The EMBO Journal Peer Review Process File - EMBO-2014-88466
Manuscript EMBO-2014-88466
Tubulin glycylases are required for primary cilia, control of
cell proliferation and tumor development in colon
Cecilia Rocha, Laura Papon, Wulfran Cacheux, Patricia Marques Sousa, Valeria Lascano, Olivia
Tort, Tiziana Giordano, Sophie Vacher, Benedicte Lemmers, Pascale Mariani, Didier Meseure, Jan
Paul Medema, Ivan BiËche, Michael Hahne, Carsten Janke
Corresponding author: Carsten Janke, Institut Curie
Review timeline:
Submission date:
Editorial Decision:
Revision received:
Accept letter:
Accepted:
11 March 2014
10 April 2014
17 July 2014
30 July 2014
30 July 2014
Editor: Thomas Schwarz-Romond
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.)
1st Editorial Decision
10 April 2014
Thank you very much for submitting your study describing a potential crucial role of glycylases in
primary cilia formation/maintenance for consideration to The EMBO Journal editorial office.
You will find two sets of consistent and constructive comments from two expert scientists enclosed
below.
As you will recognize, both scientists are aware of potential merit and general value of the data,
though also outline necessary and relevant further experimentation that would have to be provided
to substantiate the major conclusions. Specifically, both request:
-complementary immunohistochemistry to bolster claims about glycylase expression;
-assess presence of cilia in the CRC-lines upon confluent culture conditions and extended
starvations;
-re-expression of Ttll3 to check for sufficiency of this glycylase for cilia formation;
-place the results on nuclear CyclinD1 rather in the context of general proliferation regulation than
actual Wnt-signaling;
-extend/substantiate the proliferation results in single versus compound TTLL3/8 Mefs as
recommended by ref2 point 6;
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Conditioned on these rationale and constructive amendments, I am delighted to invite a revised
version of your dataset for final assessment/eventual publication. I realize that some of these present
possibly challenging demands. However, I certain you have to agree that these would dramatically
improve the currently rather indicative, to a definitive and much stronger paper.
_____
REFEREE REPORTS:
Referee #1:
In this study by Rocha and colleagues, the authors make several interesting observations. They first
demonstrate that two separate tubulin glycylases are expressed in MEFs and required for ciliation:
the first demonstration of a role for tubulin acylases in non-motile cilia. They then show that only
one of these glysylases, ttll3 is expressed in colon tissue, and that in mice deficient for ttll3, the total
number of cilia is reduced, tissue is more proliferative, and the colon is more prone to tumorigenesis
upon exposure to a mutagen. Given the very limited characterization to date of any kind regarding
cilia in the colon, these are useful advances. The study is hence novel, and potentially a valuable
contribution to the literature. At present, there are some weaknesses in the data and interpretation
that need to be addressed to justify the authors' claims.
Major Critiques.
1. All claims regarding expression of TTLL3 and TTLL8 are based on RT-PCR. A quick check of
Biocompare and other sources indicates that there are antibodies to ttll proteins available that are at
minimum functional in human tissues, and may also be usable in mice. These should be used to
bolster the claims about expression.
2. Figure 3 should be supplemented with antibody TAP952, to confirm absence of glysylated tubulin
in ttll33-/- CECs.
3. Development of the digestive tract is highly dependent on Hedgehog signaling, which in turn is
highly dependent on intact cilia. It is very surprising that a mutation that greatly reduces ciliation in
colon crypts is not causing extensive abnormalities. The authors should look at basic hallmarks of
Hedgehog signaling in ttll3-/- colons, and determine whether CECs are no longer responsive to
Hedgehog ligand.
4. The authors base their claim of altered Wnt signaling on increased cyclin D in ttll3-/- cells. This is
a very non-specific marker of Wnt signaling; indeed, the fact of greater cycling by ttll3-/- cells
makes it inevitable that cyclin D will be elevated. To confirm differences in Wnt signaling, the
authors need to show greater levels of nuclear beta-catenin, or Tcf1/Lef. In terms of Wnt activation,
it somewhat argues against the author's point that Supp Fig S3C does not show elevated nuclear
beta-catenin in ttll3-/- normal tissue, versus control normal tissue.
5. The results of cilia in CCD 841 cells, and absolutely no cilia in DLD1 or SW480 cells, are
somewhat surprising. The stated methods note that cells were starved for 24 or 48 hours for
ciliogenesis, which is a very short period of time, particularly for transformed cell lines. Based on
the images supplied in Figure 6B, the cells were not confluent, which also does not support optimal
ciliogenesis. The authors need to repeat these experiments, starving cells at least 72 hours at high
confluency, then count ciliary frequency rather than show a single image of ciliated or non-ciliated
cells.
Minor points.
1. In the interest of saving space, some of the figures of cells are very large, and can be reduced in
scale. In addition, Figures 5E and 6D can be moved to supplemental figures, as they do not add
significantly to the study.
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Referee #2:
The manuscript by Rocha et al. presents interesting evidence for the hypothesis that tubulin
glycylation plays an essential role in the maintenance of primary cilia and indirectly in cell cycle
control, based on the analysis of defects in colon epithelial cells of the TTLL3 knockout mouse. The
authors show that tubulin glycylation occurs in primary cilia from overlapping activities of the
tubulin glycine ligases, TTLL3 and TTLL8, and that complete loss of glycylation affects primary
cilia formation or stability.
While the work is interesting and novel, it suffers from over-interpretation of the data and some
internal logical inconsistencies. To fully support all the claims made in the paper, some additional
experiments would be required.
Comments
1. The biggest discrepancy seems to be the claim that presence or absence of cilia is a defining
regulator of intestinal cell proliferation. Yet the authors show that >20% of wildtype crypts have no
detectable cilia and the largest fraction of crypts show only 1 ciliated cell (figure 3). Perhaps another
antibody staining (acetylated tubulin?) would help resolve whether or not the majority of crypt cells
are actually ciliated. Otherwise how could loss of cilia affect crypt cell proliferation (if many are not
ciliated to start with)? Also: perhaps the authors could comment on the cytoplasmic staining for
Arl13b in the Ttll3 mutant (figure 3A).
2. The fact that 20% of ttll3-/- CEC cells still had cilia (figure 3E) means that glycylation is not
absolutely required for ciliogenesis. When the authors conclude that "our results support a model in
which the majority of cilia, primary as well as motile, depend on the presence of glycylation
catalyzed by either TTLL3, TTLL8 or both enzymes" it is not very clear what they mean. I think it
would be safe to say that absence of glycylation affects the efficiency of ciliogenesis but that it is
not absolutely required.
3.With regard to the absence of cilia in the SW480 cell line and the statement that "These
observations confirm that absence of glycylating enzymes result in absence of primary cilia": there
are many other explanations for why SW480 cells might lack primary cilia other than the absence of
Ttll3; at best this is a correlation and does not confirm their hypothesis. The authors would have to
re-express Ttll3 in the SW480 cells and show rescue of ciliogensis to make this claim.
4. Presence of nuclear Cyclin D1 is a marker of proliferating cells; it is not specific to Wnt
activation of proliferation. So the statement that "This strongly suggests that the increased cell
proliferation ttll3-/- colon epithelium is initiated by an upregulation of the Wnt signaling pathway"
really has no support in the data shown. It may well be that Wnt signaling is driving the observed
proliferation, as it normally drives crypt cell proliferation, but the authors have not shown data in
support of this.
5. Inspection of Fig.3A indicates that a majority of the cilia are present in the top compartment
where the cells are more differentiated. Reduced presence of primary cilia on CECs of TTLL3
knockout mice could also be explained on account of delayed or incomplete differentiation prior to
their accelerated shedding. Can the authors comment on this alternative hypothesis?
6. The in vitro analysis of cell proliferation and presence of cilia in CECs from different mice in
Fig.5S, suggests that reduced stability of CEC primary cilia, induced by loss of TTLL3
(glycylation), favors cell proliferation. Do MEFs with compound deficiency of TTLL3 and TTLL8
(figure 1F) also have higher in vitro proliferation rates compared to wildtype MEFs or those
deficient in TTLL3 or TTLL8 alone?
Minor points:
In Fig. 1 C it is not clear what the n values represent.
The sizes of mouse TTLL3 RT PCR amplicons shown in Fig. 1A are different than those shown in
Fig.2B and in testis control (Fig. 6A). It is not clear whether different primer pairs were used for
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amplification or the position of the marker is shifted.
The images of tumor histopathology shown in Fig.5E are not clear in the printed size. These might
appear clear in online views and could be provided as supplemental data.
Reference to Figure S5 is missing in the results section. This includes important details related to in
vitro proliferation potential of CECs.
Typographic errors: The word "adherens", describing the epithelial junction has been misspelled as
"adherent" or "adherence"; extra word in sentence " can is present" (page 5 paragraph 2); "positivie"
(page 10, line 2).
1st Revision - authors' response
17 July 2014
Referee #1:
In this study by Rocha and colleagues, the authors make several interesting observations. They first
demonstrate that two separate tubulin glycylases are expressed in MEFs and required for ciliation:
the first demonstration of a role for tubulin acylases in non-motile cilia. They then show that only
one of these glysylases, ttll3 is expressed in colon tissue, and that in mice deficient for ttll3, the total
number of cilia is reduced, tissue is more proliferative, and the colon is more prone to
tumorigenesis upon exposure to a mutagen. Given the very limited characterization to date of any
kind regarding cilia in the colon, these are useful advances. The study is hence novel, and
potentially a valuable contribution to the literature. At present, there are some weaknesses in the
data and interpretation that need to be addressed to justify the authors' claims.
Major Critiques.
1. All claims regarding expression of TTLL3 and TTLL8 are based on RT-PCR. A quick check of
Biocompare and other sources indicates that there are antibodies to ttll proteins available that are
at minimum functional in human tissues, and may also be usable in mice. These should be used to
bolster the claims about expression.
We fully agree with the referee. However, we encountered several difficulties with commercially
advertised antibodies. For example, we have tried several times to order an anti-TTLL3 antibody
from Sigma, and were told each time that it is not deliverable. When we inspected the datasheet of
the anti-TTLL3 antibody from Abcam, we noticed that this antibody recognizes a protein of about
10 kDa in the immuno blot, which does not correspond to the expected molecular weight of TTLL3
of about 90 kDa. Finally, several antibodies commercially available only recognize the antigen when
overexpressed.
We were able to raise some antibodies for TTLLs in our lab (including an anti-TTLL8 antibody, but
unfortunately no TTLL3-antibody). For all antibodies we could confirm their specificity to their
respective antigens when overexpressed, but none of them was able to detect the endogenous
proteins, not even in cilia (where these proteins should be accumulated). A possible explanation for
this is that despite the upregulation of glutamylation and glycylation during ciliogenesis, the total
protein levels of the modifying enzymes are too low to be detectable by the presently available
antibodies. For these reasons we decided to use qRT-PCR for analysis of expression levels.
2. Figure 3 should be supplemented with antibody TAP952, to confirm absence of glycylated tubulin
in ttll33-/- CECs.
Currently, the unique antibody available for the staining of monoglycylation is the antibody
TAP952. This antibody has been initially raised against glycylated tubulin from Paramecium, but
has later been shown to nicely label motile cilia of many organisms, however barely primary cilia.
This difficulty in detecting primary cilia is most likely related to lower glycylation levels as
compared to motile cilia, but could also be related to the different use of modification sites (which
are all clustered within the tubulin tail domain, see Bulinski & Janke, 2011).
We have tried many different conditions to label glycylation in primary cilia in colon epithelium in
both, tissue sections and cultured colon epithelial cells, but never managed to obtain a specific
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labeling. However it is important to notice that even well-established cilia markers of tubulin
modifications, such as acetylation, are difficult to use in colon. This point is discussed in more detail
in the response to referee 2.
3. Development of the digestive tract is highly dependent on Hedgehog signaling, which in turn is
highly dependent on intact cilia. It is very surprising that a mutation that greatly reduces ciliation in
colon crypts is not causing extensive abnormalities. The authors should look at basic hallmarks of
Hedgehog signaling in ttll3-/- colons, and determine whether CECs are no longer responsive to
Hedgehog ligand.
We followed the suggestion of the referee and have analyzed the expression of some Hh-related
genes (Shh, Ihh, Dhh, Ptch1, Ptch2, Smo, Gli1, Gli2, Gli3) by qRT-PCR. Though some of these
genes tended to have altered expression levels in ttll3-/- colon, the differences were not statistically
significant, and we therefore decided to not include these data in the current manuscript.
4. The authors base their claim of altered Wnt signaling on increased cyclin D in ttll3-/- cells. This
is a very non-specific marker of Wnt signaling; indeed, the fact of greater cycling by ttll3-/- cells
makes it inevitable that cyclin D will be elevated. To confirm differences in Wnt signaling, the
authors need to show greater levels of nuclear beta-catenin, or Tcf1/Lef. In terms of Wnt activation,
it somewhat argues against the author's point that Supp Fig S3C does not show elevated nuclear
beta-catenin in ttll3-/- normal tissue, versus control normal tissue.
We agree with the concern of the referee to use cyclin D1 expression as a marker for Wnt signaling
and we have thus changed the interpretation of the cyclin D1 data accordingly in the paper.
However, these data nicely confirm the data obtained with BrdU incorporation, thus strengthening
the point of increased proliferation, which is now clearly stated in the text on page 11. As illustrated
for Hh signaling in the previous point our data indicate that the changes in signaling pathways in
colonic crypts of ttll3-/- mice are rather subtle, which might also explain the difficulty to detect
significantly elevated levels of nuclear beta-catenin by immunohistochemistry.
5. The results of cilia in CCD 841 cells, and absolutely no cilia in DLD1 or SW480 cells, are
somewhat surprising. The stated methods note that cells were starved for 24 or 48 hours for
ciliogenesis, which is a very short period of time, particularly for transformed cell lines. Based on
the images supplied in Figure 6B, the cells were not confluent, which also does not support optimal
ciliogenesis. The authors need to repeat these experiments, starving cells at least 72 hours at high
confluency, then count ciliary frequency rather than show a single image of ciliated or non-ciliated
cells.
Following the suggestion of the referee we have performed these experiments several times with
longer starvation times (72 h, as suggested by the referee). We could not detect ciliation in the two
colon cancer cell lines, however surprisingly and not coherently with the findings reported in
Lancaster et al., 2011, we found that only a small percentage of CCD 841 cells grew cilia, and that
this varied from experiment to experiment, while in all experiments, MDCK cells, a standard cell
line for ciliogenesis, grow reliable cilia in the same experimental conditions.
This observation indicates that different versions of the CCD 841 cells exist, and that their
ciliogenesis might be altered. We thus decided to remove the ciliogenesis data on colon cancer cell
lines from the manuscript, and used this opportunity also to re-focus this experiment to the key
message, which is that absence of TTLL3 expression is observed in all cell lines of colon cancer
origin, but not in a cell line that was initially derived from normal colon tissue. The reasons CCD
841 might have partially lost the ability to grow cilia might be multiple, especially because cell
lines, even if derived from normal tissue, undergo several transformations that could have a range of
effects.
We therefore conclude that glycylation is required for primary cilia, but is not sufficient to generate
them and have the clarified throughout the revised manuscript.
Minor points.
1. In the interest of saving space, some of the figures of cells are very large, and can be reduced in
scale. In addition, Figures 5E and 6D can be moved to supplemental figures, as they do not add
significantly to the study.
We agree with the referee concerning Fig. 5E (which was also mentioned by referee 2). This figure
has now been transferred to the supplement. As for Fig. 6D, we believe that it is important to show
this histopathology in the main figure, as it is an important representation of the tissue we have
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analyzed, and allows both, the non-specialist and the specialist reader to understand the differences
between the different tumor types. We have therefore left this part of the figure in the main text.
Referee #2:
The manuscript by Rocha et al. presents interesting evidence for the hypothesis that tubulin
glycylation plays an essential role in the maintenance of primary cilia and indirectly in cell cycle
control, based on the analysis of defects in colon epithelial cells of the TTLL3 knockout mouse. The
authors show that tubulin glycylation occurs in primary cilia from overlapping activities of the
tubulin glycine ligases, TTLL3 and TTLL8, and that complete loss of glycylation affects primary
cilia formation or stability.
While the work is interesting and novel, it suffers from over-interpretation of the data and some
internal logical inconsistencies. To fully support all the claims made in the paper, some additional
experiments would be required.
Comments
1. The biggest discrepancy seems to be the claim that presence or absence of cilia is a defining
regulator of intestinal cell proliferation. Yet the authors show that >20% of wildtype crypts have no
detectable cilia and the largest fraction of crypts show only 1 ciliated cell (figure 3). Perhaps
another antibody staining (acetylated tubulin?) would help resolve whether or not the majority of
crypt cells are actually ciliated.
We agree with the referee that it is not fully satisfying to see only such a small number of cilia per
crypt, however we have tried many different antibodies and experimental conditions and we were
not able to improve this. The major limitation is that Arl13b is the only antibody that reliably detects
primary cilia in the colon. No specific staining was obtained in the colonic epithelium using
acetylated tubulin antibody, which is in agreement with a previous report (Saqui-Salces et al., 2012).
To make the logic of our conclusions easier to follow, we have swapped the panels in Fig. 3 and the
text (page 8-9) such that we first show the ciliation rate of colon epithelia cells in culture, which are
much higher than what we were able to see in the colon crypts. We also discuss that the low number
of cilia in colon tissue are rather the result of a technical limitations (page 9).
Notably, to our knowledge, we are the first to report primary cilia in colon tissue and isolated colon
cells, which could be taken as an indication for the difficulty to see cilia in this tissue.
Otherwise how could loss of cilia affect crypt cell proliferation (if many are not ciliated to start
with)?
From the cultured colon epithelia cells, we assume that most of the epithelial cells in the colon crypt
are ciliated, but due to the technical hurdles described above, we reason to detect only a part of
them. This has now been clarified in the text on page 9
Also: perhaps the authors could comment on the cytoplasmic staining for Arl13b in the Ttll3 mutant
(figure 3A).
We fully agree with the referee that there is some cytoplasmic staining for Arl13b, which could be
in the stroma and is likely to be non-specific, as Arl13b is associated to the ciliary membrane (Cevik
et al, 2010). We have mentioned this in the legend to Fig. 3C.
2. The fact that 20% of ttll3-/- CEC cells still had cilia (figure 3E) means that glycylation is not
absolutely required for ciliogenesis. When the authors conclude that "our results support a model in
which the majority of cilia, primary as well as motile, depend on the presence of glycylation
catalyzed by either TTLL3, TTLL8 or both enzymes" it is not very clear what they mean. I think it
would be safe to say that absence of glycylation affects the efficiency of ciliogenesis but that it is not
absolutely required.
We fully agree with the referee, and we have changed the text accordingly.
3. With regard to the absence of cilia in the SW480 cell line and the statement that "These
observations confirm that absence of glycylating enzymes result in absence of primary cilia": there
are many other explanations for why SW480 cells might lack primary cilia other than the absence of
Ttll3; at best this is a correlation and does not confirm their hypothesis. The authors would have to
re-express Ttll3 in the SW480 cells and show rescue of ciliogensis to make this claim.
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We agree with the referee, and as already mentioned in the response of referee 1, we have decided to
remove the interpretation of the ciliogenesis data of the cell lines (see comments to referee 1, point
5). We have carefully re-read the manuscript and made sure that we do not over-interpret our data,
and that the interpretation that glycylation is important for primary cilia, but glycylation alone is not
sufficient to grow cilia is clear throughout the text. Indeed, expressing glycylase in cells that
otherwise not grow primary cilia did never lead to ciliogenesis in our hands (we have tried this
experiment with many different non-ciliated cell lines and different glycylation enzymes). This
could be due to the fact that overexpression of glycylases leads to massive modification of normally
non-glycylated cytosolic microtubules and perturbs the cells.
4. Presence of nuclear Cyclin D1 is a marker of proliferating cells; it is not specific to Wnt
activation of proliferation. So the statement that "This strongly suggests that the increased cell
proliferation ttll3-/- colon epithelium is initiated by an upregulation of the Wnt signaling pathway"
really has no support in the data shown. It may well be that Wnt signaling is driving the observed
proliferation, as it normally drives crypt cell proliferation, but the authors have not shown data in
support of this.
We thank the referee for pointing this out. The same point has been also raised by referee 1 and we
have now used the increased cyclin D1 levels to confirm our BrdU data on proliferation, and not to
draw conclusions on the signaling pathway (see response to point 4 of referee 1).
5. Inspection of Fig.3A indicates that a majority of the cilia are present in the top compartment
where the cells are more differentiated. Reduced presence of primary cilia on CECs of TTLL3
knockout mice could also be explained on account of delayed or incomplete differentiation prior to
their accelerated shedding. Can the authors comment on this alternative hypothesis?
Due to the difficulties in detecting cilia in immunohistochemistry, only a small number of ciliated
cells are seen in each crypt (see statistics in Fig. 3B). In this figure we have chosen an image in
which many cilia can be seen, but this was rather rare. In some other cases, ciliated cells were also
detected in the lower compartments, but none of our pictures could capture the presence of primary
cilia in all compartments. Thus we have chosen one of the best images that shows many cilia. We
have clarified this in the text page 8, 9, and in the figure legends.
The differentiation of the colon cells seems to be not significantly affected, as we did not observe
changes in differentiated cells such as goblet cells that can be visualized by Alcian Blue and PAS
staining as shown in Fig. S1. This has now been clarified in the text on page 10.
6. The in vitro analysis of cell proliferation and presence of cilia in CECs from different mice in
Fig.5S, suggests that reduced stability of CEC primary cilia, induced by loss of TTLL3 (glycylation),
favors cell proliferation. Do MEFs with compound deficiency of TTLL3 and TTLL8 (figure 1F) also
have higher in vitro proliferation rates compared to wildtype MEFs or those deficient in TTLL3 or
TTLL8 alone?
The referee has raised a very important point here. To address the referee’s question, we have first
tested if we could detect altered proliferation and cell cycle in MEFs transfected with shRNA for
TTLL8 used in the experiment shown in Fig. 1E and F. We repeated the experiment using different
transfection reagents, and analyzed the cells subsequently by flow cytometry. Unfortunately, using
this rather straightforward approach we are not able to obtain reliable data pointing towards altered
cell proliferation.
We thus decided to generate lentiviruses expressing the different control and TTLL8-specific
shRNAs in order to transfect the MEFs in a more homogenous and reproducible manner. The
viruses were successfully generated and tested, and were able to efficiently transduce the MEFs (as
confirmed by the expression of GFP from the virus genome).
Next we established a protocol that should take into account the timely order of events. It was
particularly important to deplete TTLL8-expression before ciliogenesis, and once ciliogenesis has
been induced (which is usually done by serum starvation and thus cell cycle arrest). Thus we used
the following protocol:
-
For lentiviral production, 293T cells were transfected with the virus constructs coding for the
respective shRNA and GFP, the VSV-G envelope-encoding plasmid and the packaging
plasmid. Supernatants were collected after 24 h.
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-
For infection, MEFs from control and ttll3-/- mice were seeded in 6-cm dishes and infected for
24 h using 1 ml of virus supernatant completed with polybrene (10 µg/ml). The transduction
efficiency was tested by GFP fluorescence (100%).
-
Cells were then washed in PBS and used for cell cycle experiments one passage after infection.
For this, the transduced MEFs were plated at a density of 200,000 cells/well into 12 welldishes.
-
After 24 h, the culture medium was changed to either normal medium (containing 10% FCS)
or starvation medium (containing 1% FCS).
-
Cells were cultured for an additional 48 h and then fixed in ethanol, stained with propidium
iodide, and analyzed for DNA content by FACS using a FACS Canto II (BD) machine.
Following this protocol we analyzed MEFs generated from six control and ttll3-/- mice under
starvation and normal cell culture conditions, however no consistent change in the partitioning of
MEFs into cycling (S and M phase) and resting (G1/G0 phase) has been found (we also controlled
the viability of the MEFs in these experiments by MTS assay and found no differences between
control and ttll3-/- cells). This might reflect the fact that the regulation of cell cycle progression by
primary cilium differs between cell types, as it might depend on additional factors. Thus, further
work is necessary to understand precisely how this regulation works.
It is already known that primary cilia can have opposite roles in different types of cancers, for
instance, in medulloblastoma or basal cell carcinoma, both, ciliary loss or overgrowth can be
associated with cancer progression, depending on the origin of the cancer (Han et al., 2009; Wong et
al, 2009). Establishing the precise mechanism of primary cilia and glycylation in other cell types
will thus be project that goes beyond the scope of the present manuscript. According to the
suggestions of the referees we have carefully re-written the manuscript to avoid overinterpretations
of our findings (including the title) and made clear that our data are relevant for the colon, and that
the question of the general mechanisms that link glycylating enzymes with primary cilia, and how
these control the cell cycle and cancer progression need to be investigated.
Minor points:
In Fig. 1 C it is not clear what the n values represent.
We have now added the numbers of the figure legend: number of experiments, and total number of
cells (at least 50 cells per experiment): control n=4 (total 200 cells); ttll3-/- n=4 (total 200 cells).
The sizes of mouse TTLL3 RT PCR amplicons shown in Fig. 1A are different than those shown in
Fig.2B and in testis control (Fig. 6A). It is not clear whether different primer pairs were used for
amplification or the position of the marker is shifted.
We thank the referee for having pointed this out. We indeed used different pairs of primers in Fig. 1
and 2 to amplify mouse TTLL3. In Fig. 1A, mTTLL3 is amplified with TTLL3-F1263 and TTLL3R2106, generating a 842-bp product. The PCR in Fig. 2B has been performed with
mTTLL3_C2028-F1 and TTLL3-R855, the product is 536 bp. Fig. 6A has now been removed. We
have clarified this in the supplementary material, page 12.
The images of tumor histopathology shown in Fig.5E are not clear in the printed size. These might
appear clear in online views and could be provided as supplemental data.
We agree with the referee and have transferred these images to the supplement.
Reference to Figure S5 is missing in the results section. This includes important details related to in
vitro proliferation potential of CECs.
This figure (now Fig. S6) is a control with little importance for the main text. It is cited in the
method section.
Typographic errors: The word "adherens", describing the epithelial junction has been misspelled as
"adherent" or "adherence"; extra word in sentence " can is present" (page 5 paragraph 2);
"positivie" (page 10, line 2).
We would like to thank the referee for carefully reading the text and pointing out these mistakes,
which have been corrected and for which we apologize.
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Accept letter
30 July 2014
Thank you very much for the revised study that has been carefully assessed by the original referees.
I am therefore pleased to accept the paper for publication in The EMBO Journal, pending minor
amendments to the text that should address the minor remarks in the attached comments.
-----------------------------------------------Referee #1:
This revised manuscript from Rocha et al has improved significantly since the first submission.
Many data and interpretations that were problematic were removed. The authors have clearly
demonstrated a role for TTLL3 in colon proliferation and predisposition to colon cancer. They have
for the first time shown that some monocilia stain positively for glycylation markers, and that
manipulation of TTLL proteins affects the ability of cells to form monocilia. The quality of the data
provided is high.
Remaining weaknesses in the study arise from the technical problems that the authors are unable to
directly measure TTLL3 or TTLL8 protein in cells or in vivo, and the authors are only able to detect
a very small number of cilia in colon tissue; and the general question as to why the authors do not
detect significant changes in Hedgehog signaling in the colon upon manipulation of TTLL3, even
though this signaling depends on cilia and is known to be particularly important in colon
differentiation versus carcinogenesis. Based on the entire pre-existing literature of TTLL proteins, it
is understandable that the authors tend to ascribe all actions of TTLL proteins to actions at cilia.
However, given the above issues, one wonders if it possible that these proteins also have some nonciliary action, generally affecting microtubule dynamics? These points might be added to the
discussion.
Minor point.
The authors state on page 15. "Using Arl13b, a primary-cilia specific marker, we were able to detect
primary cilia in colon tissue, however we do certainly not detect all cilia present in this tissue." This
would better be phrased either as "we likely do not detect all cilia" or "however, we do not detect
cilia on all cells found in this tissue"
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