authors show that another septin, Cdc12, is required for Cdc3 localization to endosomes. This seems to argue for the anchoring of septins to the endosomes, but does not necessarily support an endosome-based translation model. What happens to cdc3 mRNA localization in the absence of cdc12? Is that affected?? Correct, according to our preliminary results cdc12 mRNA is also transported on Rrm4-positive endosomes. Thus, most likely assembly takes place on endosomes and without Cdc12, the septin Cdc3 cannot be stably anchored on endosomes. However, in accordance with Referee #1 these results are beyond the scope of this work. Referee #3: I am happy with the thorough response of the authors to the reviews and feel this work should be published in EMBO. © European Molecular Biology Organization 18 EMBO reports - Peer Review Process File - EMBOR-2013-38037 1st Editorial Decision - after transfer to EMBO reports 26 September 2013 Thank you for the transfer of your revised manuscript to EMBO reports. Given that after crosscommenting on each others' reports, two referees clearly support publication of the study and acknowledge that this is an interesting paper, I am happy to tell you that we can offer to publish it. However, the remaining referee concerns need to be addressed in the manuscript text, the main manuscript text needs to be substantially shortened and the number of main figures must be reduced. As the referees request, it needs to be mentioned in the main text that alternative models for cdc3 translation and protein transport cannot be ruled out with certainty, as for example, the endosomal transport of pre-synthesized Cdc3 protein in the FRAP experiment. It would be good to more clearly work out what exactly can be concluded from each experiment and whether alternative explanations are possible. Regarding figure 4, it is not exactly clear to me whether the data indeed show that cdc3 mRNA is essential for protein localization on endosomes, or whether the absence of mRNA simply reduces the amount of transported protein. This needs to be clarified in the text. It also needs to be clearly stated that the FRAP experiment shows that while the transport of Cdc3 protein and mRNA contributes to fluorescence recovery it is not crucial. I further agree with referee 2 that the cycloheximide experiments are not really conclusive, especially the one with the very long treatment. This could be taken out. While it is interesting that the shorter treatment reduces the transport of ribosomes, 80 min is still a long time and may cause a number of defects. Given that EMBO reports only allows a maximum of 5 main figures, I suggest that the cycloheximide experiments and the data on cdc12 could be moved to the supplementary information, in addition to all confirmatory and redundant data. We usually also only allow 5 supplementary figures, but can make exceptions. However, please reduce the number of supplementary figures as much as possible (some can potentially be combined). The main text has currently 66.700 characters, and we only allow a maximum of 30.000 (including spaces, figure legends, references). EMBO reports papers have a numbered reference style, and changing to this style will help in shortening the text. We also allow the combination of the results and discussion sections, which may help to eliminate some redundancy that is inevitable when discussing the same experiments twice. Parts of the materials and methods can further be moved to the supplemental information, but please note that the materials and methods essential for the understanding of the experiments described in the main manuscript file must remain in the main methods section. Commonly used methods can be moved to the supplementary file. For all quantifications and statistical analyses, please mention the number n of how many experiments were performed, and specify the bars and error bars and the tests used to calculate pvalues in the relevant figure legends. This information is currently incomplete and must be provided in the legends of both main and supplementary figures. Finally, I would like to suggest to include the most interesting hypothesis of septin translation on endosomes in the title: Endosomal transport of septin mRNA and protein indicates local translation on endosomes and is required for correct septin filamentation I also have some suggestions for changes to the abstract: Endosomes transport lipids and proteins over long distances by shuttling along microtubules. They also carry mRNAs on their surface, but the precise molecular function of this trafficking process is unknown. By live cell imaging of polarized fungal hyphae, we show microtubule-dependent transport of septin mRNA and encoded septin protein on the same shuttling endosomes. Consistent with the hypothesis that septin mRNA is translated on endosomes, the accumulation of septin protein on endosomes is tightly linked to the recruitment of septin mRNA. Furthermore, ribosomal proteins co-localize with shuttling endosomes, but only if they are translationally active and if mRNA is present. Importantly, endosomal trafficking is essential for an efficient delivery of septin © European Molecular Biology Organization 19 EMBO reports - Peer Review Process File - EMBOR-2013-38037 protein to filaments at growth poles, a process necessary to establish unipolar growth. Thus, we propose that local mRNA translation loads endosomes with septins for assembly and for an efficient delivery to septin filaments. I look forward to seeing a new revised version of your manuscript as soon as possible. Please let me know if you have any further questions or comments. 1st Revision - authors' response 10 October 2013 Revised version received. 2nd Editorial Decision 14 October 2013 I am very pleased to accept your manuscript for publication in the next available issue of EMBO reports. Thank you for your contribution to our journal. 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