Download Endosomal transport of septin mRNA and protein indicates local

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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
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26 September 2013
26 September 2013
10 October 2013
14 October 2013
Editor: Esther Schnapp
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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.
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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.
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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:
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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.
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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
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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:
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“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.
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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.
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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).
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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.
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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.
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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
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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
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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.,
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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
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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
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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
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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.
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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.
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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
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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
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