Ms. Ref. No.: EJCB-D-12-00056
Title: A rod domain sequence in segment 1B triggers dimerisation of the two small Branchiostoma IF proteins B2 and A3
European Journal of Cell Biology
Received
Decision
Revision received
Accepted
Mar 28, 2012
May 08, 2012
May 27, 2012
Jun 05, 2012
Decision letter
Dear Dr. Karabinos,
Your manuscript has been seen by two referees whose comments are enclosed. As you will see from their reports, they are, in
principle, in favour of publication, though their level of enthusiasm is understandably moderate. The reason is that while the data
appear to be solid and worth reporting, the way they are presented, and the clarity of presentation, leave much to be desired.
Both reviewers provide you with detailed and specific comments on how to improve the manuscript, and it will be mandatory that
you heed their advice in all points. In addition to improving the scientific aspects of your paper, it will also be necessary to work
on the logic of argumentation and the intelligibility of the narrative. In this respect I would ask you to consult a native English
speaker. Should you feel in a position to meet all the criticisms raised, I will be happy to receive a suitably revised manuscript in
due course.
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 authors characterize sequences in the Branchiostoma IF proteins B2 and A3 that are required for heteromerization, using
overlay assays. The data are solid and help to understand the IF system in this organism. What is needed to make the
manuscript understandable and attractive to a wider audience is clarity in writing and shortening in parts. In many places, it is
not clear to a reader unfamiliar with the field what the aims are. Regarding the data, can one rigorously exclude homodimer
formation of biotinylated species used in overlay assays? If this happens for the small B2 fragment, it may explain its apparent
failure to bind to A3 under assay conditions?
Abstract:
Branchiostoma expresses 5 IF proteins, the pairing of which is not fully understood. While B1 can homopolymerize, its close
relative B1 forms heterodimers with A3. What is the state regarding A1 and A2?... What do you mean by "muscle tails"?
The abstract should be concise. What is the aim, the approach, the results and the conclusion? Not all details need to be listed
in the abstract.
Here, we characterized sequences in A3 and B2 required for heterodi(?)merization, using recombinant protein fragments as
probes in overlay assays dedicated to reveal protein interactions….What do you mean by "should have the potential"? What is
new in your findings and this conclusion?
Introduction: Given that Branchiostoma is not widely used, it would be helpful to briefly introduce the expression pattern of the
known IF proteins and to clarify which are bona fide heteropolymers and which are homopolymers. In the context of the
manuscript, evolutionary aspects are less important, but the open questions should be clear at the end of the introduction.
Results
P8 middle para: In order to …sentence not clear
P8 lower para: not clear how the assay is done from the text. Did you pre-mix B2 plus A3 to enhance B2 solubility? End of para:
why do you expect that presence of full length B2 prevents aggregation of B2-c1?
Discussion
It is not clear to me why one should expect a trigger motif common to all IF proteins? Clearly, keratins do not mix with any of the
other types and even A and B type lamins form distinct oligomers.
Figures
Shorten legends where possible. Keep methods to methods section. Figure 3: what does A, B,…k, Y,X mean?
Recommendation: Publish following improvement in clarity, style and language.
Reviewer 2
Karabinos et. al. present an article about the heteropolymeric intermediate filament (IF) A3/B2 from lancelets. The
Cephalochordata to which lancelets belong, diverged from the vertebrates more than 500 million years ago and comparisons
between these two subphyla of the chordata might tell us much about the evolutionary development of humans and other
vertebrates. The recently discovered IF variants A1, A2, A3, B1 and B2 cannot be classified in any of the hitherto defined IF
types I-VI and they are, to our current knowledge, specific to lancelets. The current work follows the publication from Karabinos
et. al. in JMB from 2002 and analyzes the early interactions during filament assembly between the A3 and B2 IF proteins in
more detail. They are using a buffer containing 4M urea to solubilize the protein and use technique based on Western blotting
they call "Blot overlay". One of the protein interaction partner is immobilized on a nitrocellulose membrane, then incubated with
its biotinylated binding partner and binding is detected by a horse-radish peroxidase-conjugated streptavidine. They generated a
smart combination of A3 and B2 mutants with coil 1 or coil 2 of the filament rod deleted. They also generated a series of
mutations where they swapped the coil 1 or coil 2 region with its homologue from the B1 IF variant which forms homopolymeric
filaments as controls. They further present a CD spectrum of the B2-c2 mutant to prove that the observed absence of binding is
not due to misfolding of the fragment. They then further confine the interaction region of B2/B3 by chemical cleavage of the
protein fragments. Finally they analyze the salt-bridge pattern within the identified binding region and propose the presence of
salt-bridge pattern similar to the coiled-coil trigger sequence found in cortexillin but without providing system specific
experimental data that these ionic interactions are indeed the initial interactions that trigger coiled-coil formation and allow
intermediate filaments to form.
The experiments are well designed, the results are convincing and the Blot overlay data alone merit publication.
I recommend the paper for publication. There are a few issues the authors should address:
? Title: "of the two small Branchiostoma IF proteins". Please comment on "small", this is not discussed in the introduction.
? Abstract: "can form heteropolymeric IF with A3 essential in the formation, based on ...". Do you mean "formation of
filaments?"
? Please state that the sequences were derived from Branchiostoma floridae somewhere in the introduction. There are
approximately 22 different species. (I finally found the information in M&M).
? Circular dichroism in Tris buffer and <beta>-mercaptoethanol is tricky due to its high absorbance. Can you show the
standard deviations of three measurements in the spectrum? What was the cuvette length? How was the <alpha>-helicity
calculated? (Just indicate the base values used for 100% helicity/random coil, the reference Greenfield 1969 is provided). The
blot overlay was done in presence of 4M urea, but the CD spectrum was not. Can you comment on that? How does the
spectrum of B2-c2 look in the urea-containing Blot overlay buffer? How did you solubilize A3 for the CD study?
? B2-c2 seems to be quite soluble (Fig. 4B). Did you do a Blot overlay (as in Fig. 2 bottom) in urea-free buffer also and how
does it look like? Did the urea denature the protein fragment and prevent binding? Please comment more on how the 4M urea
that was used to solubilize the proteins affects the binding essay.
? The possibly disruptive effect caused by the biotin label (which binds to lysines!), SDS denaturation or adsorption to
nitrocellulose membrane regarding the observed absence of binding of B2-c2 to A3 is discussed by the authors and due caution
is urged in the interpretation of the result. -> Fair enough! (just a comment, not an issue)
? The Urea containing Blot overlay buffer had a pH of 7.5, the urea-free blotting buffer had pH 9.0, the CD buffer had pH 9.0.
What was the reason to choose different pHs?
? Why is there is no salt in the urea-containing Blot overlay buffer. Can you comment on that?
? Page 7: "In these chimeras both the B2 ... it is present in the deletion mutants, described above (...)." I have a hard time to
understand these two sentences. What does "it" refer to? Are you trying to say that the domain swap was performed in a way
that kept the heptad repeat pattern undisrupted?
? Fig6A description: "Arrows pointing down mark the start of segment 1B". Can't see any other arrows than those marking the
ionic interactions.
? M&M Nucleic acid techniques: Construct B2-c2/t is mentioned in the first sentence. I cannot find any reference to this
construct in the text. Do you mean B2-c2?
? Consider to move the cloning primers into supplementary information (just a suggestion).
? M&M Protein techniques: The ECL chemiluminescence kit is now produced and sold by GE and Pierce products are now
marketed by Thermo Fisher. (Bwt.: Chemiluminescence is misspelled in the text as "Chemiluminiscence".)
? Discussion: The authors state: "In general, however, we think that there need not be a trigger sequence motif common to all
IF." I agree with the authors.
--------------------------------------------------------------------------------------------------------------------------------------------------------Authors response letter
Dear Dr. Schliwa,
Please find enclosed a revised version of our manuscript entitled, „A rod domain sequence
in segment 1B triggers dimerisation of the two small Branchiostoma IF proteins B2 and
A3“ (MS EJCB-D-12-00056), authored by myself and my colleagues J. Schünemann and
David. A.D. Parry. This version has been read and corrected by a native speaking scientist to
improve the presentation as suggested by both reviewers. We have also followed all of their
other recomendations and made changes, as described below, with respect to each of them.
The changes in the revised paper are indicated in yellow.
Reviewer 1:
1. „Regarding the data, can one rigorously exclude homodimer formation of biotinylated
species used in overlay assays? If this happens for the small B2 fragment, it may explain its
apparent failure to bind to A3 under assay conditions?“
Yes, it would be an interesting possibility to explain the binding failure of the B2-c2 but, in
our many overlay experiments, we have never seen homotypic interactions of the protein B2
or its fragments.
2.„Abstract:
Branchiostoma expresses 5 IF proteins, the pairing of which is not fully understood. While B1
can homopolymerize, its close relative B1 forms heterodimers with A3. What is the state
regarding A1 and A2?“
The A1 and A2 proteins exhibit 93% identity over their rod domains. In addition, both show a
very close relationship to the protein A3, sharing with it about 90% identity in the rod.
Moreover, the Ab specific to both A1 and A3 proteins revealed the same expression pattern
as documented for the corresponding B2 specific Ab (Karabinos et al., 2002). Finally, our
unpublished results have demonstrated specific interactions between the A1 and B2 rod
fragments in blot overlays. Thus, we believe that the A1 and A2 proteins form obligatory
heteropolymeric filaments with B2 in a similar manner to that demonstrated here and in our
previous paper for A3 (Karabinos et al., 2002) and that the A1, A2, A3 and B2 Branchiostoma
proteins form another heteropolymeric keratin-like IF system (Karabinos et al., 2002).
3. „What do you mean by "muscle tails"?“
In has been shown by means of electron micrographs that, in common with nematodes and
echinoderms, Amphioxus possesses muscle fibres or muscle tails which make contact between
the muscle and the nerve cord by means of fine cytoplasmic extensions. These structures are
of mesodermal origin and reveal, in contrast to the axial muscle, expression of the IF protein
B1 (Karabinos et al., 2001).
4. „The abstract should be concise. What is the aim, the approach, the results and the
conclusion? Not all details need to be listed in the abstract.“
We have now rewritten the abstract to describe more clearly the aim, the approach, the
results and the conclusion of the study.
5. „What do you mean by "should have the potential"? What is new in your findings and this
conclusion?
We replaced this sentence in the Abstract by: „Thus, a common and essential feature of
trigger sequences with different primary structures found so far in IF and other coiled coil
proteins seems to be their ability to form multiple inter-chain ionic interactions which brings
the chains close to one another and allows coiled coil formation to propagate accordingly.“.
The new finding is that trigger elements in coiled coil proteins relate not to a conserved
sequence motif but to the ability of the sequence to make multiple inter-chain ionic
interactions.
6. „Introduction: Given that Branchiostoma is not widely used, it would be helpful to briefly
introduce the expression pattern of the known IF proteins and to clarify which are bona fide
heteropolymers and which are homopolymers. In the context of the manuscript, evolutionary
aspects are less important, but the open questions should be clear at the end of the
introduction.“
We included a short paragraph into the Introduction describing the expression patterns and
polymerisation properties of 13 currently known IF proteins from Branchiostoma.
7.„Results
„P8 middle para: In order to directly exclude possibilities that the A3-binding incompetence
of the B2-c2 fragment, documented in the experiments described above, is not due to nonnative protein folding, the B2-c2 polypeptide was investigated by circular dichroism (CD)
spectroscopy. .sentence not clear“
We revised this sentence as follows: „In order to directly exclude the possibility that the
incompetence of the B2-c2 fragment to bind A3, documented above, is not due to a wrong
protein folding, the B2-c2 polypeptide was investigated by circular dichroism (CD)
spectroscopy.“
8. „P8 lower para: not clear how the assay is done from the text. Did you pre-mix B2 plus A3
to enhance B2 solubility?“
Yes exactly, the pre-mixing of B2 or B2-h/c1 (both are insoluble in the used buffer) with A3
dramatically increases the solubility of both B2 proteins.
9. „End of para: why do you expect that presence of full length B2 prevents aggregation of
B2-c1?“
The mixture of B2-c1 with B2 was used in the study as a negative control. This information
has now been included in the text.
10.“Discussion
It is not clear to me why one should expect a trigger motif common to all IF proteins? Clearly,
keratins do not mix with any of the other types and even A and B type lamins form distinct
oligomers.“
We fully agree with the reviewer and document it in the following sentence in the discussion:
"In general, however, we think that there need not be a trigger sequence motif common to all
IF."
11. „Shorten legends where possible. Keep methods to methods section. Figure 3: what does
A, B,.k, Y,X mean?
We have now shortened the figure legends without a loss in content. We also explained the
meaning of the „letters:A, B,.k, Y,X ...“ in the figure legend to Fig. 3.
Reviewer 2:
1. „Title: "of the two small Branchiostoma IF proteins". Please comment on "small", this
is not discussed in the introduction.“
The following sentence was inclued in the Introduction to explain the term „small“: „
Interestingly, both IF proteins A3 and B2, which have been previously cloned from the
Branchiostoma floridae, essentially lack a tail domain and are therefore designated as
„small” (Karabinos et al., 2002).“
2. „Abstract: "can form heteropolymeric IF with A3 essential in the formation, based on ...".
Do you mean "formation of filaments?"
Yes, we thank the reviewer for this remark and we now include „of filaments“ into the above
sentence in both the Abstract and the Introduction.
3. „Please state that the sequences were derived from Branchiostoma floridae somewhere in
the introduction. There are approximately 22 different species. (I finally found the
information in M&M).“
We have now mentioned the origin of both proteins in the Introduction (see the point 1
above)
4. „Circular dichroism in Tris buffer and <beta>-mercaptoethanol is tricky due to its high
absorbance. Can you show the standard deviations of three measurements in the spectrum?
What was the cuvette length? How was the <alpha>-helicity calculated? (Just indicate the
base values used for 100% helicity/random coil, the reference Greenfield 1969 is provided).
The blot overlay was done in presence of 4M urea, but the CD spectrum was not. Can you
comment on that? How does the spectrum of B2-c2 look in the urea-containing Blot overlay
buffer? How did you solubilize A3 for the CD study?“
We did several CD measurements and got almost identical results. It was probably because
we used only low concentrations of Tris and betaME thereby producing very stabile CD
spectra in the range from 200 nm upwards, which are essential for CD calculations (Fig. 4A).
However, the Bradford which we used for protein concentration measurements, defines
somehow the stringency of our CD experiments. The cuvette path length was 0.2 cm and we
have now included this information in the section Materials and Methods. The secondary
structure of the measured proteins was calculated using the k2d computer program for
protein secondary structure prediction. This information and the relevant reference have been
included in the Materials and Methods section. We never used any urea containing buffer for
CD spectroscopy and we could not therefore provide CD spectra of the B2-c2 in such buffer.
The A3 protein was dialysed for three hours at room temperature against the „CD-buffer“.
5. „B2-c2 seems to be quite soluble (Fig. 4B). Did you do a Blot overlay (as in Fig. 2 bottom)
in urea-free buffer also and how does it look like? Did the urea denature the protein fragment
and prevent binding? Please comment more on how the 4M urea that was used to solubilize
the proteins affects the binding essay.“
Unfortunately, we are not able to provide a rational explanation on exactly how urea at
different concentrations can affect solubility and/or binding properties of different IF
proteins. In our overlay experiments we usually use buffers containing different
concentrations of urea in order to prevent agregation of the analysed IF proteins. The few
experiments which we made without urea, including this one with B2-c2, were almost
completely negative.
6. „The Urea containing Blot overlay buffer had a pH of 7.5, the urea-free blotting buffer had
pH 9.0, the CD buffer had pH 9.0. What was the reason to choose different pHs?“
The overlay technique was performed in the standard buffer which was previously
successfully applied for different IF proteins incluing these of Branchiostoma and C. elegans
(Hatzfeld et al., 1987, Karabinos et al., 2002, 2003). Generally, however, the blot overlay
system looks very robust accepting buffers of different ionic strengths as well as ureaconcentrations ranging from 1 to 5-6 M. For our CD experiments we used the buffer which
was very similar to that which we used in our urea-free binding experiments, mainly because
this urea-free buffer keeps both proteins B2-c2 and A3 soluble.
7. „Why is there is no salt in the urea-containing Blot overlay buffer. Can you comment on
that?“
Please look at our discussion in point 6.
8. „Page 7: "In these chimeras both the B2 coil 1 and B2 coil 2 segments, respectively, were
put within the context of the full length IF molecule. This, it is proposed, will provide better
conditions for optimal folding of the analysed heptad repeat-containing long helices as it is
present in the deletion mutants, described above." I have a hard time to understand these two
sentences. What does "it" refer to? Are you trying to say that the domain swap was performed
in a way that kept the heptad repeat pattern undisrupted?“
In order to clarify the meaning of this sentence we changed it as follows“ In these chimeras
both the B2 coil 1 and B2 coil 2 segments, respectively, were put within the context of the full
length IF molecule. This domain swap kept the heptad repeat containing helices undisrupted
and gives them better conditions for optimal folding.“
9. „Fig6A description: "Arrows pointing down mark the start of segment 1B". Can't see any
other arrows than those marking the ionic interactions.“
We apologize for this mistake and provide a new Fig.6 containing the missing arrows.
10. „M&M Nucleic acid techniques: Construct B2-c2/t is mentioned in the first sentence. I
cannot find any reference to this construct in the text. Do you mean B2-c2?“
We thank the reviewer for this remark. We corrected the wrong name of the construct B2-c2
in two places in the M&M section.
11. „Consider to move the cloning primers into supplementary information (just a
suggestion).“
We agree with this suggestion and completely removed the primer sequences from the M&M
section using the comment : „The primers used for preparation of the deletion and chimeric
mutants, described above, are available upon request. “
12. M&M Protein techniques: The ECL chemiluminescence kit is now produced and sold by
GE and Pierce products are now marketed by Thermo Fisher. (Bwt.: Chemiluminescence is
misspelled in the text as "Chemiluminiscence".)“
We replaced the old company name by „Thermo Fisher Scientific Inc., Waltham, MA, USA“
and corrected also the spelling of the product.
With best regards,
Sincerely yours,
Anton Karabinos