Ms. Ref. No.: EJCB-D-12-00050
Title: Genes encoding cytoplasmic intermediate filament proteins of vertebrates revisited: identification of a cytoplasmic
intermediate filament protein in the sea anemone Nematostella
European Journal of Cell Biology
Received
Decision
Revision received
Accepted
Mar 25, 2012
May 16, 2012
Jul 31, 2012
Aug 03, 2012
Decision letter
Dear Thomas,
Your manuscript has now been seen by three referees whose comments are enclosed. As you will see from their reports, all
three think that this study makes a valuable contribution to the evolutionary history of the intermediate filament protein family.
Thus I am happy to say that I will be glad to receive a revised version of your manuscript. This revision will, however, require
some additional work. The reviewers really did an excellent job in making helpful suggestions on how to improve the manuscript.
Their comments mainly revolve around the way the data are presented and what to include in the phylogenetic comparison. I
suggest that you address the points they raised and revise and amend the manuscript accordingly.
Please submit a list of changes or a rebuttal against each point which is being raised when you submit the revised manuscript.
Yours sincerely,
Manfred Schliwa
Editor
p.p. Dagmar Gebauer
Editorial Office
European Journal of Cell Biology
Reviewers' comments:
Reviewer 1
The manuscript by Zimek et al. reports the sequence of a cytoplasmic intermediate filament
protein of the anthozoan Nematostella vectensis. After the discovery of nematocilins A and B
in Hydra vulgaris, this is the second report of a cytoplasmic IF protein in cnidarians, a basic
group of Eumetazoa. This manuscript will contribute to the elucidation of the evolutionary
origin of the IF proteins. This justifies publication in the EJCB.
Prior to the publication, the manuscript should, however, be thoroughly revised. Even a quick
database search revealed that the impact of this manuscript could be improved significantly.
Criticism and suggestions:
p5: 'Position of introns 2, 4, 8 and 10 are conserved with those in Nematostella lamin...' Reinspection
revealed that eight out of the 12 introns match in position with those of the
Nematostella lamin. These introns correspond to eight out of the nine archetypal introns in
lamin genes, highlighting the high degree of conservation between these two Nematostella
genes. The marking of intron positions in Figure 2 is hardly visible and is inaccurate
(accidentally slipped?). As in previous publications, the marks should exactly indicate, in
which position with respect to the codon the introns are located. Highlighting the archetypal
introns in the Figure would make this point clearer to the reader. In contrast, listing the size of
the introns is of little relevance in the context of evolutionary issues.
p5-6: '...and has no recognizable immunoglobulin-like domain (Fig. 1)' This must be a
printing error. In our opinion, there is clear evidence for presence of an Ig-like domain in
cytovec: The sequence alignments particularly in Figures 2 and 4 show significant sequence
similarities. Visual inspection of conserved residues present in lamin Ig-domains show a large
number of conserved residues in the cytovec sequence. Last but not least, protein structure
prediction of the relevant cytovec sequence (residues 478-582) on the Phyre server (Kelley
LA and Sternberg MJE 2009. Protein structure prediction on the web: a case study using the
Phyre server. Nature Protocols 4, 363 - 371) predicts an Ig-like beta-sandwich which matches
that of lamin tail domains (100% confidence, 98% coverage) (details on request). Similar
results were obtained when the I-TASSER program was used (Roy A , Kucukural A, Zhang
Y. I-TASSER. 2010. A unified platform for automated protein structure and function
prediction. Nature Protocols 5, 725-738; Zhang, Y. 2008. I-TASSER server for protein 3D
structure prediction. BMC Bioinformatics, 9, 40).
p4: 'In our work, we identify the cytoplasmic protein cytovec, a putative IF-forming protein
with the highest degree of similarity to the cytosolic IF proteins nematocilin A and B from
Hydra vulgaris...' EST sequences from public data banks reveal that cytoplasmic IF proteins
from another Anthozoan, Acropora digitifera, and a Hydrozoan, Clytia hemisphaerica, are
much closer to cytovec than the Hydra nematocilins (see attached alignment). Nematostella
cytovec and the Clytia protein share 44.8% identical amino acid residues in a region of 627
residues, which covers the head, rod and the entire tail domain of the Clytia protein.
Nematostella cytovec has a C-terminal extension of 135 extra residues, which has no
counterpart in the Clytia and Acropora sequences.
Figure 4: Figure 4 shows a sequence alignment of cytovec with six lamins. The choice of the
Nematostella lamin for this comparison is immediately apparent. The reasons for the selection
of the other lamin protein sequences is not entirely clear to me: (i) The genomes of C. elegans
and Drosophila are among those, that show a very strong sequence drift. (ii) Vertebrate Atype
lamins are the most derived members of the vertebrate lamin family. (iii) Why three Atype
lamins are included? I would expect that a comparison between cytovec and lamins
could be more meaningful when also representatives of more basic phylogenetic groups were
included (Porifera, Placozoa) and when lamins from species with less genetic drift would be
selected. Instead of three A-type lamins as representatives of the vertebrate lamins, one could
include one representative from each of the four different vertebrate lamin subtypes, e.g.
lamin B1, B2, LIII, and lamin A.
Figure 5: Why are lamins and cytoplasmic IF proteins combined in a single phylogenetic tree?
Nematostella lamin and cytovec are members of two different protein (sub)-families. Such
phylogenetic trees are useful if they are calculated for proteins of a particular type. Trees
created for different proteins can then be compared. The combination of two types of proteins
from which we assume that they have been separated early in evolution (lamins and
cytoplasmic IF proteins) in a single tree is not clear to me.
Minor points:
- Figure 5 is not mentioned in the text.
- A sizeable number of Nematostella EST sequences are available, which cover the entire coding sequence of the
cytovec gene (details on request). Mentioning this fact would be beneficial.
- p6: coil 1A (consistent with capital letters)
- p7, 2nd line: Correct the spelling of species names.
- Colour coding of identical residues in the sequence alignments is not consistent at some positions.
- Marks of intron positions in the alignment are hardly visible and do not fit in several positions (see above).
In conclusion: This manuscript should be carefully re-edited and the points mentioned above
should be corrected before publication in EJCB. The reviewer(s) are willing to provide the
data mentioned above.
Reviewer 2
Zimek et al. used a similarity search against the genome of the sea anemone Nematostella vectensis to identify an intermediate
filament protein which is similar to the protein nematocilin from another cnidarian, Hydra vulgaris. They then present a series of
alignments and a small phylogenetic tree which they interpret as showing that the protein ("cytovec") is a cytoplasmic IF, and as
evidence that the family of bilaterian cytoplasmic IF proteins were present in the common ancestor of Cnidaria and Bilateria (I
assume this is what is meant by "already present in cnidarians").
Hwang et al. [2008, Mol Biol Evol 25:2009-17] have previously presented some great work on nematocilin in Hydra, which they
show is a lamin-like IF protein (lacking NLS and farnesylation sites) localised to the cnidocil (a specialised cilium). They further
showed that the gene arose through lineage-specific gene duplication of lamin, but only found orthologues in Cnidaria|Hydrozoa
and not Cnidaria|Anthozoa (including Nematostella).
The key questions here are: 1) how does this work relate to that of Hwang et al.? 2) why was this protein missed in that study
(new gene model? improved coverage?)? and 3) what evidence is there that this is an orthologue of bilaterian cytoplasmic IFs
(rather than being a straight orthologue of nematocilin)? While I found the work here to be potentially very interesting, none of
these questions has yet been properly addressed.
Since the authors are presenting no biological data, the definition of this protein as "cytoplasmic" is based purely on sequence
similarity to experimentally characterised proteins. This is extremely important, as all the data seem to point to this being a
lamin-like protein: shared intron structure; stronger sequence similarity to lamins rather than bilaterian cytoplasmic IFs (Fig 1/4
vs. 2/3); phylogenetic affinity to lamins (allbeit in a tree with only 1 bilaterian cyto IF, Fig 5). Which makes me conclude that the
authors have simply identified the lamin-like orthologue of nematocilin in Nematostella (in which case, the new name "cytovec"
should not be used). This would be interesting, but certainly isn't evidence that bilaterian cytoplasmic IFs predate Bilateria.
All of this could be resolved by a simple, bootstrapped (or otherwise supported) phylogeny including lamins and also bilaterian
cytoplasmic IFs. This would also make most of the sequence alignments and waffle about percentage identity redundant,
considerably improving the manuscript, in my opinion.
Table 1 provides no useful information for the analysis and should be removed.
Reviewer 3
The authors present a very interesting finding concerning the presence of a gene encoding an intermediate filament protein in
the sea anemone Nematostella. Using as a probe, an intermediate filament gene from Saccoglossus kowalevskii, they searched
the genome assembly of the sea anemone and identified a putative intermediate filament, which they call "cytovec".
In Figure 1, the authors show the alignment of cytovec with Nematostella lamin and with Hydra v. nematocilin A and B. I find
that the dark blue color makes it difficult to see the actual letters representing amino acids. Also, the authors do not highlight the
YRKLLEGEE sequence, and I was not able to locate it in Figure 1.
The phylogenetic tree analysis provides convincing data that cytovec forms a sister group with the hydra nematocilins A and B.
This group is monophylectic with the Ifs of Saccoglosssus, Strongylocentrotus, and Ciona. Furthermore, Nematostella lamin
clusters with other lamins including those found in fish, amphibian, and mammals. I understand why isomin was used as the
outgroup, and why hydra, Saccoglossus, Strongylocentrotus, and Ciona were used, but it would be interesting to include a
vertebrate IF in the tree since vertebrate lamins are included. Perhaps the authors have done this and could comment on their
observations in the discussion section.
--------------------------------------------------------------------------------------------------------------------------------------------------------Authors response letter
Reviewer 1:
We thank the reviewer for his helpful comments which we followed in the revised version. P5 and later: Intron positions are now
labeled black on white background in all figures to mark them very clearly. Marking of 2 amino acids indicate intron position in
the middle without codon interruption. Marking of 1 amino acid indicates that the exon-intron boundary interrupts the
corresponding codon.
P5-6: we agree on the presence of an Ig –like domain and have included a new Figure 2 that models the position accordingly as
suggested.
P4: we agree on the similarity of the putative cytoplasmic cytovec protein with other Anthozoan proteins from Acropora digitifera
and the Hydrozoan Clytia h.. This has been changed in the text and the sequences have been included in the new Figure 3. We
point out that the genome project of Clytia h. is incomplete and that at present one can’t exclude the occurrence of a second IF
protein.
Figure 4: we have chosen the aligment of the selected lamins to illustrate the limited but distinct extent of conservation among
these lamins.
Figure 5: As one can see from the comments of all reviewers, the views on whether to in- or exclude cytoplasmic IF from the
phylogenetic tree differ widely. We have chosen to preserve a single tree in order to accommodate the fact that all these
proteins share most likely one common ancestor. In the revised version, we have now marked 3 groups, namely the putative
cytoplasmic “cytovec” cluster, the lamin cluster and one that contains select cytoplasmic IF proteins from other species. In this
way, the figure illustrates well the various relationships.
We now mention the Figure 5 in the text.
The Nematost. EST sequences are mentioned in the text.
P6 and p7: we have corrected spelling.
Colour coding: we have made it consistent.
Intron positions: see above comments, have been clearly marked.
Reviewer 2:
We thank the reviewer for his insightful suggestions.
We have clarified what we mean by “already present”… You are right in your assumption.
Regarding the work of Hwang, which we had cited in the first version of our manuscript, we point out that the sequence we
present here was not contained in the version used by these authors. The reviewer is correct that at present, without
experimental data, one can’t be certain whether cytovec which we identified is a true cytoplasmic IF protein. We are fully aware
of this and were cautious in using the term “putative”. As one can see now from the inclusion of additional sequences of
cytoplasmic IF proteins from Acropora digitifera and the Hydrozoan Clytia h., cytovec is considerably closer to these than to its
lamin counterpart. This and the absence of a recognizable NLS add to the likelihood of cytovec being cytoplasmic.
The revised version of the phylogenetic tree further adds to the grouping of cytovec.
We would like to maintain Table 1 in the manuscript because it relates data from invertebrate IF proteins to corresponding
vertebrate keratins, presented in a seminal paper by Strnad P, Usachov V, Debes C, Gräter F, Parry DA, Omary MB., J Cell Sci.
2011 Dec 15;124(Pt 24):4221-32. Epub 2012 Jan 3; “Unique amino acid signatures that are evolutionarily conserved distinguish
simple-type, epidermal and hair keratins”.
Reviewer 3:
We thank the reviewer for his positive comments. To improve readability, we have changed the colour coding of all figures.
We agree on the need to highlight the YRKLLEGEE sequence and have marked it in all figures by a black line.
We thank the reviewer for the proposal to include a vertebrate IF sequence in the phylogenetic tree. In the revised version, the
groupings of sequences have been clearly marked and in analogy to human lamins, we have included human keratins K8 and
K18.